CN109479903B - Isaria javanicus conidium oil suspending agent and application thereof - Google Patents
Isaria javanicus conidium oil suspending agent and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- A—HUMAN NECESSITIES
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- A01N25/02—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing liquids as carriers, diluents or solvents
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Abstract
The invention belongs to the technical field of biological pesticides and biological pest control, and relates to a conidium oil suspending agent of Isariajavanica and application thereof. The isaria javanicus conidium oil suspending agent comprises the following components in percentage by mass: 3-10% of conidium pure powder of Isaria javanicus, 3-10% of dispersant, 3-10% of thickener and 80-90% of oil base. The Isaria javanicus of the present invention belongs to Fungi, Dikarya binuclear, Ascomycota, Pezizomycotina, Sordariomyces, Hypocryyceae, Hypocryycetales, Hypocryales, Cordyceps, Cordycipicaceae, Isaria, in Ascomycota taxonomy. The oil suspending agent provided by the invention is used for preventing and treating prodenia litura, and the pathogenicity to third-instar larvae of the prodenia litura is up to more than 80%.
Description
Technical Field
The invention belongs to the technical field of biological pesticides and biological pest control, relates to a fungal pesticide, and particularly relates to a conidium oil suspending agent of Isariajavanica and application thereof.
Background
Isaria javanica is a common entomopathogenic fungus, is widely distributed in nature, is not safe to people and livestock, does not cause environmental pollution, is a biological pesticide with a contact killing function due to invasion of pest epidermis to cause insect morbidity and death, and does not cause insect resistance, so that the Isaria javanica is more and more concerned, and has the potential of developing into a fungal pesticide. However, since the fungal pesticide is a living body, and is sensitive to environmental factors such as high temperature, drought and ultraviolet rays, the field application is restricted, so that conidia of the fungi need to be processed into a proper dosage form to improve the field application effect.
The oil suspending agent is a suspension liquid formulation formed by dispersing solid pesticide active ingredients which are insoluble in an oil solvent in a non-aqueous medium, namely oil, and forming high dispersion stability by virtue of a surfactant, and the oil suspending agent mainly comprises the pesticide active ingredients, an oil base, a dispersing agent, an emulsifying agent, a thickening agent and the like. The fungal conidium oil suspending agent has the following advantages: the medium is natural oil generally, has no environmental pollution, can avoid the problems of flammability, explosiveness, poisoning and the like in production, does not generate dust, has large specific gravity, small packaging volume, good dispersibility and spreadability, is easy to adhere to the surfaces of insects and plants, resists rain wash and has high drug effect. Therefore, the oil suspending agent of the fungal conidium is more and more valued. Some of the presently disclosed invention patents relate to oil suspensions of fungal conidia:
the invention has the following patent: a fungal spore oil suspension (application No. 200810069704.9) is prepared from beauveria bassiana, Metarrhizium anisopliae, Lecanicillium lecanii, and Trichoderma.
The invention has the following patent: a Paecilomyces lilacinus fungal spore oil suspending agent and its preparation method and application (application No. 201210256521.4) are provided.
The invention has the following patent: a trichoderma oil suspending agent and its preparation method (application No. 201610905079.1).
Although the oil suspending agent has various advantages, the oil suspending agent also successfully overcomes the defects that fungal spores are difficult to use in a dry environment and the insecticidal effect is influenced by the humidity in the environment, but the oil suspending agent has the following problems: (1) the cost of the preparation oil is high, and the preparation oil needs to be diluted by oil or special diluent and then sprayed during field pesticide application, so that the preparation oil is not suitable for industrial production; (2) the entomogenous fungi spore has low germination rate and poor storage stability in the preparation oil; these greatly restrict the use of entomogenous fungi. Therefore, the research of the new fungal oil-based formulation with low cost, good storage stability and small environmental influence becomes a hotspot of fungal preparation research.
Disclosure of Invention
The invention aims to provide the entomogenous fungus conidium oil suspending agent which is low in application cost and convenient to use by combining the advantages of entomogenous fungi and the oil suspending agent.
The invention also aims to provide a preparation method of the oil suspending agent.
The invention further aims to provide application of the oil suspending agent in prevention and control of crop pests, and particularly provides application of the oil suspending agent in prevention and control of prodenia litura.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
an Isaria javanicus conidium oil suspending agent comprises the following components in percentage by mass: 3-10% of conidium pure powder of Isaria javanicus, 3-10% of dispersant, 3-10% of thickener and 80-90% of oil base.
Preferably, 5% of pure powder of conidium of the Isaria javanicus, 5% of dispersing agent, 5% of thickening agent and 85% of oil base.
Preferably, the conidium pure powder of Isaria javanicanis has the spore content of more than or equal to 1.5 x 1010Each spore per gram, the water content is less than or equal to 3 percent, and the active spore rate is more than or equal to 95 percent.
Preferably, the ratio of the dispersant to thickener is 1: 1.
Preferably, the dispersing agent is one or more selected from fatty alcohol-polyvinyl ether, sodium methylene dinaphthalene sulfonate, abietic acid, sodium dodecyl sarcosinate and sodium butyl naphthalenesulfonate.
Preferably, the thickening agent is selected from one or two of xanthan gum and aluminum stearate.
Preferably, the oil base is selected from one or more of blend oil, peanut oil, soybean oil and corn oil.
More preferably, the dispersing agent is fatty alcohol-polyvinyl ether; the thickening agent is xanthan gum or aluminum stearate; the oil base is corn oil.
The preparation method of the oil suspending agent comprises the following steps: mixing the pure powder of the conidium of the isaria javanica with a dispersant and a thickener, then adding an oil base, and fully shaking the mixed solution on a shaking instrument to fully disperse the spore powder, the added dispersant and the thickener in the oil base to prepare the conidium oil suspending agent of the isaria javanica.
Provides the application of the Isaria javanicus conidium oil suspending agent in the aspect of preventing and controlling prodenia litura.
Compared with the prior art, the invention has the following beneficial effects:
(1) the Isaria javanicus conidium oil suspending agent provided by the invention has good storage stability and long storage period at normal temperature and low temperature of-4 ℃; particularly, 5 percent of conidium pure powder of the isaria javanica, 5 percent of fatty alcohol-polyvinyl ether, 5 percent of xanthan gum and 85 percent of corn oil are preferably selected, the stability of the isaria javanica stored for three months at normal temperature is the best, and the stability of the isaria javanica stored for three months at the low temperature of-4 ℃ is the best by selecting 5 percent of conidium pure powder of the isaria javanica, 5 percent of fatty alcohol-polyvinyl ether, 5 percent of aluminum stearate and 85 percent of corn oil.
(2) The oil suspending agent provided by the invention has good activity and high poisoning effect; the pesticide is used for preventing and treating prodenia litura, the indoor toxicity of the pesticide is increased along with the increase of time after the pesticide is used for treating third-instar larvae of the prodenia litura, and the prevention effect is as high as more than 80%.
(3) The oil suspending agent has simple preparation method and wide raw material source of products, and is suitable for industrial production.
(4) The Isaria javanicus conidium oil suspending agent provided by the invention can be used for large-scale field pest control.
Drawings
FIG. 1 conidia oil suspending agent No. 1-28 after storage for 100 days at normal temperature;
FIG. 2-4 deg.C storage of No. 1-8 conidia oil suspension after 100 days;
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more concise and clear, the present invention is described with the following specific embodiments, but the present invention is by no means limited to these embodiments. The following description is only a preferred embodiment of the present invention, and is only for the purpose of describing the present invention, and should not be construed as limiting the scope of the present invention. It should be understood that any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Example 1: preparation and quality detection of Isaria javanicus conidium pure powder
1. Preparation method of Isaria javanicus conidium pure powder
(1) First-stage solid culture, inoculating conidium from Isaria javanicus slant strain on sterilized PDA (potato 200g, glucose 20g, agar 20g, water 1000mL) plate, culturing in 25 deg.C incubator for about 2 weeks, and scraping conidium for use.
(2) And (2) secondary liquid culture, namely suspending the cultured conidia in a sterile 0.02% (v/v) Tween-80 solution, inoculating the conidia into a PD liquid culture medium (200 g of potatoes, 20g of glucose and 1000mL of water) filled with the sterilized PD, and performing shake culture at the temperature of 25 +/-1 ℃ and 170r/min for 48 hours to serve as seed liquid for later use.
(3) And performing three-stage rice fermentation and spore production culture, soaking rice in clear water for 8h, then placing the rice into polypropylene plastic bags, sterilizing the bags at high temperature and high pressure, inoculating 50mL of the seed solution into each bag, culturing the bags at 25 +/-1 ℃ for 14d in the dark, then collecting spores by using a spore separator to obtain pure powder of Isaria javanicus conidia, and storing the powder in a refrigerator at 4 ℃ for short-term use.
2. Quality detection of Isaria javanicus conidium pure powder
(1) Determination of spore content
Accurately weighing 10mg of conidium pure powder into 1mL of 0.02% (v/v) Tween-80 solution, fully and uniformly mixing, diluting by a certain multiple, measuring the spore concentration by adopting a blood counting chamber, repeating for 3 times, and calculating the spore content of the spore powder, wherein the results are shown in Table 1.
(2) Determination of spore water content
The spore water content was determined using the baking method: drying a clean weighing bottle in a drying oven at 120 ℃ for 10min, and weighing the bottle after cooling to obtain a bottle weight m 1; weighing 1g of spore powder, and placing the spore powder in a weighing bottle to weigh m 2; and then placing the spore in a drying oven at 120 ℃ for drying for 2h, covering a bottle cap, weighing to obtain m3, calculating the water content of the spores, repeating the steps for 5 times, taking an average value, and calculating the water content of the spores, wherein the results are shown in Table 1.
(3) Measurement of conidium germination rate
Weighing 1mg of spore powder, adding 10mL of autoclaved 0.05% Tween-80 aqueous solution, fully and uniformly mixing the spore powder with a vortex mixer, recording the number of spores under a microscope, diluting the spore suspension to ensure that each 10 mu l of spore suspension contains no more than 100 spores, connecting 10 mu l of spore suspension to a PDA plate, observing the number of fungus germination on the PDA after three days, calculating the spore germination rate, repeating the steps for three times, and taking an average value, wherein the results are shown in Table 1.
TABLE 1 quality test of Isaria javanicus conidium pure powder
The data in Table 1 show that the spore powder content obtained by the rice fermentation method was 1.73X 1010The spore/g has the water content of 3.03 percent and the 72-hour active spore rate of 95.46 percent, and can be used for subsequent oil suspending agent research
Example 2: screening of Isaria javanicus conidium oil suspending agent auxiliary agent
Test spore powder: conidial pure powder prepared in example 1.
Dispersant for test: sodium methylenedinaphthalenesulfonate, abietic acid, sodium dodecylsarcosinate, sodium butylnaphthalenesulfonate, and fatty alcohol-polyoxyethylene ether, all available from Guangzhou Kohao Biotech, Inc.
Test thickeners: xanthan gum and aluminum stearate were purchased from Guangzhou Konha Biotech, Inc.
The test oil base: blend oil, peanut oil, soybean oil and corn oil.
1. Preparation of oil suspension agent
Taking 1.5mL of a centrifuge tube, adding collected 5% Isaria javanicus conidium pure powder, then adding 5% of a dispersing agent and 5% of a thickening agent, finally adding 85% of an oil base to supplement to 1mL, fully shaking the centrifuge tube on a shaking instrument to fully disperse the spore powder and the added auxiliary agent in the oil base, totally 28 formulas are provided, each formula is provided with six repetitions, three tubes are respectively placed at normal temperature and-4 ℃, and in the 28 formulas, the dispersing agent, the thickening agent and the oil base are selected and shown in tables 2 and 3.
2. Character detection of oil suspending agent
(2) Storage stability
The suspension heights of the oil suspension concentrates stored for 100 days at normal temperature and in an environment of-4 ℃ in the above 28 formulations were measured, and it was judged that the storage stability was "+++" for a height of less than 1.00cm, "+++++++" for a height in the range of 1.0cm to 1.50cm and "++++" for a height of more than 1.50cm, wherein more "+" indicates better storage stability. The storage stability results at room temperature and-4 deg.C in 28 formulations are shown in figure 1-2, wherein 1-28 represents storage at room temperature for 100 days; 1 '-28' represents storage at-4 ℃ for 100 days. The storage stability of FIGS. 1-2 was converted to "+" and the results are shown in tables 2 and 3.
(1) Measurement of conidium germination rate
The prepared oil suspension preparation is shaken uniformly again, 1mL of oil solution is diluted by 1000 times, the oil solution is fully and uniformly mixed by a vortex mixer, the number of spores is recorded under a microscope, the spore suspension is diluted so that each 10 microliter contains no more than 100 spores, 10 microliter is connected to a PDA plate, the number of the conidia germinated on the PDA is observed after three days, the spore germination rate is calculated, the operation is repeated for three times, the average value is taken, and pure spore powder without any auxiliary agent is used as a blank control, and the result is shown in Table 4.
TABLE 2 storage stability of each oil suspension group at room temperature for 100 days
TABLE 3 storage stability of oil suspensions of each group at Low temperature (-4 ℃ C.) for 100 days
The storage stability of the oil suspending agent was judged by measuring the suspension height, and the results shown in FIGS. 1 to 2 show that the suspension height of the oil suspending agent stored at ordinary temperature was varied from 0.849 to 1.801, the height of the oil suspending agent stored at a low temperature of-4 ℃ was varied from 1.079 to 1.838, the storage stability was "++++" when the storage height was less than 1.00cm, the storage stability was "+++++" when the storage height was 1.0cm to 1.50cm, and the storage stability was "+++++" when the storage height was more than 1.50 cm.
From the results shown in tables 2 and 3, it can be seen that 10 formulations of Isaria javanicus conidium oil suspensions having relatively good storage stability for 100 days at room temperature and-4 ℃ are provided, and the formulations are numbered 2: 5% Isaria javanicus conidium pure powder, 85% blend oil, 5% abietic acid and 5% xanthan gum; number 6: 5% of Isaria javanicus conidium pure powder, 85% of peanut oil, 5% of sodium lauryl sarcosine and 5% of xanthan gum; number 6: 5% Isaria javanicus conidium pure powder, 85% blend oil, 5% abietic acid and 5% xanthan gum; number 8: 5% Isaria javanicus conidium pure powder, 85% soybean oil, 5% abietic acid and 5% xanthan gum; number 14: 5% of Isaria javanicus conidium pure powder, 85% of blend oil, 5% of fatty alcohol-polyvinyl ether and 5% of xanthan gum; number 16: 5% of Isaria javanicus conidium pure powder, 85% of blend oil, 5% of fatty alcohol-polyvinyl ether and 5% of aluminum stearate; number 18: 5% of Isaria javanicus conidium pure powder, 85% of peanut oil, 5% of fatty alcohol-polyvinyl ether and 5% of xanthan gum; number 20: 5% of Isaria javanicus conidium pure powder, 85% of peanut oil, 5% of fatty alcohol-polyvinyl ether and 5% of aluminum stearate; number 24: 5% of Isaria javanicus conidium pure powder, 85% of soybean oil, 5% of fatty alcohol-polyvinyl ether and 5% of aluminum stearate; number 26: 5% of Isaria javanicus conidium pure powder, 85% of corn oil, 5% of fatty alcohol-polyvinyl ether and 5% of xanthan gum; number 28: 5 percent of Isaria javanicus conidium pure powder, 85 percent of corn oil, 5 percent of fatty alcohol-polyvinyl ether and 5 percent of aluminum stearate.
TABLE 4 conidium germination Rate for each group of oil-based suspension Agents
The conidium germination rate of the isaria javanica conidium oil suspending agent in the 28 formulas is shown in the results in table 4, and the conidium germination rate of the isaria javanica conidium oil suspending agent stored at normal temperature is higher than that of the control formula, namely the formula with the higher germination rate is numbered 26: under the formula, the germination rate of the conidia is up to 94.27 percent and is higher than 86.38 percent of the control by 5 percent of Isaria javanicus conidia pure powder, 85 percent of corn oil, 5 percent of fatty alcohol-polyvinyl ether and 5 percent of xanthan gum; compared with a control, the formulation with higher germination rate of the Isaria javanicus conidium oil suspending agent stored in the environment of-4 ℃ is numbered 28: the germination rate of the conidia is up to 87.78% and is slightly lower than the germination rate of a control by 89.52% under the formula of 5% isaria javanicus conidia pure powder, 85% corn oil, 5% fatty alcohol-polyvinyl ether and 5% aluminum stearate.
Combining the results in tables 2 and 4, the formulation with good stability after being stored for 100 days at normal temperature and high conidium germination rate of the Isaria javanicus conidium oil suspending agent is No. 26: 5% of Isaria javanicus conidium pure powder, 85% of corn oil, 5% of fatty alcohol-polyvinyl ether and 5% of xanthan gum.
Combining the results in tables 3 and 4, the formulation with good stability after 100 days of storage at-4 ℃ and high conidium germination rate of the Isaria javanicus conidium oil suspension is No. 28: 5 percent of Isaria javanicus conidium pure powder, 85 percent of corn oil, 5 percent of fatty alcohol-polyvinyl ether and 5 percent of aluminum stearate.
Example 3: isaria javanicus conidium oil suspending agent indoor toxicity test and field efficacy test
1. Test insects:
the prodenia litura, adult insect is collected from southern China agriculture university farm in Guangzhou, raised with mulberry leaf to lay eggs, and after hatching, the 3 rd larva with consistent growth is selected for indoor toxicity measurement.
2. Test preparations:
treatment group: preparing 1mL of 5% Isaria javanicus conidium oil suspending agent according to 28 formulas, adding 100 mL0.02% Tween-80 aqueous solution, mixing well by vortex oscillator, adjusting with blood counting plate to obtain 1.0 × 107Per mL ofA conidia suspension; control group: the pure spore powder concentration is 1.0 × 107Suspension per mL; clear water was used as a blank control.
3. The treatment method comprises the following steps:
taking out the Isaria javanicus conidium oil suspending agent which is placed at the normal temperature and the temperature of minus 4 ℃ for 100 days, shaking uniformly, and preparing into 1 multiplied by 107Each head of the larva is soaked in the prepared corresponding bacterial liquid for 10s, 5 third instar larvae are placed in each culture dish, and the larva is cultured in a 14:10(L: D) illumination incubator with the relative humidity of 70 +/-10% at the temperature of 25 +/-1 ℃. And (3) checking the body condition of the spodoptera litura under a dissecting mirror every 12h, if the polyporptera is shriveled and becomes withered and yellow in color, considering the polyporptera to be dead, repeating each treatment for 3 times, taking the larvae soaked in the Tween as a blank control group, and calculating the death rate of the spodoptera litura, wherein the results are shown in tables 5 and 6.
TABLE 5 mortality rate of Prodenia litura after treatment with various groups of oil suspending agents at room temperature
The mortality rate of the prodenia litura treated by 28 formulations of isaria javanica conidia oil suspending agent stored at room temperature for 100d is shown in table 5, and the data result shows that the mortality rate of the third instar larvae of the prodenia litura increases with the increase of time from the 5 th d to the 9 th d after the treatment, the mortality rate of the control group is 86.67% at the 9 th d after the treatment, and the formulations of the treatment group with mortality rate higher than or equal to that of the control group are numbered 13, 14, 15, 16, 17, 26 and 28, and the mortality rate is 86.67%, 100%, 93.33%, 86.67% and 93.33%, respectively.
TABLE 6-4 deg.C mortality of Spodoptera litura treated with various groups of oil suspending agents
The mortality rate of the prodenia litura treated by 28 formulation isaria javanica conidia oil suspending agent stored at-4 ℃ for 100 days is shown in table 6, and the data result shows that the mortality rate of the third instar larvae of the prodenia litura increases with the time from the 5 th day to the 9 th day after the treatment, the mortality rate of the control group is 86.67% after the 9 th day after the treatment, and the mortality rate of only the numbers 26 and 28 of the treatment groups reaches 80%.
In summary, the following steps: 28 different Isaria javanicus oil suspending agents are prepared, the three times are respectively carried out at normal temperature and-4 ℃, the Isaria javanicus oil suspending agents are placed for 100 days and then taken out to observe the suspension situation, the obvious difference of the suspension situation of spores in different formulas is found, and the storage stability under the low-temperature condition is higher. And combining the storage stability, the spore germination rate and the biological activity of each group of oil suspending agents to obtain two groups of oil suspending agents with the best effect, wherein the two groups of oil suspending agents are respectively numbered 26 and 28, and the formulas are respectively as follows: 5% of isaria javanica conidia, 85% of corn oil, 5% of fatty alcohol-polyvinyl ether and 5% of xanthan gum; 5% of isaria javanica conidia, 85% of corn oil, 5% of fatty alcohol-polyvinyl ether and 5% of aluminum stearate.
In light of the foregoing description of the preferred embodiment of the present invention, it is to be understood that various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.
Claims (4)
1. The Isaria javanicus oil suspending agent is characterized by comprising the following components: 5% of Isaria javanicus conidia, 85% of corn oil, 5% of fatty alcohol-polyvinyl ether and 5% of xanthan gum.
2. The Isaria javanicus oil suspending agent is characterized by comprising the following components: 5% of isaria javanica conidia, 85% of corn oil, 5% of fatty alcohol-polyvinyl ether and 5% of aluminum stearate.
3. The Isaria javanicus oil suspending agent according to claim 1 or 2, wherein the conidium pure powder of Isaria javanicus has a spore content of not less than 1.5 x 1010The spore/g, impurity content is less than or equal to 0.1%, water content is less than or equal to 3%, and the active spore rate is more than or equal to 95%.
4. The use of isaria javanica oil suspending agent according to claim 1 or 2 for controlling prodenia litura.
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| CN101514325A (en) * | 2008-11-25 | 2009-08-26 | 华南农业大学 | Isaria javanicanicus mould strain and application thereof in preventing and controlling prodenia litura |
| CN105685024A (en) * | 2016-03-22 | 2016-06-22 | 广东中迅农科股份有限公司 | Labour-saving oil suspension and preparation method thereof |
| CN106479943A (en) * | 2017-01-03 | 2017-03-08 | 南京林业大学 | The Java Isaria BE01 of one plant height effect preventing and treating fall webworms and its application |
| CN106489932A (en) * | 2016-10-17 | 2017-03-15 | 上海万力华生物科技有限公司 | A kind of trichoderma oil-suspending agent and preparation method thereof |
| CN108865900A (en) * | 2018-07-16 | 2018-11-23 | 海南大学 | A kind of Java Isaria bacterial strain and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101514325A (en) * | 2008-11-25 | 2009-08-26 | 华南农业大学 | Isaria javanicanicus mould strain and application thereof in preventing and controlling prodenia litura |
| CN105685024A (en) * | 2016-03-22 | 2016-06-22 | 广东中迅农科股份有限公司 | Labour-saving oil suspension and preparation method thereof |
| CN106489932A (en) * | 2016-10-17 | 2017-03-15 | 上海万力华生物科技有限公司 | A kind of trichoderma oil-suspending agent and preparation method thereof |
| CN106479943A (en) * | 2017-01-03 | 2017-03-08 | 南京林业大学 | The Java Isaria BE01 of one plant height effect preventing and treating fall webworms and its application |
| CN108865900A (en) * | 2018-07-16 | 2018-11-23 | 海南大学 | A kind of Java Isaria bacterial strain and its application |
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