CN111848277A - Research method of sorghum pesticide-fertilizer composite seed coating agent - Google Patents

Abstract

The invention discloses a research method of a sorghum pesticide-fertilizer composite seed coating agent in the field of sorghum pesticide-fertilizer, which specifically comprises the following steps: s1: screening a seed coating pesticide formula: s1.1: aiming at the sorghum head smut and main underground pests, screening out proper bactericides and insecticides by using methods such as indoor toxicity measurement, seed safety test and the like, and determining the optimal dosage of the bactericides and the insecticides; s1.2: screening out a proper growth regulator by utilizing an indoor seed germination method according to the growth rule of sorghum, and determining the optimal using amount of the growth regulator; s2: screening a seed coating agent micro-fertilizer formula; s3: screening a seed coating agent auxiliary agent formula; s4: detecting the technical indexes of the seed coating agent; s5: the biological effect evaluation of the seed coating agent can research the sorghum seed coating agent which can promote seed germination and seedling growth, prevent and control seed transmission and main plant diseases and insect pests in the seedling stage and reduce the application amount of chemical fertilizers and pesticides.

Description

Research method of sorghum pesticide-fertilizer composite seed coating agent

Technical Field

The invention relates to the technical field of sorghum insecticide-fertilizer, in particular to a research method of a sorghum insecticide-fertilizer composite seed coating agent.

Background

The seed coating agent is developed by traditional seed soaking and dressing, is prepared by processing pesticide, chemical fertilizer, plant growth regulator, film forming agent and other auxiliary agents by adopting modern technical equipment, is coated on the surface of plant seeds, and is a standardized and commercialized special medicament form (Zhaolii, Leili and the like, 2009). It has been shown that seed coating with seed coating can significantly increase crop yield and prevent the occurrence of plant diseases and insect pests (songming et al, 1989) seed coating is of great interest as a special pesticide formulation because of its better protection, systemic and sustained release effects (municipality et al, 1998). And due to the characteristic of accurate pesticide application, the use amount of pesticides can be greatly reduced, so that the pollution of the pesticides to the atmosphere and the ecological environment is reduced (Wuling cloud, etc. 2007).

The research and popularization of four crops of wheat, corn, rice and cotton are used as the key points of seed engineering projects by China department of agriculture as early as the period of 'nine five' and 'fifteen'.

Sorghum is one of the important dry field crops in China, and is a main crop for resisting drought, preventing flood and relieving disaster due to the characteristics of multiple resistance, high photosynthetic effect, strong heterosis and the like. (xu ben, 2009). But at present, few researches and reports about sorghum seed coating agents at home and abroad are available. The active ingredients of the existing seed coating agent are mainly concentrated in carbofuran, thiram, carbendazim and the like, and the using amount in the agricultural production process is reduced year by year due to the resistance and toxicity problems; and the compounds with higher activity such as tebuconazole, imidacloprid, prochloraz and the like can be popularized and applied (Shiyafei, 2011). Therefore, the research on the novel, multiple-effect and environment-friendly sorghum seed coating agent becomes a problem to be solved in the current agricultural development.

Based on the research, the invention designs a research method of the sorghum pesticide-fertilizer composite seed coating agent, and aims to research the sorghum seed coating agent which can promote seed germination and seedling growth, prevent and control seed transmission and main plant diseases and insect pests in the seedling stage, and reduce the application amount of fertilizer and pesticide. Thereby contributing to the enrichment and development of the sorghum cultivation technology and the seed coating theory and technology, the quality safety of agricultural products, the ecological environment protection and the sustainable and stable development of agriculture.

Disclosure of Invention

The invention aims to provide a research method of a sorghum pesticide-fertilizer composite seed coating agent, and aims to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme: a research method of a sorghum pesticide-fertilizer composite seed coating agent specifically comprises the following steps:

s1: screening a seed coating pesticide formula:

s1.1: aiming at the sorghum head smut and main underground pests, screening out proper bactericides and insecticides by using methods such as indoor toxicity measurement, seed safety test and the like, and determining the optimal dosage of the bactericides and the insecticides;

s1.2: screening out a proper growth regulator by utilizing an indoor seed germination method according to the growth rule of sorghum, and determining the optimal using amount of the growth regulator;

s2: screening a seed coating agent micro-fertilizer formula:

aiming at the soil characteristics of a main sorghum planting area and the growth rule of sorghum, measuring the physiological characteristics of the sorghum by using a potting method, screening out proper trace elements and determining the optimal proportion;

s3: screening a seed coating agent auxiliary agent formula: according to the requirements of the seed coating formulation and the requirements of GB/T17768-1999 standard, screening out the auxiliary agent which meets the quality standard of the seed coating, and determining the optimal dosage;

s4: detecting the technical indexes of the seed coating agent: according to the GB/T17768-1999 quality standard requirement of the seed coating, the seed coating which is researched is technically detected, and the indexes mainly comprise dispersibility, stability, film forming property, shedding rate and the like;

s5: seed coating biological effect evaluation: the researched seed coating agent is biologically evaluated by pot culture and field test, and the indexes of germination rate, root length, enzyme activity, pest control effect, yield and the like are measured.

Preferably, the S1 specifically includes the following steps:

(1) selection of test materials:

(1.1) test organisms:

the strain is as follows: the sorghum head smut is provided by the plant pathology laboratory of the college of agriculture of Guizhou university;

testing insects: grubs (Scarabaeoidea), provided by the institute of insects at the college of agriculture of the university of Guizhou;

seed: sorghum seeds are Jinliangluo No. 1, and are purchased from Zhuohao agriculture science and technology Limited in Zunyi, Guizhou province;

(1.2) reagent for test:

carbendazim powder (Wuhanrong Brilliant Biotech Co., Ltd.);

triadimenol powder (Wuhanrong Brilliant Biotech Co., Ltd.);

tebuconazole raw powder (Wuhanrong Brilliant Biotech limited);

cyantraniliprole technical material (Shanghai Dupont agrichemical Co., Ltd.);

chlorantraniliprole (Shandong Weifang Runfeng chemical Co., Ltd.);

clothianidin technical (Xianlong chemical corporation, Hubei);

(1.3) test plant growth regulators:

brassinolide original drug (scientific and technological limited lan yue, Sichuan province);

paclobutrazol (anyang quanfeng biotechnology limited);

uniconazole (anyang quanfeng biotechnology limited);

(2) the test method for screening the bactericide and the optimal dosage thereof comprises the following steps:

(2.1) indoor biological virulence determination:

(2.1.1) activation of bacterial species

The preserved strain of Sphacelotheca reiliana (Kuhn.) Clint.) was taken out of the refrigerator at 4 ℃. Picking hyphae with a small amount of culture medium from a test tube on a superclean bench, putting the hyphae into a plate culture medium to be inoculated, sticking a label after inoculation, inversely placing the hyphae into a constant temperature incubator at 28 ℃ for culture, and waiting until the diameter of the bacterial colony grows to 2/3 of the diameter of a culture dish for later use;

(2.1.2) preparation of drug-containing Medium

Dissolving raw powder of the medicament by using methanol and 0.1 percent TWEEN-80, diluting the raw powder by using sterile distilled water to prepare a mother solution of 10g/L, preparing 5 series of concentrations by using the sterile distilled water on the basis of a pre-test, sterilizing a PDA culture medium, cooling the sterilized PDA culture medium to about 50 ℃, adding 1mL of prepared medicament into 9mL of the culture medium, fully shaking the mixture to obtain a medicament-containing culture medium containing the medicaments of each series of concentrations, adding equivalent sterile water as a reference, and repeating the treatment for 3 times;

(2.1.3) virulence determination

Measured by a hyphal growth rate method. Placing pre-cultured test strain colonies on an ultra-clean workbench, punching a bacterial dish along the same circumference of the edges of the bacterial colonies by using a puncher with the diameter of 5mm, then sticking one surface of the bacterial dish containing hyphae to the center of a medicament-containing culture medium, inversely placing the bacterial dish in a constant-temperature incubator at 25 ℃ for culturing for 72 hours, and measuring the diameter of the bacterial colonies treated by each medicament by using a cross method, wherein the diameter of the bacterial colonies not treated by the medicament is used as a reference;

(2.1.4) data processing and statistical methods

Converting the hypha growth inhibition rate into an inhibition rate value Y, converting the medicament concentration into a concentration log X, obtaining virulence formulas of different bactericides according to a regression method of the concentration log and the probability value, calculating the inhibition medium concentration (EC50) value of each medicament on the ustilago sorghum smut and the rhizoctonia sorghum seedling by using the virulence formulas, selecting one medicament with the best effect to be matched with the pesticide in a combined manner, setting 3 different proportions, and judging whether the two medicaments influence each other;

(2.2) seed safety test

Adopting a seed germination rate test, selecting a pesticide with the best effect on the basis of a pre-test, and setting 5 concentrations; soaking the seeds for 24 hours, placing the sorghum seeds soaked in the seeds in groups in a culture dish with 2 layers of soaked gauze at the bottom, marking the medicament, the concentration and the date, placing the sorghum seeds in a constant-temperature incubator at 25 ℃ for 12 hours under illumination, culturing for 4 days, observing the germination condition and the growth vigor, and taking the treatment without the medicament as a blank control;

(2.3) selection of the amount of Fungicide

On the basis of screening the effective medicament in the previous period, a spore germination inhibition method is adopted to further select the optimal dose of the medicament for preventing and treating the sorghum head smut and the sorghum seedling blight,

(2.3.1) preparation of spore suspension

Washing pathogenic fungal spores on a culture medium with 89% sterile water and 1% TWEEN-80, filtering the washing liquid with quantitative filter paper, centrifuging for 5min at a speed of 100r/min, pouring out the supernatant, adding deionized water, and centrifuging to obtain spore suspension;

(2.3.2) treatment with pharmaceutical agent

Setting 5 solutions of the medicament with different concentrations, sequentially sucking 0.5mL of liquid medicine from low concentration to high concentration by using a pipette gun, respectively adding the liquid medicine into small test tubes, sucking 0.5mL of prepared spore suspension, uniformly mixing the liquid medicine and the spore suspension in equal amount, sucking 1uL of mixed liquid by using the pipette gun, dripping the mixed liquid on a concave glass, placing the concave glass in a culture dish with shallow sterile water, covering and culturing in an incubator at a proper temperature, repeating the treatment for 3 times each time, and taking the treatment without the medicament as a blank control;

(2.3.3) investigation and calculation

When the germination rate of the blank control spores reaches more than 90%, checking the germination condition of the treated spores, randomly observing more than 3 visual fields in each treatment repetition, investigating the total number of the spores to be not less than 200, and respectively recording the germination number and the total number of the spores;

(4) the pesticide and the optimal dosage screening test method comprise the following steps:

(3.1) indoor biological virulence determination:

(3.1.1) preparation of test insects

Selecting grub 3-instar larvae with normal physiological conditions and consistent growth for toxicity determination;

(3.1.2) treatment with drugs

Diluting the preparation with acetone to 5 concentrations, drilling sorghum leaf into leaf with diameter of 2cm, sucking 10uL of medicinal liquid with a liquid-transferring gun, dripping the medicinal liquid on the leaf from low to high, and calculating the dose (mg/cm) of each round leaf according to the dosage design of the test scheme²). Coating gelatin on round leaves without drug, making two phases involutive to obtain toxic leaf, and setting no drug treatment control, placing 10 pieces in 10 culture dishes, placing hunger test insect 1 head in the dish, and placing moist cotton on the other side for keeping moisture;

(3.1.3) survey calculation

Counting the number of dead insects after 1d and 2d, calculating an LD50 value, selecting a medicament with the best effect to be combined with the bactericide, setting 3 different proportions, and judging whether the two medicaments influence each other;

(3.2) seed safety test

Selecting a pesticide with the best effect on the basis of a pre-test by adopting a seed germination rate test method, setting six concentrations, soaking the seeds for 24 hours, placing the sorghum seeds soaked in the seeds in groups in a culture dish with 2 layers of soaked gauze at the bottom, marking the medicament, the concentration and the date, placing the sorghum seeds in a constant-temperature incubator at 25 ℃ for 12 hours under illumination, and observing the germination condition and the growth vigor of the sorghum seeds after the sorghum seeds are cultured for 4 days;

(3.3) selection of insecticide dosage

Adopting a seed germination rate and stomach toxicity method, selecting a pesticide with the best effect on the basis of a previous test, soaking 20 sorghum seeds according to a certain concentration, putting the treated seeds and 20 grubs starved for 24 hours into a soil-carrying pest culturing box at the same time for 48 hours, observing the germination condition of the seeds and the death condition of the grubs, and screening the dosage of the pesticide according to the germination condition of the seeds and the death condition of the grubs;

(4) plant growth regulator and its optimal dosage screening

Selecting uniconazole, brassinolide and paclobutrazol to carry out a pot experiment, setting 5 concentrations for each treatment by taking a blank experiment without adding a plant growth regulator as a control, repeating the treatment for three times, and carrying out the experiment after soaking the seeds for 24 hours; the method is carried out in a pot culture mode, firstly, four fifths of volume of organic black soil is added into each pot, equal amount of water is poured thoroughly, then, 10 coated sorghum seeds are uniformly sown on the surface of the soil, then, an equal volume of the soil is covered with a soil layer with the thickness of 2cm, the soil is placed outdoors, the other fields are managed, after 30 days, indexes of 15 mung bean seedlings are randomly measured in each treatment and comparison, and the measurement indexes comprise plant height, stem thickness, root length, lateral root number, underground dry weight and chlorophyll content.

Preferably, the S2 specifically includes the following steps:

(1) selection of test materials

Borax (dazzling chemical plant of Tianjin city);

molybdic acid press (dazzling chemical plant of Tianjin city);

zinc sulfate (dazzling chemical plant of Tianjin city);

(2) test method

The method comprises the steps of selecting borax, molybdic acid and zinc sulfate to carry out pot culture tests according to different proportions of low, medium and high, taking a blank test without adding trace element fertilizers as a control, repeating the test for three times every treatment in a pot culture mode, firstly adding four fifths of volume of organic black soil into each pot, respectively adding 10g of micro-fertilizers with different proportions into each treatment, uniformly and thoroughly watering with water, then uniformly sowing 10 sorghum seeds on the soil surface, covering a soil layer with the thickness of 2cm in an equal volume, placing the soil layer outside, managing the same field for the rest, carrying out index determination on 15 random sorghum seedlings in each treatment and control after 30d, and measuring indexes comprising plant height, stem thickness, root length, lateral root number, underground dry weight and chlorophyll content.

Preferably, the S3 specifically includes the following steps:

(1) selection of test materials

Wetting and dispersing: agricultural emulsion 600, dispersant NNO, sodium dodecyl sulfate and sodium dodecyl benzene sulfonate;

thickening agent: xanthan gum, magnesium aluminum silicate and sodium alginate;

film-forming agent: carboxymethyl cellulose, polyvinyl alcohol, hydroxyethyl cellulose;

(2) test method

(2.1) selection of wetting dispersant

Screening wetting dispersant by adopting a flow point method, preparing a wetting agent into an aqueous solution with the mass fraction of 5%, putting the aqueous solution into a volumetric flask for later use, putting a 50mL beaker and a glass rod together on an electronic balance for weighing, adding 5g of raw pesticide, dripping the aqueous solution of the wetting agent into the beaker by using a rubber head dropper, grinding and stirring the beaker by using the glass rod while dripping until the slurry can freely drip from the glass rod, stopping dripping, weighing again, recording the weight of the dripping solvent, screening the wetting agent with a lower flow point as the wetting agent,

dropping 5% dispersant water solution/test material into the dispersing agent;

(2.2) selection of thickening Agents

In the test, the viscosities of three thickeners, namely xanthan gum, sodium alginate and aluminum magnesium silicate, are measured by using a rotational viscometer, and the heat storage stability and the pourability are detected, so that a better thickener is selected;

(2.3) screening of film Forming Agents

The experiment screens the film forming agents such as carboxymethyl cellulose, the prepared suspension coats seeds according to the proportion, and the film forming agents are screened according to three indexes of film forming time, shedding rate and pourability.

Preferably, the S4 specifically includes the following steps:

(1) testing of dispersibility and stability

99.5mL of standard hard water is filled in a 100mI conical measuring cylinder, 0.5mL of water suspending agent sample to be measured is absorbed by an injector, the sample is dripped into water from a position 5cm away from the water surface of the measuring cylinder, the dispersion state is observed, and the sample is divided into excellent, good and inferior grades according to the quality of the dispersion performance,

the superior grade: automatically dispersing in water in a cloud form without visible particles sinking;

good grade: can automatically disperse in water, and the particles sink, and the sinking particles can slowly disperse or lightly disperse

Dispersing after micro-shaking;

inferior grade: can not automatically disperse in water, has flocculent particles which sink, and can only be done after strong shaking

Dispersing;

inverting the diluent for 30 times in water of the postnatal seed, standing for 1h, and if no floating object exists on the postnatal seed and no precipitate exists on the postnatal seed, the dilution stability is qualified;

(2) determination of the centrifugal stability

Adding 5mL sample to be tested into 5mL conical tube with scale, centrifuging at 3000rpm for 30min, taking out, observing and recording water separating and precipitating conditions, dividing into superior, good and inferior stages according to the amount of water separating and precipitating,

the superior grade: the volume of the separated water and the precipitate is less than 1 percent or no separated water and precipitate exists,

good grade: the water precipitation and precipitation volume is more than 1% but less than 5%,

inferior grade: the volume of water precipitation and precipitation is more than 5 percent;

(3) measurement of viscosity

Fully shaking a sample to be detected uniformly, putting about 400mL of the sample to be detected in a beaker, standing in a thermostatic water bath at 25 ℃ for 1h, debugging a viscometer normally, installing a rotor, slowly inserting the rotor into the sample after the rotation speed of the rotor is adjusted to (30 +/-1) rpm, enabling the liquid level to just submerge a groove on the rotor, starting an instrument, and immediately reading a viscosity value after 1 min;

(4) measurement of film Forming Property

Weighing 100g of sorghum seeds in a culture dish, sucking 2mL of a sample by using an injector, injecting the sample into the culture dish which is continuously shaken for 5 times within 1min, shaking the culture dish for 5min, pouring the coated seeds into a watch glass, flatly spreading to form a film, standing for 20min, stirring the seeds by using a glass rod, observing the surfaces of the seeds, and if the seed coating agents on the surfaces of all the seeds are solidified into a film, determining that the film forming property is qualified;

(5) measurement of coating uniformity

Randomly taking 20 coated seeds for measuring film forming property, respectively placing the coated seeds into 10mL centrifuge tubes with covers, accurately adding 5mL of ethanol into a pipette in each centrifuge tube, shaking and extracting for 15min by covering and shaking, standing and centrifuging to obtain clear red liquid, measuring absorbance Aa by taking ethanol as reference under the wavelength of 550nm, arranging the measured 20 absorbance data from small to large, calculating the average absorbance value as Aa, and calculating the coating uniformity (x) of the sample according to the following formula:

x＝5n

n is the number of coating seeds with the absorbance A measured in the range of 0.7-1.3 Aa;

(6) determination of the shedding Rate

Weighing two parts of about 10g (accurate to 0.002g) of coated seeds, respectively, putting one part of the seeds into a 250m1 triangular flask, accurately adding 100mL of ethanol, plugging the seeds into an ultrasonic cleaner, oscillating for 10min to fully dissolve the seed coating agent on the outer surface of the seeds, taking out the seeds, standing for 10min, taking 10mL of supernatant, putting the supernatant into a 50mL volumetric flask, diluting the supernatant to a scale with ethanol, and shaking up to obtain a solution A; placing the other part of the coated seeds in an oscillator, oscillating for 10min, carefully taking out the seeds, placing the seeds in another 250mL triangular flask, and obtaining a solution B according to the treatment method of the solution A; measuring the absorbance of the solution A and the solution B at the wavelength of 550nm by taking ethanol as a reference;

M0A preparation solution A is used to measure the mass of the coated seed,

M1A preparation solution B is used to measure the mass of the coated seed,

a0 the absorbance of solution a,

a1 absorbance of solution B;

(7) determination of the suspension Rate

Weighing 5g of each of the two samples A and B in a 200mL beaker, adding 50mL of standard hard water, performing circular motion at a speed of 120 times/min by hand for about 2min, moving the suspension into a 250mL measuring cylinder, diluting the suspension to a scale with 100mL of standard hard water, covering a plug, and turning the measuring cylinder upside down within 1min for 30 times by taking the bottom of the measuring cylinder as an axis; immediately removing the 9/10 suspension in 10-15s from the sample A by using a pipette, ensuring that the opening of the pipette is always a few millimeters below the liquid surface, transferring the residual 25m1 residue to a beaker with 100m1 and dried to constant weight, adding 1mL of ethanol when the water is removed to about 2mL in a constant-temperature water bath at 80-90 ℃, continuing to remove the water in the water bath until the weight is constant, and weighing to obtain the mass m of the residue; opening the glass plug of the measuring cylinder B, vertically placing the measuring cylinder B into a constant-temperature water bath (3011 ℃) without vibration, standing for 30min, removing 9/10 (namely 225mL) floating liquid of the content in 10-155 by using a suction pipe without stirring or stirring sediment in the measuring cylinder, ensuring that the opening of the suction pipe is always a few millimeters below the liquid level, treating the same sample A with the residual 25mL to obtain residual substance m,

ma-suspension 25m1 remaining at the bottom of the A cylinder evaporates to a constant weight,

mb-suspension liquid remained at the bottom of the B measuring cylinder and 25m1 was evaporated to constant weight;

(8) determination of Heat storage stability

Injecting about 30mL of sample into a clean ampoule bottle by using an injector, sealing the ampoule bottle by using high-temperature flame, sealing at least 3 bottles, respectively weighing, placing the sealed ampoule bottle into a metal container, placing the metal container into a thermostat of 54 degrees and 2 degrees or a thermostatic water bath, taking out the metal container after placing for 14 days, weighing the ampoule bottle, weighing the sample with unchanged mass, and measuring the content of effective components within 24 hours;

(9) determination of content of active ingredient

The test adopts high performance liquid chromatography to detect the effective components of the sample, and methanol-water is used as 9: taking 1 as a mobile phase, measuring 0.04g of a bactericide standard substance in a volumetric flask with 50mI at a flow rate of 0.2m1/min and a wavelength of 230nm and a sample introduction amount of 10uL, metering to a scale with methanol to completely dissolve the bactericide standard substance, and shaking up to obtain a standard sample solution A; weighing 0.05 pesticide standard substance in another 50mI volumetric flask, transferring 5ml standard solution A in the volumetric flask by a pipette, metering to a certain volume by using methanol until the volume reaches a scale, completely dissolving the standard solution A, and shaking up to obtain solution B; weighing 1ml of sample, placing the sample in a 50mI volumetric flask, adding about 40m1 methanol, carrying out ultrasonic oscillation in a water bath at 40 ℃ for 10min to completely dissolve the sample, placing the sample to room temperature, using the methanol to fix the volume to the scale, and shaking up; then filtered by a 0.45um filter, and clear liquid is left for testing. Under the operating conditions, after the baseline of the instrument is stable, continuously injecting a plurality of needle standard sample solutions, calculating the repeatability of the relative response value of each needle, measuring according to the sequence of the standard sample solutions, the sample solutions and the standard sample solutions, respectively averaging the peak areas in the two measured needle sample solutions and the two needle standard sample solutions before and after the sample, and calculating the mass fraction;

(10) determination of pH

Weighing 1g of sample in a 100mL beaker, adding 100mL of water, stirring vigorously for 1min, inserting an electrode into the sample solution, measuring the pH value of the sample solution, measuring the pH value at least three times in parallel, wherein the absolute difference value of the measurement result is less than 0.1, taking the arithmetic mean value of the absolute difference value as the pH value of the sample, and standing for 1 min.

Preferably, S5 specifically includes the following steps:

(1) indoor safety test of seed coating agent

The test adopts a wet culture method in a dish, seeds are selected according to the proportion of the test design for coating treatment and are aired for standby, a proper amount of cotton is firstly added into a culture dish, embryos of the coated seeds are uniformly sowed upwards into the culture dish, then a layer of fine sand is coated, 10 seeds are sowed in each dish, after the seeds are cultured for 7 days at the temperature of 27-30 ℃, the whole seedling is taken out, the sand is slightly washed away by water, the emergence rate is calculated, the plant height and the main root length are measured, and the number of beard roots is recorded. Each treatment was repeated 3 times;

(2) test of field drug effect

(2.1) control target

The field pesticide effect test control objects are as follows: soil pests, sorghum head smut;

(2.2) treatment with drugs

The suspension seed coating agent is coated, and the drug-seed ratio is 1: 50;

(2.3) field design

Two fields are selected for experiments, the prevention and control effects of the maize head smut and the underground pests are respectively researched, 5 treatments are arranged in each field, the treatments are repeated for 4 times, 20 cells are arranged in random blocks, the area of each cell is about 20m2, and a protection row is arranged;

(2.4) investigation method and analysis

The artificial coating is carried out 2 days before sowing according to the treatment dosage, the medicament is required to be uniformly adhered to the seeds, the uncoated seeds are used as contrast,

insect pest investigation: investigating the base number of underground pests before sowing, setting 1 square meter as one point, and the depth of 30cm, investigating 25 points in total, investigating after sowing for 30 days, checking the gnawing condition of sorghum stalks, and calculating the control effect;

disease investigation: and (3) adopting a 5-point random sampling method, investigating when symptoms are obvious after the ears are full, calculating the prevention and treatment effect and the disease investigation, and calculating the disease index and the prevention effect.

Compared with the prior art, the invention has the beneficial effects that: through the research method of the sorghum pesticide-fertilizer composite seed coating agent, the sorghum seed coating agent capable of promoting seed germination and seedling growth, preventing and controlling seed transmission and main plant diseases and insect pests in a seedling stage and reducing the application amount of chemical fertilizer and pesticide can be researched, and the research method makes contributions to the enrichment and development of a sorghum cultivation technology and seed coating agent theory and technology, the quality safety of agricultural products, the ecological environment protection and the sustainable and stable development of agriculture.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a flow chart of the research method of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1, the present invention provides a technical solution: a research method of a sorghum pesticide-fertilizer composite seed coating agent specifically comprises the following steps:

s1: screening a seed coating pesticide formula:

s1.1: aiming at the sorghum head smut and main underground pests, screening out proper bactericides and insecticides by using methods such as indoor toxicity measurement, seed safety test and the like, and determining the optimal dosage of the bactericides and the insecticides;

s1.2: screening out a proper growth regulator by utilizing an indoor seed germination method according to the growth rule of sorghum, and determining the optimal using amount of the growth regulator;

s2: screening a seed coating agent micro-fertilizer formula:

aiming at the soil characteristics of a main sorghum planting area and the growth rule of sorghum, measuring the physiological characteristics of the sorghum by using a potting method, screening out proper trace elements and determining the optimal proportion;

s3: screening a seed coating agent auxiliary agent formula: according to the requirements of the seed coating formulation and the requirements of GB/T17768-1999 standard, screening out the auxiliary agent which meets the quality standard of the seed coating, and determining the optimal dosage;

s4: detecting the technical indexes of the seed coating agent: according to the GB/T17768-1999 quality standard requirement of the seed coating, the seed coating which is researched is technically detected, and the indexes mainly comprise dispersibility, stability, film forming property, shedding rate and the like;

s5: seed coating biological effect evaluation: the researched seed coating agent is biologically evaluated by pot culture and field test, and the indexes of germination rate, root length, enzyme activity, pest control effect, yield and the like are measured.

Wherein, S1 specifically includes the following steps:

(1) selection of test materials:

(1.1) test organisms:

the strain is as follows: the sorghum head smut is provided by the plant pathology laboratory of the college of agriculture of Guizhou university;

testing insects: grubs (Scarabaeoidea), provided by the institute of insects at the college of agriculture of the university of Guizhou;

seed: sorghum seeds are Jinliangluo No. 1, and are purchased from Zhuohao agriculture science and technology Limited in Zunyi, Guizhou province;

(1.2) reagent for test:

carbendazim powder (Wuhanrong Brilliant Biotech Co., Ltd.);

triadimenol powder (Wuhanrong Brilliant Biotech Co., Ltd.);

tebuconazole raw powder (Wuhanrong Brilliant Biotech limited);

cyantraniliprole technical material (Shanghai Dupont agrichemical Co., Ltd.);

chlorantraniliprole (Shandong Weifang Runfeng chemical Co., Ltd.);

clothianidin technical (Xianlong chemical corporation, Hubei);

(1.3) test plant growth regulators:

brassinolide original drug (scientific and technological limited lan yue, Sichuan province);

paclobutrazol (anyang quanfeng biotechnology limited);

uniconazole (anyang quanfeng biotechnology limited);

(2) the test method for screening the bactericide and the optimal dosage thereof comprises the following steps:

(2.1) indoor biological virulence determination:

(2.1.1) activation of bacterial species

The preserved strain of Sphacelotheca reiliana (Kuhn.) Clint.) was taken out of the refrigerator at 4 ℃. Picking hyphae with a small amount of culture medium from a test tube on a superclean bench, putting the hyphae into a plate culture medium to be inoculated, sticking a label after inoculation, inversely placing the hyphae into a constant temperature incubator at 28 ℃ for culture, and waiting until the diameter of the bacterial colony grows to 2/3 of the diameter of a culture dish for later use;

(2.1.2) preparation of drug-containing Medium

Dissolving raw powder of the medicament by using methanol and 0.1 percent TWEEN-80, diluting the raw powder by using sterile distilled water to prepare a mother solution of 10g/L, preparing 5 series of concentrations by using the sterile distilled water on the basis of a pre-test, sterilizing a PDA culture medium, cooling the sterilized PDA culture medium to about 50 ℃, adding 1mL of prepared medicament into 9mL of the culture medium, fully shaking the mixture to obtain a medicament-containing culture medium containing the medicaments of each series of concentrations, adding equivalent sterile water as a reference, and repeating the treatment for 3 times;

(2.1.3) virulence determination

Measured by a hyphal growth rate method. Placing pre-cultured test strain colonies on an ultra-clean workbench, punching a bacterial dish along the same circumference of the edges of the bacterial colonies by using a puncher with the diameter of 5mm, then sticking one surface of the bacterial dish containing hyphae to the center of a medicament-containing culture medium, inversely placing the bacterial dish in a constant-temperature incubator at 25 ℃ for culturing for 72 hours, and measuring the diameter of the bacterial colonies treated by each medicament by using a cross method, wherein the diameter of the bacterial colonies not treated by the medicament is used as a reference;

(2.1.4) data processing and statistical methods

Converting the hypha growth inhibition rate into an inhibition rate value Y, converting the medicament concentration into a concentration log X, obtaining virulence formulas of different bactericides according to a regression method of the concentration log and the probability value, calculating the inhibition medium concentration (EC50) value of each medicament on the ustilago sorghum smut and the rhizoctonia sorghum seedling by using the virulence formulas, selecting one medicament with the best effect to be matched with the pesticide in a combined manner, setting 3 different proportions, and judging whether the two medicaments influence each other;

(2.2) seed safety test

Adopting a seed germination rate test, selecting a pesticide with the best effect on the basis of a pre-test, and setting 5 concentrations; soaking the seeds for 24 hours, placing the sorghum seeds soaked in the seeds in groups in a culture dish with 2 layers of soaked gauze at the bottom, marking the medicament, the concentration and the date, placing the sorghum seeds in a constant-temperature incubator at 25 ℃ for 12 hours under illumination, culturing for 4 days, observing the germination condition and the growth vigor, and taking the treatment without the medicament as a blank control;

(2.3) selection of the amount of Fungicide

On the basis of screening the effective medicament in the previous period, a spore germination inhibition method is adopted to further select the optimal dose of the medicament for preventing and treating the sorghum head smut and the sorghum seedling blight,

(2.3.1) preparation of spore suspension

Washing pathogenic fungal spores on a culture medium with 89% sterile water and 1% TWEEN-80, filtering the washing liquid with quantitative filter paper, centrifuging for 5min at a speed of 100r/min, pouring out the supernatant, adding deionized water, and centrifuging to obtain spore suspension;

(2.3.2) treatment with pharmaceutical agent

Setting 5 solutions of the medicament with different concentrations, sequentially sucking 0.5mL of liquid medicine from low concentration to high concentration by using a pipette gun, respectively adding the liquid medicine into small test tubes, sucking 0.5mL of prepared spore suspension, uniformly mixing the liquid medicine and the spore suspension in equal amount, sucking 1uL of mixed liquid by using the pipette gun, dripping the mixed liquid on a concave glass, placing the concave glass in a culture dish with shallow sterile water, covering and culturing in an incubator at a proper temperature, repeating the treatment for 3 times each time, and taking the treatment without the medicament as a blank control;

(2.3.3) investigation and calculation

When the germination rate of the blank control spores reaches more than 90%, checking the germination condition of the treated spores, randomly observing more than 3 visual fields in each treatment repetition, investigating the total number of the spores to be not less than 200, and respectively recording the germination number and the total number of the spores;

(5) the pesticide and the optimal dosage screening test method comprise the following steps:

(3.1) indoor biological virulence determination:

(3.1.1) preparation of test insects

Selecting grub 3-instar larvae with normal physiological conditions and consistent growth for toxicity determination;

(3.1.2) treatment with drugs

Diluting the preparation with acetone to 5 concentrations, drilling sorghum leaf into leaf with diameter of 2cm, sucking 10uL of medicinal liquid with a liquid-transferring gun, dripping the medicinal liquid on the leaf from low to high, and calculating the dose (mg/cm) of each round leaf according to the dosage design of the test scheme²). Coating gelatin on round leaves without drug, making two phases involutive to obtain toxic leaf, and setting no drug treatment control, placing 10 pieces in 10 culture dishes, placing hunger test insect 1 head in the dish, and placing moist cotton on the other side for keeping moisture;

(3.1.3) survey calculation

Counting the number of dead insects after 1d and 2d, calculating an LD50 value, selecting a medicament with the best effect to be combined with the bactericide, setting 3 different proportions, and judging whether the two medicaments influence each other;

(3.2) seed safety test

Selecting a pesticide with the best effect on the basis of a pre-test by adopting a seed germination rate test method, setting six concentrations, soaking the seeds for 24 hours, placing the sorghum seeds soaked in the seeds in groups in a culture dish with 2 layers of soaked gauze at the bottom, marking the medicament, the concentration and the date, placing the sorghum seeds in a constant-temperature incubator at 25 ℃ for 12 hours under illumination, and observing the germination condition and the growth vigor of the sorghum seeds after the sorghum seeds are cultured for 4 days;

(3.3) selection of insecticide dosage

Adopting a seed germination rate and stomach toxicity method, selecting a pesticide with the best effect on the basis of a previous test, soaking 20 sorghum seeds according to a certain concentration, putting the treated seeds and 20 grubs starved for 24 hours into a soil-carrying pest culturing box at the same time for 48 hours, observing the germination condition of the seeds and the death condition of the grubs, and screening the dosage of the pesticide according to the germination condition of the seeds and the death condition of the grubs;

(4) plant growth regulator and its optimal dosage screening

Selecting uniconazole, brassinolide and paclobutrazol to carry out a pot experiment, setting 5 concentrations for each treatment by taking a blank experiment without adding a plant growth regulator as a control, repeating the treatment for three times, and carrying out the experiment after soaking the seeds for 24 hours; the method is carried out in a pot culture mode, firstly, four fifths of volume of organic black soil is added into each pot, equal amount of water is poured thoroughly, then, 10 coated sorghum seeds are uniformly sown on the surface of the soil, then, an equal volume of the soil is covered with a soil layer with the thickness of 2cm, the soil is placed outdoors, the other fields are managed, after 30 days, indexes of 15 mung bean seedlings are randomly measured in each treatment and comparison, and the measurement indexes comprise plant height, stem thickness, root length, lateral root number, underground dry weight and chlorophyll content.

Wherein, S2 specifically includes the following steps:

(1) selection of test materials

Borax (dazzling chemical plant of Tianjin city);

molybdic acid press (dazzling chemical plant of Tianjin city);

zinc sulfate (dazzling chemical plant of Tianjin city);

(2) test method

The method comprises the steps of selecting borax, molybdic acid and zinc sulfate to carry out pot culture tests according to different proportions of low, medium and high, taking a blank test without adding trace element fertilizers as a control, repeating the test for three times every treatment in a pot culture mode, firstly adding four fifths of volume of organic black soil into each pot, respectively adding 10g of micro-fertilizers with different proportions into each treatment, uniformly and thoroughly watering with water, then uniformly sowing 10 sorghum seeds on the soil surface, covering a soil layer with the thickness of 2cm in an equal volume, placing the soil layer outside, managing the same field for the rest, carrying out index determination on 15 random sorghum seedlings in each treatment and control after 30d, and measuring indexes comprising plant height, stem thickness, root length, lateral root number, underground dry weight and chlorophyll content.

Wherein, S3 specifically includes the following steps:

(1) selection of test materials

Wetting and dispersing: agricultural emulsion 600, dispersant NNO, sodium dodecyl sulfate and sodium dodecyl benzene sulfonate;

thickening agent: xanthan gum, magnesium aluminum silicate and sodium alginate;

film-forming agent: carboxymethyl cellulose, polyvinyl alcohol, hydroxyethyl cellulose;

(2) test method

(2.1) selection of wetting dispersant

Screening wetting dispersant by adopting a flow point method, preparing a wetting agent into an aqueous solution with the mass fraction of 5%, putting the aqueous solution into a volumetric flask for later use, putting a 50mL beaker and a glass rod together on an electronic balance for weighing, adding 5g of raw pesticide, dripping the aqueous solution of the wetting agent into the beaker by using a rubber head dropper, grinding and stirring the beaker by using the glass rod while dripping until the slurry can freely drip from the glass rod, stopping dripping, weighing again, recording the weight of the dripping solvent, screening the wetting agent with a lower flow point as the wetting agent,

dropping 5% dispersant water solution/test material into the dispersing agent;

(2.2) selection of thickening Agents

In the test, the viscosities of three thickeners, namely xanthan gum, sodium alginate and aluminum magnesium silicate, are measured by using a rotational viscometer, and the heat storage stability and the pourability are detected, so that a better thickener is selected;

(2.3) screening of film Forming Agents

The experiment screens the film forming agents such as carboxymethyl cellulose, the prepared suspension coats seeds according to the proportion, and the film forming agents are screened according to three indexes of film forming time, shedding rate and pourability.

Wherein, S4 specifically includes the following steps:

(1) testing of dispersibility and stability

99.5mL of standard hard water is filled in a 100mI conical measuring cylinder, 0.5mL of water suspending agent sample to be measured is absorbed by an injector, the sample is dripped into water from a position 5cm away from the water surface of the measuring cylinder, the dispersion state is observed, and the sample is divided into excellent, good and inferior grades according to the quality of the dispersion performance,

the superior grade: automatically dispersing in water in a cloud form without visible particles sinking;

good grade: can automatically disperse in water, and the particles sink, and the sinking particles can slowly disperse or lightly disperse

Dispersing after micro-shaking;

inferior grade: can not automatically disperse in water, has flocculent particles which sink, and can only be done after strong shaking

Dispersing;

inverting the diluent for 30 times in water of the postnatal seed, standing for 1h, and if no floating object exists on the postnatal seed and no precipitate exists on the postnatal seed, the dilution stability is qualified;

(2) determination of the centrifugal stability

Adding 5mL sample to be tested into 5mL conical tube with scale, centrifuging at 3000rpm for 30min, taking out, observing and recording water separating and precipitating conditions, dividing into superior, good and inferior stages according to the amount of water separating and precipitating,

the superior grade: the volume of the separated water and the precipitate is less than 1 percent or no separated water and precipitate exists,

good grade: the water precipitation and precipitation volume is more than 1% but less than 5%,

inferior grade: the volume of water precipitation and precipitation is more than 5 percent;

(3) measurement of viscosity

Fully shaking a sample to be detected uniformly, putting about 400mL of the sample to be detected in a beaker, standing in a thermostatic water bath at 25 ℃ for 1h, debugging a viscometer normally, installing a rotor, slowly inserting the rotor into the sample after the rotation speed of the rotor is adjusted to (30 +/-1) rpm, enabling the liquid level to just submerge a groove on the rotor, starting an instrument, and immediately reading a viscosity value after 1 min;

(4) measurement of film Forming Property

Weighing 100g of sorghum seeds in a culture dish, sucking 2mL of a sample by using an injector, injecting the sample into the culture dish which is continuously shaken for 5 times within 1min, shaking the culture dish for 5min, pouring the coated seeds into a watch glass, flatly spreading to form a film, standing for 20min, stirring the seeds by using a glass rod, observing the surfaces of the seeds, and if the seed coating agents on the surfaces of all the seeds are solidified into a film, determining that the film forming property is qualified;

(5) measurement of coating uniformity

Randomly taking 20 coated seeds for measuring film forming property, respectively placing the coated seeds into 10mL centrifuge tubes with covers, accurately adding 5mL of ethanol into a pipette in each centrifuge tube, shaking and extracting for 15min by covering and shaking, standing and centrifuging to obtain clear red liquid, measuring absorbance Aa by taking ethanol as reference under the wavelength of 550nm, arranging the measured 20 absorbance data from small to large, calculating the average absorbance value as Aa, and calculating the coating uniformity (x) of the sample according to the following formula:

x＝5n

n is the number of coating seeds with the absorbance A measured in the range of 0.7-1.3 Aa;

(6) determination of the shedding Rate

Weighing two parts of about 10g (accurate to 0.002g) of coated seeds, respectively, putting one part of the seeds into a 250m1 triangular flask, accurately adding 100mL of ethanol, plugging the seeds into an ultrasonic cleaner, oscillating for 10min to fully dissolve the seed coating agent on the outer surface of the seeds, taking out the seeds, standing for 10min, taking 10mL of supernatant, putting the supernatant into a 50mL volumetric flask, diluting the supernatant to a scale with ethanol, and shaking up to obtain a solution A; placing the other part of the coated seeds in an oscillator, oscillating for 10min, carefully taking out the seeds, placing the seeds in another 250mL triangular flask, and obtaining a solution B according to the treatment method of the solution A; measuring the absorbance of the solution A and the solution B at the wavelength of 550nm by taking ethanol as a reference;

M0A preparation solution A is used to measure the mass of the coated seed,

M1A preparation solution B is used to measure the mass of the coated seed,

a0 the absorbance of solution a,

a1 absorbance of solution B;

(7) determination of the suspension Rate

Weighing 5g of each of the two samples A and B in a 200mL beaker, adding 50mL of standard hard water, performing circular motion at a speed of 120 times/min by hand for about 2min, moving the suspension into a 250mL measuring cylinder, diluting the suspension to a scale with 100mL of standard hard water, covering a plug, and turning the measuring cylinder upside down within 1min for 30 times by taking the bottom of the measuring cylinder as an axis; immediately removing the 9/10 suspension in 10-15s from the sample A by using a pipette, ensuring that the opening of the pipette is always a few millimeters below the liquid surface, transferring the residual 25m1 residue to a beaker with 100m1 and dried to constant weight, adding 1mL of ethanol when the water is removed to about 2mL in a constant-temperature water bath at 80-90 ℃, continuing to remove the water in the water bath until the weight is constant, and weighing to obtain the mass m of the residue; opening the glass plug of the measuring cylinder B, vertically placing the measuring cylinder B into a constant-temperature water bath (3011 ℃) without vibration, standing for 30min, removing 9/10 (namely 225mL) floating liquid of the content in 10-155 by using a suction pipe without stirring or stirring sediment in the measuring cylinder, ensuring that the opening of the suction pipe is always a few millimeters below the liquid level, treating the same sample A with the residual 25mL to obtain residual substance m,

ma-suspension 25m1 remaining at the bottom of the A cylinder evaporates to a constant weight,

mb-suspension liquid remained at the bottom of the B measuring cylinder and 25m1 was evaporated to constant weight;

(8) determination of Heat storage stability

Injecting about 30mL of sample into a clean ampoule bottle by using an injector, sealing the ampoule bottle by using high-temperature flame, sealing at least 3 bottles, respectively weighing, placing the sealed ampoule bottle into a metal container, placing the metal container into a thermostat of 54 degrees and 2 degrees or a thermostatic water bath, taking out the metal container after placing for 14 days, weighing the ampoule bottle, weighing the sample with unchanged mass, and measuring the content of effective components within 24 hours;

(9) determination of content of active ingredient

The test adopts high performance liquid chromatography to detect the effective components of the sample, and methanol-water is used as 9: taking 1 as a mobile phase, measuring 0.04g of a bactericide standard substance in a volumetric flask with 50mI at a flow rate of 0.2m1/min and a wavelength of 230nm and a sample introduction amount of 10uL, metering to a scale with methanol to completely dissolve the bactericide standard substance, and shaking up to obtain a standard sample solution A; weighing 0.05 pesticide standard substance in another 50mI volumetric flask, transferring 5ml standard solution A in the volumetric flask by a pipette, metering to a certain volume by using methanol until the volume reaches a scale, completely dissolving the standard solution A, and shaking up to obtain solution B; weighing 1ml of sample, placing the sample in a 50mI volumetric flask, adding about 40m1 methanol, carrying out ultrasonic oscillation in a water bath at 40 ℃ for 10min to completely dissolve the sample, placing the sample to room temperature, using the methanol to fix the volume to the scale, and shaking up; then filtered by a 0.45um filter, and clear liquid is left for testing. Under the operating conditions, after the baseline of the instrument is stable, continuously injecting a plurality of needle standard sample solutions, calculating the repeatability of the relative response value of each needle, measuring according to the sequence of the standard sample solutions, the sample solutions and the standard sample solutions, respectively averaging the peak areas in the two measured needle sample solutions and the two needle standard sample solutions before and after the sample, and calculating the mass fraction;

(10) determination of pH

Weighing 1g of sample in a 100mL beaker, adding 100mL of water, stirring vigorously for 1min, inserting an electrode into the sample solution, measuring the pH value of the sample solution, measuring the pH value at least three times in parallel, wherein the absolute difference value of the measurement result is less than 0.1, taking the arithmetic mean value of the absolute difference value as the pH value of the sample, and standing for 1 min.

Wherein, S5 specifically includes the following steps:

(1) indoor safety test of seed coating agent

The test adopts a wet culture method in a dish, seeds are selected according to the proportion of the test design for coating treatment and are aired for standby, a proper amount of cotton is firstly added into a culture dish, embryos of the coated seeds are uniformly sowed upwards into the culture dish, then a layer of fine sand is coated, 10 seeds are sowed in each dish, after the seeds are cultured for 7 days at the temperature of 27-30 ℃, the whole seedling is taken out, the sand is slightly washed away by water, the emergence rate is calculated, the plant height and the main root length are measured, and the number of beard roots is recorded. Each treatment was repeated 3 times;

(2) test of field drug effect

(2.1) control target

The field pesticide effect test control objects are as follows: soil pests, sorghum head smut;

(2.2) treatment with drugs

The suspension seed coating agent is coated, and the drug-seed ratio is 1: 50;

(2.3) field design

Two fields are selected for experiments, the prevention and control effects of the maize head smut and the underground pests are respectively researched, 5 treatments are arranged in each field, the treatments are repeated for 4 times, 20 cells are arranged in random blocks, the area of each cell is about 20m2, and a protection row is arranged;

(2.4) investigation method and analysis

The artificial coating is carried out 2 days before sowing according to the treatment dosage, the medicament is required to be uniformly adhered to the seeds, the uncoated seeds are used as contrast,

insect pest investigation: investigating the base number of underground pests before sowing, setting 1 square meter as one point, and the depth of 30cm, investigating 25 points in total, investigating after sowing for 30 days, checking the gnawing condition of sorghum stalks, and calculating the control effect;

disease investigation: and (3) adopting a 5-point random sampling method, investigating when symptoms are obvious after the ears are full, calculating the prevention and treatment effect and the disease investigation, and calculating the disease index and the prevention effect.

The existing research proves that the seed coating agent has good control effect on underground pests, seed-borne diseases and seedling diseases; the composite seed coating agent is also available on the market; therefore, the research is feasible for researching and developing the compound seed coating agent on the basis and determining the effects of the compound seed coating agent on the prevention and control of the main plant diseases and insect pests of sorghum, the seed germination and the plant growth, and the experimental conditions are also provided.

EXAMPLE 1 preparation of a suspended seed coating

Putting 0.6% tebuconazole, 9% cyantraniliprole, 9% thiamethoxam, 1% borax, 0.5% zinc sulfate, 0.5% molybdic acid, 0.0026mg/L brassinolide, 1.5% sodium alginate, 3% NNO, 1% Tween 80, 1% acid scarlet and 72.9% water into an electromagnetic stirring machine, stirring and mixing uniformly, putting the mixture into a high-speed emulsification shearing machine, and shearing and emulsifying to obtain a finished product.

Example 2 method for making coated sorghum seeds

Placing 500g of carefully selected sorghum seeds into an aseptic dry wide-mouth conical flask, pouring 10mL of the suspension seed coating agent prepared in the embodiment 1 into the aseptic dry wide-mouth conical flask, shaking the mixture evenly by hand, placing the mixture into an oscillator for 10min, and then placing the mixture in a ventilated place for drying.

Example 3 indoor germination test of coated sorghum seeds

The germination rate was determined after culturing 20 coated sorghum seeds in a petri dish with three layers of wetted filter paper at 28 ℃ under 85% relative humidity for seven days, L: D ═ 12:12, as shown in table 1.

TABLE 1 germination rates of coated sorghum seeds

Example 4 field control effect experimental data for coated sorghum seeds

The experiment is carried out on experiment farm of Guizhou university in Huaxi district, Guiyang city, Guizhou province, the coating treatment is 2kg of the coating agent in the example 1 for every 100kg of seeds, and the statistical control effect is shown in the table 2.

TABLE 2 field control Effect of coated sorghum seeds

Treatment of	Mole cricket	Head smut of sorghum
			CK-1	71.9	66.9
CK-2	73.1	67.1
			CK-3	71.0	68.0
BY-1	83.9	76.3
			BY-2	85.4	74.2
BY-3	86.2	75.9

In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims (6)

1. A research method of a sorghum pesticide-fertilizer composite seed coating agent is characterized by comprising the following steps: the method specifically comprises the following steps:

s1: screening a seed coating pesticide formula:

s1.1: aiming at the sorghum head smut and main underground pests, screening out proper bactericides and insecticides by using methods such as indoor toxicity measurement, seed safety test and the like, and determining the optimal dosage of the bactericides and the insecticides;

s1.2: screening out a proper growth regulator by utilizing an indoor seed germination method according to the growth rule of sorghum, and determining the optimal using amount of the growth regulator;

s2: screening a seed coating agent micro-fertilizer formula:

aiming at the soil characteristics of a main sorghum planting area and the growth rule of sorghum, measuring the physiological characteristics of the sorghum by using a potting method, screening out proper trace elements and determining the optimal proportion;

s3: screening a seed coating agent auxiliary agent formula: according to the requirements of the seed coating formulation and the requirements of GB/T17768-1999 standard, screening out the auxiliary agent which meets the quality standard of the seed coating, and determining the optimal dosage;

s4: detecting the technical indexes of the seed coating agent: according to the GB/T17768-1999 quality standard requirement of the seed coating, the seed coating which is researched is technically detected, and the indexes mainly comprise dispersibility, stability, film forming property, shedding rate and the like;

s5: seed coating biological effect evaluation: the researched seed coating agent is biologically evaluated by pot culture and field test, and the indexes of germination rate, root length, enzyme activity, pest control effect, yield and the like are measured.

2. The research method of the sorghum fertilizer composite seed coating agent according to claim 1, characterized in that: the S1 specifically includes the following steps:

(1) selection of test materials:

(1.1) test organisms:

the strain is as follows: the sorghum head smut is provided by the plant pathology laboratory of the college of agriculture of Guizhou university;

testing insects: grubs, provided by the institute of insects at the college of agriculture of the university of Guizhou;

seed: sorghum seeds are Jinliangluo No. 1, and are purchased from Zhuohao agriculture science and technology Limited in Zunyi, Guizhou province;

(1.2) reagent for test:

carbendazim raw powder;

triadimenol raw powder;

tebuconazole raw powder;

cyantraniliprole raw drug;

chlorantraniliprole raw drug;

clothianidin bulk drug;

(1.3) test plant growth regulators:

brassinolide technical;

paclobutrazol crude drug;

a raw uniconazole drug;

(2) the test method for screening the bactericide and the optimal dosage thereof comprises the following steps:

(2.1) indoor biological virulence determination:

(2.1.1) activation of bacterial species

And taking out the preserved strain of the sorghum head smut from a refrigerator at 4 ℃. Picking hyphae with a small amount of culture medium from a test tube on a superclean bench, putting the hyphae into a plate culture medium to be inoculated, sticking a label after inoculation, inversely placing the hyphae into a constant temperature incubator at 28 ℃ for culture, and waiting until the diameter of the bacterial colony grows to 2/3 of the diameter of a culture dish for later use;

(2.1.2) preparation of drug-containing Medium

Dissolving raw powder of the medicament by using methanol and 0.1 percent TWEEN-80, diluting the raw powder by using sterile distilled water to prepare a mother solution of 10g/L, preparing 5 series of concentrations by using the sterile distilled water on the basis of a pre-test, sterilizing a PDA culture medium, cooling the sterilized PDA culture medium to about 50 ℃, adding 1mL of prepared medicament into 9mL of the culture medium, fully shaking the mixture to obtain a medicament-containing culture medium containing the medicaments of each series of concentrations, adding equivalent sterile water as a reference, and repeating the treatment for 3 times;

(2.1.3) virulence determination

Measured by a hyphal growth rate method. Placing pre-cultured test strain colonies on an ultra-clean workbench, punching a bacterial dish along the same circumference of the edges of the bacterial colonies by using a puncher with the diameter of 5mm, then sticking one surface of the bacterial dish containing hyphae to the center of a medicament-containing culture medium, inversely placing the bacterial dish in a constant-temperature incubator at 25 ℃ for culturing for 72 hours, and measuring the diameter of the bacterial colonies treated by each medicament by using a cross method, wherein the diameter of the bacterial colonies not treated by the medicament is used as a reference;

(2.1.4) data processing and statistical methods

Converting the hypha growth inhibition rate into an inhibition rate value Y, converting the medicament concentration into a concentration log X, obtaining virulence formulas of different bactericides according to a regression method of the concentration log and the probability value, calculating the inhibition medium concentration values of each medicament on the ustilago sorghum and the rhizoctonia sorghum by using the virulence formulas, selecting one medicament with the best effect to be combined with the pesticide, setting 3 different proportions, and judging whether the two medicaments influence each other;

(2.2) seed safety test

Adopting a seed germination rate test, selecting a pesticide with the best effect on the basis of a pre-test, and setting 5 concentrations; soaking the seeds for 24 hours, placing the sorghum seeds soaked in the seeds in groups in a culture dish with 2 layers of soaked gauze at the bottom, marking the medicament, the concentration and the date, placing the sorghum seeds in a constant-temperature incubator at 25 ℃ for 12 hours under illumination, culturing for 4 days, observing the germination condition and the growth vigor, and taking the treatment without the medicament as a blank control;

(2.3) selection of the amount of Fungicide

On the basis of screening the effective medicament in the previous period, a spore germination inhibition method is adopted to further select the optimal dose of the medicament for preventing and treating the sorghum head smut and the sorghum seedling blight,

(2.3.1) preparation of spore suspension

Washing pathogenic fungal spores on a culture medium with 89% sterile water and 1% TWEEN-80, filtering the washing liquid with quantitative filter paper, centrifuging for 5min at a speed of 100r/min, pouring out the supernatant, adding deionized water, and centrifuging to obtain spore suspension;

(2.3.2) treatment with pharmaceutical agent

Setting 5 solutions of the medicament with different concentrations, sequentially sucking 0.5mL of liquid medicine from low concentration to high concentration by using a pipette gun, respectively adding the liquid medicine into small test tubes, sucking 0.5mL of prepared spore suspension, uniformly mixing the liquid medicine and the spore suspension in equal amount, sucking 1uL of mixed liquid by using the pipette gun, dripping the mixed liquid on a concave glass, placing the concave glass in a culture dish with shallow sterile water, covering and culturing in an incubator at a proper temperature, repeating the treatment for 3 times each time, and taking the treatment without the medicament as a blank control;

(2.3.3) investigation and calculation

When the germination rate of the blank control spores reaches more than 90%, checking the germination condition of the treated spores, randomly observing more than 3 visual fields in each treatment repetition, investigating the total number of the spores to be not less than 200, and respectively recording the germination number and the total number of the spores;

(3) the pesticide and the optimal dosage screening test method comprise the following steps:

(3.1) indoor biological virulence determination:

(3.1.1) preparation of test insects

Selecting grub 3-instar larvae with normal physiological conditions and consistent growth for toxicity determination;

(3.1.2) treatment with drugs

Diluting the preparation with acetone to 5 concentrations, drilling sorghum leaf into leaf with diameter of 2cm, sucking 10uL of medicinal liquid with liquid-transfering gun from low to low according to dosage design of test schemeDripping the medicinal liquid on the leaves, and calculating the dose (mg/cm) of each round leaf²). Coating gelatin on round leaves without drug, making two phases involutive to obtain toxic leaf, and setting no drug treatment control, placing 10 pieces in 10 culture dishes, placing hunger test insect 1 head in the dish, and placing moist cotton on the other side for keeping moisture;

(3.1.3) survey calculation

Counting the number of dead insects after 1d and 2d, calculating an LD50 value, selecting a medicament with the best effect to be combined with the bactericide, setting 3 different proportions, and judging whether the two medicaments influence each other;

(3.2) seed safety test

Selecting a pesticide with the best effect on the basis of a pre-test by adopting a seed germination rate test method, setting six concentrations, soaking the seeds for 24 hours, placing the sorghum seeds soaked in the seeds in groups in a culture dish with 2 layers of soaked gauze at the bottom, marking the medicament, the concentration and the date, placing the sorghum seeds in a constant-temperature incubator at 25 ℃ for 12 hours under illumination, and observing the germination condition and the growth vigor of the sorghum seeds after the sorghum seeds are cultured for 4 days;

(3.3) selection of insecticide dosage

Adopting a seed germination rate and stomach toxicity method, selecting a pesticide with the best effect on the basis of a previous test, soaking 20 sorghum seeds according to a certain concentration, putting the treated seeds and 20 grubs starved for 24 hours into a soil-carrying pest culturing box at the same time for 48 hours, observing the germination condition of the seeds and the death condition of the grubs, and screening the dosage of the pesticide according to the germination condition of the seeds and the death condition of the grubs;

(4) plant growth regulator and its optimal dosage screening

Selecting uniconazole, brassinolide and paclobutrazol to carry out a pot experiment, setting 5 concentrations for each treatment by taking a blank experiment without adding a plant growth regulator as a control, repeating the treatment for three times, and carrying out the experiment after soaking the seeds for 24 hours; the method is carried out in a pot culture mode, firstly, four fifths of volume of organic black soil is added into each pot, equal amount of water is poured thoroughly, then, 10 coated sorghum seeds are uniformly sown on the surface of the soil, then, an equal volume of the soil is covered with a soil layer with the thickness of 2cm, the soil is placed outdoors, the other fields are managed, after 30 days, indexes of 15 mung bean seedlings are randomly measured in each treatment and comparison, and the measurement indexes comprise plant height, stem thickness, root length, lateral root number, underground dry weight and chlorophyll content.

3. The research method of the sorghum fertilizer composite seed coating agent according to claim 2, characterized in that: the S2 specifically includes the following steps:

(1) selection of test materials

Borax;

molybdic acid;

zinc sulfate;

(2) test method

The method comprises the steps of selecting borax, molybdic acid and zinc sulfate to carry out pot culture tests according to different proportions of low, medium and high, taking a blank test without adding trace element fertilizers as a control, repeating the test for three times every treatment in a pot culture mode, firstly adding four fifths of volume of organic black soil into each pot, respectively adding 10g of micro-fertilizers with different proportions into each treatment, uniformly and thoroughly watering with water, then uniformly sowing 10 sorghum seeds on the soil surface, covering a soil layer with the thickness of 2cm in an equal volume, placing the soil layer outside, managing the same field for the rest, carrying out index determination on 15 random sorghum seedlings in each treatment and control after 30d, and measuring indexes comprising plant height, stem thickness, root length, lateral root number, underground dry weight and chlorophyll content.

4. The research method of the sorghum fertilizer composite seed coating agent according to claim 1, characterized in that: the S3 specifically includes the following steps:

(1) selection of test materials

Wetting and dispersing: agricultural emulsion 600, dispersant NNO, sodium dodecyl sulfate and sodium dodecyl benzene sulfonate;

thickening agent: xanthan gum, magnesium aluminum silicate and sodium alginate;

film-forming agent: carboxymethyl cellulose, polyvinyl alcohol, hydroxyethyl cellulose;

(2) test method

(2.1) selection of wetting dispersant

Screening wetting dispersant by adopting a flow point method, preparing a wetting agent into an aqueous solution with the mass fraction of 5%, putting the aqueous solution into a volumetric flask for later use, putting a 50mL beaker and a glass rod together on an electronic balance for weighing, adding 5g of raw pesticide, dripping the aqueous solution of the wetting agent into the beaker by using a rubber head dropper, grinding and stirring the beaker by using the glass rod while dripping until the slurry can freely drip from the glass rod, stopping dripping, weighing again, recording the weight of the dripping solvent, screening the wetting agent with a lower flow point as the wetting agent,

dropping 5% dispersant water solution/test material into the dispersing agent;

(2.2) selection of thickening Agents

In the test, the viscosities of three thickeners, namely xanthan gum, sodium alginate and aluminum magnesium silicate, are measured by using a rotational viscometer, and the heat storage stability and the pourability are detected, so that a better thickener is selected;

(2.3) screening of film Forming Agents

The experiment screens the film forming agents such as carboxymethyl cellulose, the prepared suspension coats seeds according to the proportion, and the film forming agents are screened according to three indexes of film forming time, shedding rate and pourability.

5. The research method of the sorghum fertilizer composite seed coating agent according to claim 1, characterized in that: the S4 specifically includes the following steps:

(1) testing of dispersibility and stability

99.5mL of standard hard water is filled in a 100mI conical measuring cylinder, 0.5mL of water suspending agent sample to be measured is absorbed by an injector, the sample is dripped into water from a position 5cm away from the water surface of the measuring cylinder, the dispersion state is observed, and the sample is divided into excellent, good and inferior grades according to the quality of the dispersion performance,

the superior grade: automatically dispersing in water in a cloud form without visible particles sinking;

good grade: can automatically disperse in water, and the particles sink, and the sinking particles can slowly disperse or lightly disperse

Dispersing after micro-shaking;

inferior grade: can not automatically disperse in water, has flocculent particles which sink, and can only be done after strong shaking

Dispersing;

inverting the diluent for 30 times in water of the postnatal seed, standing for 1h, and if no floating object exists on the postnatal seed and no precipitate exists on the postnatal seed, the dilution stability is qualified;

(2) determination of the centrifugal stability

Adding 5mL sample to be tested into 5mL conical tube with scale, centrifuging at 3000rpm for 30min, taking out, observing and recording water separating and precipitating conditions, dividing into superior, good and inferior stages according to the amount of water separating and precipitating,

the superior grade: the volume of the separated water and the precipitate is less than 1 percent or no separated water and precipitate exists,

good grade: the water precipitation and precipitation volume is more than 1% but less than 5%,

inferior grade: the volume of water precipitation and precipitation is more than 5 percent;

(3) measurement of viscosity

Fully shaking a sample to be detected uniformly, putting about 400mL of the sample to be detected in a beaker, standing in a thermostatic water bath at 25 ℃ for 1h, debugging a viscometer normally, installing a rotor, slowly inserting the rotor into the sample after the rotation speed of the rotor is adjusted to (30 +/-1) rpm, enabling the liquid level to just submerge a groove on the rotor, starting an instrument, and immediately reading a viscosity value after 1 min;

(4) measurement of film Forming Property

Weighing 100g of sorghum seeds in a culture dish, sucking 2mL of a sample by using an injector, injecting the sample into the culture dish which is continuously shaken for 5 times within 1min, shaking the culture dish for 5min, pouring the coated seeds into a watch glass, flatly spreading to form a film, standing for 20min, stirring the seeds by using a glass rod, observing the surfaces of the seeds, and if the seed coating agents on the surfaces of all the seeds are solidified into a film, determining that the film forming property is qualified;

(5) measurement of coating uniformity

Randomly taking 20 coated seeds for measuring film forming property, respectively placing the coated seeds into 10mL centrifuge tubes with covers, accurately adding 5mL of ethanol into a pipette in each centrifuge tube, shaking and extracting for 15min by covering and shaking, standing and centrifuging to obtain clear red liquid, measuring absorbance Aa by taking ethanol as reference under the wavelength of 550nm, arranging the measured 20 absorbance data from small to large, calculating the average absorbance value as Aa, and calculating the coating uniformity (x) of the sample according to the following formula:

x＝5n

n is the number of coating seeds with the absorbance A measured in the range of 0.7-1.3 Aa;

(6) determination of the shedding Rate

Weighing two parts of about 10g of coated seeds respectively, placing one part of the two parts in a 250m1 triangular flask, accurately adding 100mL of ethanol, plugging the triangular flask, placing the triangular flask in an ultrasonic cleaner, oscillating for 10min to fully dissolve the seed coating agent on the surface of the seeds, taking out the seeds, standing for 10min, taking 10mL of supernatant, placing the supernatant in a 50mL volumetric flask, diluting the supernatant to a scale with ethanol, and shaking up to obtain a solution A; placing the other part of the coated seeds in an oscillator, oscillating for 10min, carefully taking out the seeds, placing the seeds in another 250mL triangular flask, and obtaining a solution B according to the treatment method of the solution A; measuring the absorbance of the solution A and the solution B at the wavelength of 550nm by taking ethanol as a reference;

M0A preparation solution A is used to measure the mass of the coated seed,

M1A preparation solution B is used to measure the mass of the coated seed,

a0 the absorbance of solution a,

a1 absorbance of solution B;

(7) determination of the suspension Rate

Weighing 5g of each of the two samples A and B in a 200mL beaker, adding 50mL of standard hard water, performing circular motion at a speed of 120 times/min by hand for about 2min, moving the suspension into a 250mL measuring cylinder, diluting the suspension to a scale with 100mL of standard hard water, covering a plug, and turning the measuring cylinder upside down within 1min for 30 times by taking the bottom of the measuring cylinder as an axis; immediately removing the 9/10 suspension in 10-15s from the sample A by using a pipette, ensuring that the opening of the pipette is always a few millimeters below the liquid surface, transferring the residual 25m1 residue to a beaker with 100m1 and dried to constant weight, adding 1mL of ethanol when the water is removed to about 2mL in a constant-temperature water bath at 80-90 ℃, continuing to remove the water in the water bath until the weight is constant, and weighing to obtain the mass m of the residue; opening the glass plug of the B measuring cylinder, vertically placing the B measuring cylinder into a constant temperature water bath (3011 ℃) without vibration, standing for 30min, then removing 9/10 floating liquid of the content in 10-155 by using a suction pipe without stirring or stirring sediment in the measuring cylinder, ensuring that the opening of the suction pipe is always a few millimeters below the liquid level, treating the same sample A with 25mL of residue to obtain a residual substance m,

ma-suspension 25m1 remaining at the bottom of the A cylinder evaporates to a constant weight,

mb-suspension liquid remained at the bottom of the B measuring cylinder and 25m1 was evaporated to constant weight;

(8) determination of Heat storage stability

Injecting about 30mL of sample into a clean ampoule bottle by using an injector, sealing the ampoule bottle by using high-temperature flame, sealing at least 3 bottles, respectively weighing, placing the sealed ampoule bottle into a metal container, placing the metal container into a thermostat of 54 degrees and 2 degrees or a thermostatic water bath, taking out the metal container after placing for 14 days, weighing the ampoule bottle, weighing the sample with unchanged mass, and measuring the content of effective components within 24 hours;

(9) determination of content of active ingredient

The test adopts high performance liquid chromatography to detect the effective components of the sample, and methanol-water is used as 9: taking 1 as a mobile phase, measuring 0.04g of a bactericide standard substance in a volumetric flask with 50mI at a flow rate of 0.2m1/min and a wavelength of 230nm and a sample introduction amount of 10uL, metering to a scale with methanol to completely dissolve the bactericide standard substance, and shaking up to obtain a standard sample solution A; weighing 0.05 pesticide standard substance in another 50mI volumetric flask, transferring 5ml standard solution A in the volumetric flask by a pipette, metering to a certain volume by using methanol until the volume reaches a scale, completely dissolving the standard solution A, and shaking up to obtain solution B; weighing 1ml of sample, placing the sample in a 50mI volumetric flask, adding about 40m1 methanol, carrying out ultrasonic oscillation in a water bath at 40 ℃ for 10min to completely dissolve the sample, placing the sample to room temperature, using the methanol to fix the volume to the scale, and shaking up; then filtered by a 0.45um filter, and clear liquid is left for testing. Under the operating conditions, after the baseline of the instrument is stable, continuously injecting a plurality of needle standard sample solutions, calculating the repeatability of the relative response value of each needle, measuring according to the sequence of the standard sample solutions, the sample solutions and the standard sample solutions, respectively averaging the peak areas in the two measured needle sample solutions and the two needle standard sample solutions before and after the sample, and calculating the mass fraction;

(10) determination of pH

Weighing 1g of sample in a 100mL beaker, adding 100mL of water, stirring vigorously for 1min, inserting an electrode into the sample solution, measuring the pH value of the sample solution, measuring the pH value at least three times in parallel, wherein the absolute difference value of the measurement result is less than 0.1, taking the arithmetic mean value of the absolute difference value as the pH value of the sample, and standing for 1 min.

6. The research method of the sorghum fertilizer composite seed coating agent according to claim 1, characterized in that: the S5 specifically includes the following steps:

(1) indoor safety test of seed coating agent

The test adopts a wet culture method in a dish, seeds are selected according to the proportion of the test design for coating treatment and are aired for standby, a proper amount of cotton is firstly added into a culture dish, embryos of the coated seeds are uniformly sowed upwards into the culture dish, then a layer of fine sand is coated, 10 seeds are sowed in each dish, after the seeds are cultured for 7 days at the temperature of 27-30 ℃, the whole seedling is taken out, the sand is slightly washed away by water, the emergence rate is calculated, the plant height and the main root length are measured, and the number of beard roots is recorded. Each treatment was repeated 3 times;

(2) test of field drug effect

(2.1) control target

The field pesticide effect test control objects are as follows: soil pests, sorghum head smut;

(2.2) treatment with drugs

The suspension seed coating agent is coated, and the drug-seed ratio is 1: 50;

(2.3) field design

Two fields are selected for experiments, the prevention and control effects of the maize head smut and the underground pests are respectively researched, 5 treatments are arranged in each field, the treatments are repeated for 4 times, 20 cells are arranged in random blocks, the area of each cell is about 20m2, and a protection row is arranged;

(2.4) investigation method and analysis

The artificial coating is carried out 2 days before sowing according to the treatment dosage, the medicament is required to be uniformly adhered to the seeds, the uncoated seeds are used as contrast,

insect pest investigation: investigating the base number of underground pests before sowing, setting 1 square meter as one point, and the depth of 30cm, investigating 25 points in total, investigating after sowing for 30 days, checking the gnawing condition of sorghum stalks, and calculating the control effect;

disease investigation: and (3) adopting a 5-point random sampling method, investigating when symptoms are obvious after the ears are full, calculating the prevention and treatment effect and the disease investigation, and calculating the disease index and the prevention effect.

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