Locust microsporidia spore water suspending agent and preparation method and application thereof
阅读说明：本技术 蝗虫微孢子虫孢子水悬浮剂及其制备方法和应用 (Locust microsporidia spore water suspending agent and preparation method and application thereof ) 是由 张昕然 张欣杨 张宏 张鹏飞 于 2021-10-15 设计创作，主要内容包括：本发明涉及农业技术领域,尤其涉及一种蝗虫微孢子虫孢子水悬浮剂及其制备方法和应用,悬浮剂包含活性孢子、分散剂、诱导剂、稳定剂、活性物质；分散剂包含萘磺酸盐甲醛缩合物及烷基萘磺酸盐,以上组分按照一定顺序混合得孢子水悬浮剂,可以广泛用于田地、滩涂、草原和山林的蝗虫防治。本方案解决了现有技术中储存存活率低分层问题,可以长期保持静置不分层并形成保水层,在干燥情况下保持微孢子虫较长时间活性；本方案含有诱导成分,提高了虫孢子摄入量和杀虫效率；由于微孢子虫属于自然界存在微生物,不易对环境和农产品造成污染,对人畜无毒,且害虫不易产生抗药性。(The invention relates to the technical field of agriculture, in particular to a nosema locustae spore water suspending agent and a preparation method and application thereof, wherein the suspending agent comprises active spores, a dispersing agent, an inducing agent, a stabilizing agent and active substances; the dispersant comprises naphthalene sulfonate formaldehyde condensate and alkyl naphthalene sulfonate, and the spore water suspending agent is obtained by mixing the components in a certain sequence and can be widely used for locust control in fields, mudflats, grasslands and mountain forests. The scheme solves the problem of low storage survival rate and delamination in the prior art, can keep standing for a long time without delamination and form a water-retaining layer, and keeps the activity of the microsporidian for a longer time under the dry condition; the technical scheme contains the inducing component, so that the intake of the insect spores and the insect killing efficiency are improved; as the microsporidian belongs to microorganisms existing in nature, the microsporidian is not easy to pollute the environment and agricultural products, is non-toxic to people and livestock, and is not easy to cause drug resistance of pests.)
1. An aqueous suspending agent of nosema locustae spores is characterized by comprising active spores, 0.01-10 wt% of a dispersant, 0.01-5 wt% of a stabilizer and 0.01-4 wt% of active substances; the dispersant comprises a naphthalene sulfonate formaldehyde condensate and an alkyl naphthalene sulfonate.
2. The suspension according to claim 1, wherein the concentration of the active spores is 0.2 x 108-2×108/ml。
3. The suspension concentrate of claim 1, wherein the active agent comprises a mixture of one or more of glucose, sucrose, and trehalose.
4. The suspension according to claim 1, wherein the naphthalenesulfonate formaldehyde condensate has a degree of polymerization of 100-3000.
5. The suspension concentrate of claim 1, wherein the alkyl naphthalene sulfonate contains an alkyl chain of 6 to 20 carbon atoms.
6. The suspending agent as claimed in claim 1, wherein the stabilizer comprises xanthan gum, sodium carbonate, sodium lactate and salicylic acid, and the mass ratio of the xanthan gum to the sodium carbonate is 1: 1-2: 1-2: 1-2.
7. Use of an aqueous suspension of microsporidia locustae spores for the control of locusts with a suspension according to any one of claims 1 to 6.
8. The use of claim 7, wherein the locust comprises locust in fields, beaches, grasslands and forests.
9. The use according to claim 7 or 8, wherein the amount of the suspending agent is more than or equal to 20 ml/mu.
10. A process for preparing an aqueous suspending agent of microsporidia locustae, which is used for preparing the suspending agent of any one of claims 1 to 6, comprising the following steps:
(1) quantitatively weighing the active substance, adding a proper amount of water, and stirring until the active substance is completely dissolved to obtain a mixed solution I;
(2) dissolving the stabilizer in a proper amount of water, heating, dissolving and cooling to obtain a stabilizer solution, adding the dispersant and the stabilizer solution into the solution I, and uniformly mixing to obtain a mixed solution II;
(3) quantitatively weighing microsporidian spore mother liquor, adding the microsporidian spore mother liquor into the mixed solution II, quantitatively adding water, and uniformly mixing to obtain a final spore aqueous suspension agent product.
Technical Field
The invention relates to the technical field of agriculture, in particular to a nosema locustae spore water suspending agent and a preparation method and application thereof.
Background
The locust is a major pest seriously threatening the production of agriculture and animal husbandry, the existing method for preventing and controlling the pest is mainly chemical pesticide prevention and control, and the large amount of chemical pesticide can effectively control the locust, but can bring adverse consequences such as environmental pollution, overproof chemical pesticide residue of agricultural products, damage to biodiversity, increased locust drug resistance, increased prevention and control cost, frequent serious incidents causing poisoning to people and livestock and the like, so the prevention and control method capable of replacing the chemical pesticide is required to be adopted, and the existing biological preparation has strong specificity to the locust, is not easy to generate drug resistance and is harmless to people and livestock. The inventor of the invention finds that the existing biological preparation for controlling the locust has the following problems: 1. poor dispersibility and stability and easy precipitation; 2. the active ingredients have short survival time, short validity period and difficult transportation and use; 3. the disinsection efficiency is low, the concentration of active ingredients and the concentration of suspending agents need to be kept high, and the use cost is high.
Disclosure of Invention
In order to solve at least one problem mentioned in the background technology, the invention researches and preferably selects a microsporidia locustae water suspending agent system, can obtain good insecticidal effect under lower usage amount, belongs to natural substances, thus being not easy to cause pollution to environment and agricultural products, belongs to natural substances, namely special parasites of locusts, is only effective to the locusts, and the spore water suspending agent components are all nontoxic and harmless substances, thus being not easy to cause pollution to environment and agricultural products, being not easy to cause drug resistance of pests, and being nontoxic to human and livestock; the microsporidian can be stored for a long time in a normal temperature environment and the activity of the microsporidian can be kept for a long time under a drought condition.
In order to solve at least one of the problems mentioned in the background art, an object of an embodiment of the present invention is to provide a microsporidia locustae aqueous suspension comprising active spores, 0.01 to 10 wt% of a dispersant, 0.01 to 5 wt% of an inducer, 0.01 to 5 wt% of a stabilizer, 0.01 to 4 wt% of an active substance; the active substance comprises one or more of glucose, sucrose and trehalose.
Preferably, the concentration of active spores is 0.2X 108-2×108/ml。
Preferably, the dispersant comprises a naphthalene sulfonate formaldehyde condensate and an alkyl naphthalene sulfonate.
Preferably, the naphthalenesulfonate formaldehyde condensate has a degree of polymerization of 100-.
Preferably, the alkyl naphthalene sulfonate contains an alkyl chain of 6 to 20 carbon atoms.
Preferably, the stabilizer comprises xanthan gum, sodium carbonate, sodium lactate and salicylic acid, and the mass ratio of the xanthan gum to the sodium carbonate is 1: 1-2: 1-2: 1-2.
An application of microsporidia locustae water suspending agent for preventing and treating locusts.
Preferably, the locust comprises locusts in fields, beaches, grasslands and mountains.
Preferably, the dosage of the suspending agent is more than or equal to 20 ml/mu.
A preparation method of a nosema locustae spore water suspending agent is used for preparing the suspending agent and comprises the following steps:
(1) quantitatively weighing the active substance, adding a proper amount of water, and stirring until the active substance is completely dissolved to obtain a mixed solution I;
(2) dissolving a stabilizer in a proper amount of water, heating for dissolving, cooling to obtain a stabilizer solution, adding the dispersant and the stabilizer solution into the solution I, and uniformly mixing to obtain a mixed solution II;
(3) and quantitatively weighing microsporidian spore mother liquor, adding the microsporidian spore mother liquor into the mixed solution II, further quantitatively adding water, and uniformly mixing to obtain the final spore aqueous suspension agent product.
Advantageous effects
The invention provides a nosema locustae spore water suspending agent, which has the following characteristics:
1. an optimized dispersant system is adopted, a naphthalene sulfonate formaldehyde condensate and an alkyl naphthalene sulfonate composition are adopted, a hydrophobic group is combined with a spore cell membrane, and a hydrophilic naphthalene sulfonic acid group improves the affinity with water and can keep standing and stability for a long time without layering;
2. the naphthalene sulfonate formaldehyde condensate and the alkyl naphthalene sulfonate composition can form a water-retaining layer on the surface of the microsporidian in a dry environment, and keep the activity of the microsporidian for a longer time under the dry condition;
3. the active substance is added, so that necessary nutrient substances are provided during the dormancy period of the microsporidian, and the storage survival rate of the microsporidian spores is further improved;
4. the optimized combined stabilizer has good inhibition effect on microsporidian phage, and further improves the storage survival rate of microsporidian spores;
5. the active substance is simultaneously used as a sensitive inducing agent of the locust, so that the locust is induced to be fed intensively, the intake of the spores of the locust and the insecticidal efficiency are improved, and a good insecticidal effect can be obtained under a lower using amount;
6. the insecticidal spore is an obligate parasite for locust and is only effective on locust, and the spore water suspending agent components are non-toxic and harmless substances, so that the insecticidal spore does not pollute environment and agricultural products, is not easy to cause pest resistance to drugs, and is non-toxic to people and livestock.
Drawings
FIG. 1 is a graph showing the live locust attracting effect of the experiment of Experimental example 1.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all 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 scope of the present invention.
Example 1
The embodiment of the invention provides a nosema locustae spore water suspending agent, which comprises active spores, 0.01 wt% of dispersing agent, 0.01 wt% of inducer, 0.01 wt% of stabilizing agent and 0.01 wt% of active substance; the active substance is sucrose.
Preference is given toThe concentration of the active spores is 0.2X 108/ml。
Preferably, the dispersant comprises a naphthalene sulfonate formaldehyde condensate and an alkyl naphthalene sulfonate.
Preferably, the polymerization degree of the naphthalene sulfonate formaldehyde condensate is 100.
Preferably, the alkyl naphthalene sulfonate contains a 6 carbon alkyl chain.
Preferably, the stabilizer comprises xanthan gum, sodium carbonate, sodium lactate and salicylic acid, and the mass ratio of the xanthan gum to the sodium carbonate is 1: 1.2: 1.2: 1.2.
an application of microsporidia locustae water suspending agent for preventing and treating locusts.
Preferably, the locust comprises locusts in fields, beaches, grasslands and mountains.
Preferably, the dosage of the suspending agent is 40 mL/mu.
The embodiment of the invention also discloses a preparation method of the nosema locustae spore water suspending agent, which is used for preparing the suspending agent and comprises the following steps:
(1) quantitatively weighing the active substance, adding a proper amount of water, and stirring until the active substance is completely dissolved to obtain a mixed solution I;
(2) dissolving a stabilizer in a proper amount of water, heating for dissolving, cooling to obtain a stabilizer solution, adding the dispersant and the stabilizer solution into the solution I, and uniformly mixing to obtain a mixed solution II;
(3) and quantitatively weighing microsporidian spore mother liquor, adding the microsporidian spore mother liquor into the mixed solution II, further quantitatively adding water, and uniformly mixing to obtain the final spore aqueous suspension agent product.
Example 2
The embodiment of the invention provides a nosema locustae spore water suspending agent, which comprises active spores, 10 wt% of a dispersing agent, 5 wt% of an inducer, 5 wt% of a stabilizing agent and 4 wt% of active substances; the active substance comprises glucose and sucrose.
Preferably, the concentration of active spores is 0.4X 108/ml。
Preferably, the dispersant comprises a naphthalene sulfonate formaldehyde condensate and an alkyl naphthalene sulfonate.
Preferably, the polymerization degree of the naphthalene sulfonate formaldehyde condensate is 3000.
Preferably, the alkyl naphthalene sulfonate contains an alkyl chain of 20 carbon atoms.
Preferably, the stabilizer comprises xanthan gum, sodium carbonate, sodium lactate and salicylic acid, and the mass ratio of the xanthan gum to the sodium carbonate is 1: 1.5: 1.5: 1.5.
an application of microsporidia locustae water suspending agent for preventing and treating locusts.
Preferably, the locust comprises locusts in fields, beaches, grasslands and mountains.
Preferably, the dosage of the suspending agent is 50 ml/mu.
A preparation method of a nosema locustae spore water suspending agent is used for preparing the suspending agent and comprises the following steps:
(1) quantitatively weighing the active substance, adding a proper amount of water, and stirring until the active substance is completely dissolved to obtain a mixed solution I;
(2) dissolving a stabilizer in a proper amount of water, heating for dissolving, cooling to obtain a stabilizer solution, adding the dispersant and the stabilizer solution into the solution I, and uniformly mixing to obtain a mixed solution II;
(3) and quantitatively weighing microsporidian spore mother liquor, adding the microsporidian spore mother liquor into the mixed solution II, further quantitatively adding water, and uniformly mixing to obtain the final spore aqueous suspension agent product.
Example 3
The embodiment of the invention provides a nosema locustae spore water suspending agent, which comprises active spores, 5 wt% of a dispersing agent, 2 wt% of an inducer, 2 wt% of a stabilizing agent and 2 wt% of active substances; the active substance is a mixture of glucose, sucrose and trehalose.
Preferably, the concentration of active spores is 2X 108/ml。
Preferably, the dispersant comprises a naphthalene sulfonate formaldehyde condensate and an alkyl naphthalene sulfonate.
Preferably, the polymerization degree of the naphthalene sulfonate formaldehyde condensate is 1000.
Preferably, the alkyl naphthalene sulfonate contains a 10 carbon alkyl chain.
Preferably, the stabilizer comprises xanthan gum, sodium carbonate, sodium lactate and salicylic acid, and the mass ratio of the xanthan gum to the sodium carbonate is 1: 1.8; 1.8: 1.8.
an application of microsporidia locustae water suspending agent for preventing and treating locusts.
Preferably, the locust comprises locusts in fields, beaches, grasslands and mountains.
Preferably, the dosage of the suspending agent is 80 ml/mu.
A preparation method of a nosema locustae spore water suspending agent is used for preparing the suspending agent and comprises the following steps:
(1) quantitatively weighing the active substance, adding a proper amount of water, and stirring until the active substance is completely dissolved to obtain a mixed solution I;
(2) dissolving a stabilizer in a proper amount of water, heating for dissolving, cooling to obtain a stabilizer solution, adding the dispersant and the stabilizer solution into the solution I, and uniformly mixing to obtain a mixed solution II;
(3) and quantitatively weighing microsporidian spore mother liquor, adding the microsporidian spore mother liquor into the mixed solution II, further quantitatively adding water, and uniformly mixing to obtain the final spore aqueous suspension agent product.
Some optional embodiments, adopt the dispersant system of optimization, adopt naphthalene sulfonate formaldehyde condensate and alkyl naphthalene sulfonate composition, hydrophobic group and spore cell membrane are bound, the affinity with water of hydrophilic naphthalene sulfonate group improvement, can not delaminate and keep the stationary stability for a long time;
in some alternative embodiments, the naphthalene sulfonate formaldehyde condensate and alkyl naphthalene sulfonate composition can form a water-retaining layer on the surface of microsporidian in a dry environment, and maintain the activity of microsporidian for a longer period of time in the dry environment;
some alternative embodiments, with the addition of active substances, further improve the storage survival rate of microsporidian spores in order to provide the necessary nutrients during dormancy of microsporidian;
some optional embodiments adopt the optimized combined stabilizer, have good inhibition effect on microsporidian phage, and further improve the storage survival rate of microsporidian spores;
in some optional embodiments, the active substances are simultaneously used as a sensitive inducing agent for the locust, so that the locust is induced to concentrate on feeding, the ingestion of the spores of the locust and the insecticidal efficiency are improved, and a good insecticidal effect can be obtained under the condition of low usage amount;
in some alternative embodiments, the insecticidal spore belongs to a natural substance which is an obligate parasite of locust and is only effective on locust, and the spore water suspending agent components are nontoxic and harmless substances, so that the insecticidal spore does not easily cause pollution to the environment and agricultural products, does not easily cause drug resistance of pests, and is nontoxic to people and livestock.
Experimental example 1
The locust can obtain the external emitted information smell by means of special olfactory organs distributed on the tentacles of the locust, and as a result of natural selection, the locust has a feeding tendency on plants rich in nutrition, the basic reason is that the locust is sensitive to pheromones of parts of favorite plants, and substances capable of inducing the locust to approach or contact with the feeding are extracted by researching information substances in the plants.
Further research finds that the micromolecular saccharides and starch substances mainly have influence on the feeding tendency and the feeding amount of the locust, and the reason is that the starch and the micromolecular saccharides are high-efficiency energy supplying substances and are convenient for organisms to rapidly supplement energy; but the volatility is not strong, so that the locust sensed by olfaction cannot be directly influenced. The significance lies in that when the locust killing agent is used with insecticidal spores and the like, the locust feeding tendency and the toxin intake can be increased, and then the killing rate and the killing efficiency are increased.
The specific test mode is as follows: as shown in fig. 1, 20 PVC square white boards with a width of 0.5 m × 0.5 m were randomly placed in a locust-beneficial area, and the white boards were spaced apart by 10 m. Randomly smearing 5 pieces of spore suspending agent prepared in the embodiment 1 on a white board, numbering 1-5, and taking the spore suspending agent as a first test group; randomly coating 5 pieces of the spore suspending agent sample I on a white board, and replacing sucrose with glucose compared with the sample prepared in the example 1, wherein the number is 6-10, and the sample is used as a test group II; randomly coating 5 pieces of the sample II of the spore suspending agent on a white board, and replacing sucrose with trehalose compared with the sample prepared in the example 1, wherein the sample is numbered 11-15 and is used as a test group III; the final 5 sporulated suspension samples three, which contained no sucrose compared to the samples prepared in example 1, were used as controls in nos. 16-20. Respectively arranging a vertically downward camera 50 meters above the middle of 10 white boards, respectively taking pictures of the white boards every 3 hours from 8 o 'clock to 6 o' clock later, and recording the number of locusts in the pictures, wherein the results are shown in table 1: the inducing ability of cane sugar, glucose and trehalose to the locust feeding tendency is reduced in sequence; and has obvious food intake tendency inducing effect compared with a control group without adding sucrose, glucose and trehalose substances.
TABLE 1 feeding tendency test
Experimental example 2
Internal Activity assay
1. Purpose of the experiment
The activity of the nosema locustae spore water suspending agent is measured by taking 3-year nymphs of locusta migratoria (locusta nymphs) in east Asia as a test target according to an NY/T1154.2-2006 toxic leaf clamping method.
2. Test conditions
Test targets: and 3-year nymphs of locusta migratoria (locusta nymphs) in east Asia are taken as test targets.
The instrument equipment comprises:
electronic balance (minimum weighing value 0.1mg)
Hole puncher, capillary dropper
250ml transparent plastic cup
Culture dish with diameter of 9cm
Beaker
Liquid transfer device
Tweezers
Gauze, rubber band, etc
3. Design of experiments
Preparing a test material:
locusta migratoria (L.) Merr
Feeding corn leaves at a feeding temperature of 28 + -1 deg.C and humidity of 50 + -10% under 24 hr illumination.
Medicament:
test agents: locust microsporidian spore mother liquor (2X 10)10/ml)。
Control agents: 95% fipronil raw material.
4. Test procedure
Preparing a medicament:
the microsporidian locust spore mother liquor is diluted by the microsporidian locust spore aqueous suspending agent formula (example 1) of the invention and is respectively prepared into 2 multiplied by 105、2×106、2×107、2×108、2×109Spore aqueous suspension/ml.
Control agents: dissolving 95% fipronil with acetone to obtain 5mg/L solution.
Blank control: an aqueous suspension without microsporidia of locusta was used as CK.
Preparing a test insect:
selecting 3-instar locusta migratoria manilensis nymphs, starving for 8h, selecting 50 locusta nymphs, weighing by using an electronic balance, and calculating the average mass of each locusta manilensis.
Manufacturing toxic leaf blades:
taking fresh corn leaf with a puncher with diameter of 1cm, dripping 1ul of liquid medicine to be detected on the leaf from low concentration by using a liquid transfer device, and combining with another leaf butterfly coated with starch paste to prepare the toxin-containing leaf butterfly. After the manufacturing is finished, the leaf butterfly is fixed by a pin inserted with a hard triangular plate and placed in a transparent plastic cup for the locust nymph to take. Each treatment was 4 replicates, with 20 replicates per test. Control agents and blanks were also treated in this manner.
Medicament treatment:
1 locust nymph was placed in each transparent plastic beaker and cultured under normal conditions. After 4h, after the test insects completely eat the butterfly, the test insects are normally fed (feeding temperature is 28 +/-1 ℃, humidity is 50 +/-10%, illumination is carried out for 24 hours, and fresh corn leaves are fed) until investigation. Removing locusta nymphs which do not completely eat the whole butterfly.
5. Investigation
According to the action characteristics of the nosema locustae, investigation is carried out at 7d, 14d, 21d and 30d after the treatment respectively, and the total number of the nosema locustae and the number of dead nosema locustae are recorded.
6. Data statistics and analysis
The calculation method comprises the following steps:
the amount of test insects ingested and the corrected mortality for each treatment were calculated according to method 5.1 in NY/T1154.2-2006.
7. Results
After 3-instar locusta migratoria locusta nymphs are treated by microsporidia locusta with different concentrations, the ingestion amount of the test locusts and the death condition on the 30 th day are shown in table 2:
TABLE 2 locusts microspore spore aqueous suspension and control agent mortality on locusts migratoria in east Asia 30 days after treatment
Treatment concentration (/ mL)
Number of test insects
Number of dead insects (head)
Mortality rate
2×109
80
79
98.75％
2×108
79
77
97.47％
2×107
76
72
91.88％
2×106
72
58
80.56％
2×105
77
42
54.55％
CK
80
10
12.50％
Control agent
76
70
92.11％
Note: the mean weight of locusta migratoria nymphs of 3-instar east Asia is 7.5 × 10-2g/head; the corrected mortality rates for the different concentration treatments were compared using a new repolarization method with a difference level of P<0.05。
2X 10 by indoor Activity measurement test9/mL、2×l08/mL、2×l07The mortality rate of nymph cells treated by the/mL nosema locustae spore water suspending agent is more than 95 percent, which is equivalent to 96.94 percent of the mortality rate of nymph cells treated by the control medicament fipronil, which indicates that the nosema locustae spore water suspending agent is effective.
Experimental example 3
To further verify the resistance of the microsporidian suspending agent to delamination and precipitation, suspending agent a was prepared as a blank control, suspending agent a contained no dispersant, as in example 2; suspending agent B was prepared as test group four, suspending agent B containing only naphthalene sulfonate formaldehyde condensate dispersant as in example 2; suspension C was prepared as test group five, suspension C containing only alkylnaphthalene sulfonate as in example 2; example 2 samples were prepared as test group six. The turbidity (NTU) of the solution was measured by a turbidimeter for different periods of time; the selected equipment measures the turbidity, namely the turbidity degree of water, and can directly measure the concentration of trace insoluble suspended substances; the selected measurement period is 30d, and the specific test results are shown in table 3:
TABLE 3 differential drying standing time Microsporum suspending agent turbidity
Numbering
0d/NTU
5d/NTU
10d/NTU
15d/NTU
20d/NTU
25d/NTU
30d/NTU
Control group
3828
1025
500
223
198
212
199
Test group four
3728
3563
3313
3198
2912
2734
2712
Test group five
3828
3643
3238
3016
2878
2763
2668
Test group six
3625
3543
3428
3325
3199
3018
2988
According to test results, the optimized dispersant system adopts a naphthalene sulfonate formaldehyde condensate and an alkyl naphthalene sulfonate composition, hydrophobic groups are combined with spore cell membranes, hydrophilic naphthalene sulfonate groups improve the affinity with water, and suspended spores in four to six groups of test groups can keep standing and stable for a long time without layering; in contrast, the control group had a much higher rate of decrease in turbidity of the solution and suspended matter concentration than the test groups of four to six due to the absence of the dispersion system for 5 days. The difference between the test group four, the test group five and the test group six is that the test group six adopts a naphthalene sulfonate formaldehyde condensate and an alkyl naphthalene sulfonate composition, and the test group four and the test group five only contain one of the substances, and the experimental result shows that the turbidity reduction rate of the hexaspore suspension of the test group is obviously lower than that of the test group four and the test group five, so that the conclusion can be drawn: the naphthalene sulfonate formaldehyde condensate and the alkyl naphthalene sulfonate composition have better anti-precipitation and anti-layering effects than the two compositions are used independently.
Experimental example 4
In order to further verify the activity retention capacity of the microsporidian suspending agent dispersant on microsporidian in a dry environment, the microsporidian suspending agent prepared in the example 2 is selected as a test group; suspension a was prepared as a blank control, suspension a containing no dispersant, as in example 2; standing at 35-40 ℃ and 10% -20% of relative humidity for different times (0d, 5d, 10d, 15d, 20d, 25d and 30d), the method is the same as experimental example 2, and the specific results are shown in Table 4:
TABLE 4 mortality of nosema locusta treated with microsporidian suspension at different standing times
The test results show that: compared with a control group sample without the dispersant component, the test group containing the dispersant component can keep the activity for a longer time in a dry environment, and the infected locusts still have 11.25 percent of mortality rate after standing for 30 days in the dry environment; the control group has almost no insecticidal effect after 10 days; this is because the storage naphthalene sulfonate formaldehyde condensate and alkyl naphthalene sulfonate compositions containing water-absorbing polar groups can form a water-retaining layer on the microsporidian surface in a dry environment, maintaining the microsporidian activity for a longer period of time in the dry condition.
Experimental example 5
To further verify the effect of the active substance on the storage activity of microsporidian, microsporidian suspension prepared in example 1 was used as test group seven, the suspension containing the active substance sucrose; suspension D was prepared as test group eight, the suspension containing the active substance glucose, as in example 1; suspension E was prepared as test group nine, the suspension containing the active substance trehalose, the remainder being as in example 1; preparing a microsporidian suspending agent F serving as a blank control group and containing no active substances; the test method is the same as that of experimental example 2, and the specific results are shown in Table 5:
TABLE 5 mortality of locusta migratoria locusta treated with microsporidian suspensions containing different active ingredients
Numbering
Number of test insects
Number of dead insects (head)
Mortality rate
Control group
80
38
47.50％
Test group seven
76
71
93.42％
Test group eight
78
76
97.44％
Test group nine
77
75
97.40％
The test results show that: by adding the active substances, the insecticidal rates of the test groups from seven to nine are far better than that of a control group without the active substances, and the insecticidal rates of the test group from seven to nine are obviously better than that of the test group from nine and the test group from seven when the trehalose and the sucrose are independently used as the active substances, which shows that the active substances provide necessary nutrient substances for the microsporidian during dormancy, the storage survival rate of the microsporidian is further improved, and the effect of the glucose as the single active substance is more obvious.
Experimental example 6
To further verify the effect of the stabilizer on phage inhibition and thus on microsporidian storage activity, experiments were carried out with the addition of T7 phage at a concentration of 1% microsporidian: microsporidian suspension prepared in example 1 was used as test group ten; preparation of suspension G as test group eleven, the suspension stabilizer component contained only sodium carbonate, the remainder being as in example 1; suspending agent H was prepared as test group twelve, the suspending agent stabilizer component containing only sodium lactate, the remainder being as in example 1; suspension I was prepared as test group thirteen, with the suspension stabilizer component containing only salicylic acid, the remainder being as in example 1; preparing a microsporidian suspending agent J as a blank control group without any stabilizer component; the test method is the same as that of experimental example 2, and the specific results are shown in Table 6:
TABLE 6 mortality of nosema locusta treated with microsporidian suspension containing different stabilizer compositions
Numbering
Number of test insects
Number of dead insects (head)
Mortality rate
Control group
80
5
6.75％
Test group ten
76
72
94.74％
Test group eleven
78
10
12.82％
Twelve test groups
77
68
75.32％
Test group thirteen
78
70
76.92％
The test results show that: by adopting the optimized combined stabilizer, the insecticidal rate is far better than that of a control group without stabilizer components, and is obviously better than that of sodium carbonate, sodium lactate and salicylic acid which are independently used as the stabilizer, so that the compound stabilizer has a good inhibition effect on T7 bacteriophage of microsporidian, and the storage survival rate of the microsporidian is further improved.
Experimental example 7
Test of field drug effect
1. Purpose of the experiment
Part 1 according to NY/T1464.1-2007 pesticide field efficacy test guidelines; the method for preventing and controlling locusts with pesticide aims at defining the prevention and control effect of microsporidian spore suspension on uncultivated area locusts and the safety of uncultivated area plants and non-target beneficial insects, etc. with different dosages of 2000 ten thousand/ml locusts, defining the proper pesticide application period, dosage and method and laying a foundation for applying the pesticide.
2. Test conditions
Selection of test subjects, crops and varieties
Test subjects: is locustamigratoria manilensis meyen, east asia migratory locust.
And (3) test crops: for non-cultivated land, the main grass seeds are small reeds, tripper and the like, and the small reeds are the main.
Environmental or facility cultivation conditions
The test land is selected on the coastal wasteland grassland, the beach area is large, various weeds are mixed, the vegetation height is 50-70cm, and the terrain is flat; locusta migratoria occurs moderately in the test.
3. Design and arrangement of experiments
Medicament:
test agents: 2000 ten thousand/ml locust microsporidia spore water suspending agent; control agents: 100 hundred million spores/gram metarhizium anisopliae wettable powder.
Dosage and numbering of the medicaments:
TABLE 7 test design of test agents
Numbering
Medicament
Dosage for administration
A
2000 ten thousand/ml microsporidian water suspending agent
10 ml/mu
B
2000 ten thousand/ml microsporidian water suspending agent
20 ml/mu
C
2000 ten thousand/ml microsporidian water suspending agent
30 ml/mu
D
2000 ten thousand/ml microsporidian water suspending agent
40 ml/mu
E
2000 ten thousand/ml microsporidian water suspending agent
50 ml/mu
F
2000 ten thousand/ml microsporidian water suspending agent
60 ml/mu
G
2000 ten thousand/ml microsporidian water suspending agent
70 ml/mu
H
2000 ten thousand/ml microsporidian water suspending agent
80 ml/mu
I
2000 ten thousand/ml microsporidian water suspending agent
100 ml/mu
J
100 hundred million spores/gram metarhizium anisopliae wettable powder
25 g/mu
CK
Blank control
—
Adding blank control to 10 test groups of test medicament and control medicament, and totaling 11 data groups;
cell area and repetition:
cell area: 666.6 square meters.
The number of repetitions: 4 times.
The application method comprises the following steps: spraying, and uniformly spraying the agent on the surface of the weeds.
The application apparatus is as follows: an atomizer.
Capacity of use: 50Kg of liquid medicine for each mu (750 Kg/hm)2)。
The information of the pesticide for preventing and treating other diseases and insect pests: no other agents were sprayed prior to and during the test.
4. Investigation, recording and measuring method
Weather and soil data:
weather data: the medicine is cloudy at the same day, the average temperature is 24 ℃, the highest temperature is 32 ℃, the lowest temperature is 22 ℃, the relative humidity is 30-40%, and the southwest wind is 4-grade.
Soil data: the soil of the test field is coastal moist soil which is alkaline and has poor soil fertility.
Investigation method, time and number of times:
investigation time and number of times: the number of the test bases before application and the control effect were tested 7, 14 and 21 days after application, and the test was conducted 4 times.
The investigation method comprises the following steps: each cell adopts a five-point sampling method, and 5 directions in east, west, south and north are distributed. Each point is more than 30 square meters, and the insects are completely caught by a catching net in a sticking way. The number of live insects was investigated and recorded.
And (3) calculating the drug effect:
the method uses the corrected population reduction rate to express the prevention and treatment effect, and carries out statistical calculation according to a method specified by 'pesticide field efficacy test criterion (I)', wherein the calculation formula is as follows:
oral cavity decline rate (%) [ (number of living insects before application-number of living insects after application)/number of living insects before application ] × 100
The percent increase of population (%) in the control area was [ (number of live insects after application-number of live insects before application)/number of live insects after application ]. times.100
Control effect (%) [ (treatment area population reduction rate-control area population increase rate)/(1-control area population increase rate) ] × 100
Direct impact on crops:
during the test period, it was observed that each treatment with the test and control agents had no significant effect on the growth of various vegetation.
Quality and yield of the product:
no observation of crop quality and yield is required.
Influence on other organisms
Impact on other pests: the influence of the test dosage of the test reagent on other pests and diseases is not found.
Effects on other non-target organisms: no adverse effect of test doses of test agents on other non-target organisms was found.
5. Results and analysis
TABLE 82000 ten thousand/ml locust microsporidian spore water suspending agent for controlling migratory locust
Note: the control (%) in the table above is the average of each repetition.
And analyzing the test result data by a Duncan's new double offset (DMRT) method.
And (3) evaluation of the medicament: the test results are combined, the prevention and treatment effect of 2000 ten thousand/ml nosema locustae spore water suspending agent on locusts on uncultivated land is good, and when the dosage of the preparation is 20 ml/mu, the death rate of 21 days after the application can reach 68.36%; when the dosage exceeds 50 ml/mu, the death rate exceeds 80 percent, the dosage is continuously increased, and the change of the death rate is not obvious.
The recommended dose is as follows: the recommended dosage is 20-60 ml/mu.
Safety: the field observation shows that the microsporidia locustae aqueous suspension agent of 2000 ten thousand/ml has no adverse effect on the growth of grasslands, the variety and the quantity of beneficial natural enemies and the like.
It will be understood by those skilled in the art that the foregoing embodiments are specific to a particular implementation of the invention and that various changes in form and detail may be made therein without departing from the spirit and scope of the invention in its practical application.