WO2013020350A1

WO2013020350A1 - Empedobacter brevis pesticide and preparation method thereof

Info

Publication number: WO2013020350A1
Application number: PCT/CN2012/000563
Authority: WO
Inventors: 高小文
Original assignee: Gao Xiaowen
Priority date: 2011-08-09
Filing date: 2012-04-27
Publication date: 2013-02-14
Also published as: CN106359454A; CN102342299A

Abstract

The present invention is an Empedobacter brevis pesticide which can lead to plant pests dying of disease rapidly and a preparation method thereof. The Empedobacter brevis pesticide includes an Empedobacter brevis suspension and an Empedobacter brevis technical concentrate. Empedobacter brevis GXW15-4 is a pathogenic bacteria for plant pests isolated and extracted through dissecting the corpses of pests which have died of disease. The bacteria is preserved in the China Center for Type Culture Collection (deposit no.: CCTCC NO:M202046). The Empedobacter brevis suspension is manufactured by employing a fermentation method using a three-stage fermentation device, and the Empedobacter brevis technical concentrate is manufactured from the fermentation products at the first or second stages in the manufacturing process of the Empedobacter brevis suspension by high speed isolation and a three-stage freeze-drying method. The Empedobacter brevis pesticide has a good insecticidal effect against various lepidopterous plant pests. The application of the Empedobacter brevis pesticide can prevent agricultural products being polluted by chemical pesticides and is very safe for the plants themselves, natural enemies of plant pests, humans, livestock and poultry.

Description

Short-acting bacillus pesticide and preparation method thereof

Technical field

The invention relates to a biological pesticide and a preparation method thereof, in particular to a short-lived bacillus pesticide and a preparation method thereof, and belongs to the technical field of microorganisms. Background technique

The main pests of various plants are lepidopteran pests, such as Pieris rapae, Plutella xylostella, Spodoptera litura, rice leaf curler, Euphorbia, Chilo suppressalis, Chilo suppressalis, Cotton aphid, and fruit tree moth.

The application of pesticides to plants is the main measure to control plant pests. With the development of society and the progress of mankind, people are increasingly worried about chemical pollution of agricultural products, and it is urgent to replace chemical pesticides with biological pesticides. To this end, many developed countries in the world have invested considerable manpower and financial resources to develop bio-pharmaceuticals, but biocide products have been developed more and biopesticide products have been slow to develop. For the development and application of biological pesticides for major lepidopteran pests such as Plutella xylostella, Spodoptera litura, and Rice leaf roller, which are highly resistant to chemical pesticides, the main products in the domestic and international markets are still German Bernay.

(Berliner) discovered and studied the successful production of Bacillus thuringiensis insecticide (BT) 100 years ago, and there have been no other better alternatives.

In recent years, there have been some plant-derived pesticides in the domestic and international pesticide market. However, because plant-derived pesticides are limited by plant varieties and resources, industrial production is difficult, and the development cost of plant-derived pesticides is generally higher. High, reducing the enthusiasm of people choosing to use plant-derived pesticides. Summary of the invention

The technical problem to be solved by the present invention is: In view of the defects existing in the prior art, a biological pesticide and a preparation method thereof are proposed, and the pesticide is safe to use and has no pollution to the environment.

The present invention firstly provides Empedobacter brevis GXW15-4, which is a plant pest pathogen which is extracted and screened from the corpse of dead plant worms. The strain was in 2002 12 Deposited at the China Center for Type Culture Collection (CCTCC) on the 11th of the month, deposit number CCTCC

The individual morphological characteristics of GXW15-4 were: short stem shape, length 2.6-3.8μπι, width 0.8- 1.1μπι, both ends obtuse, and observed by scanning electron microscopy with a square crystal of about 1μπι3 inside.

The present invention provides a pesticide containing short-lived bacillus, which is a suspension agent, and contains a milky white suspension of a living body of 10 billion spores/ml of B. oxysporum GXW15-4. The preparation method of the suspending agent comprises the following steps:

Step 1: Inoculating the GXW15-4 strain into a test tube, culturing for 24 to 36 hours, temperature of 24 to 30 ° C, illumination of 3000 to 5000 Lx, pH of 7 to 7.2; here, the weight percentage is 97% of beef soup, The medium having 1.5% sucrose and 1.5% agar may also be LB medium.

Step 2: According to the characteristics of the colony of GXW15-4, the cultured short-lived bacterium GXW15-4 was selected by microscopic observation and inoculated into the culture flask. The culture medium of the culture flask was selected as the weight of the beef soup. 98.5% and sucrose 1.5% medium; first-stage fermentation by shaker culture method, the culture conditions are the same as step one;

Step 3: Inoculating the first-stage fermented B. oxysporum GXW15-4 into a secondary fermenter with a volume gradient of 10 times for secondary fermentation culture, and cultivating the substrate by weight percentage using the sterilized soybean powder (or beans) Milk powder) 4.5%, corn flour 4.5%, silkworm pupa powder 3%, sucrose 1.5%, calcium powder 1%, salt 0.5%, water 85%, culture conditions are the same as in step 1, fermentation to form the short-lived bacillus stock; The short-lived bacillus stock was inoculated into a three-stage fermentation apparatus with a volume gradient of 10 times. The culture substrate was 4.5% by weight of sterilized soy flour (or soy milk powder), 4.5% of corn flour, and 3% of silkworm pupa powder. Sucrose 1.5%, calcium powder 1%, salt 0.5%, water 85%, other culture conditions are the same as in step one, fermenting to form a suspension of short-lived bacillus, after being qualified for product quality inspection, and packaging, to obtain a suspension of short-lived bacillus.

The invention provides a pesticide containing short bacillus, the pesticide is a parent drug, and has a greater than A light yellow powder of 30 billion cubits/mg of B. oxysporum GXW15-4 in vivo. The parent drug can also be further processed to form other short-acting bacillus pesticide formulations, and can also be used in combination with chemical pesticides to treat other plant diseases and insect pests.

The preparation method of the parent drug comprises the following steps:

Steps 1 to 3 are the same as the short-lived suspension method, wherein the secondary fermentation medium is sterilized soybean powder (or soy milk powder) 4.5%, corn flour 4.5%, silkworm powder 3 %, sucrose 1. 5%, calcium powder 1%, salt 0.5%, water 85%;

Step 4: injecting the first-stage fermentation product or the secondary fermentation product into a centrifugal drying machine, and performing conventional centrifugal dewatering to a pickable and movable state;

Step 5: The product after centrifugal dehydration is subjected to three-stage freeze-drying treatment with a freeze dryer, and the temperature is controlled at -5 ± 2 ° C, -15 ± 2 ° C, - 75 ± 2 ° C, respectively, to prepare water. The powder containing less than 5% of the content is packaged after passing the test, and the short-acting bacillus parent drug is obtained.

Diluting the above two short-acting bacilli pesticides into a solution of 1-1.300 million spores/ml, sprayed on the plant epidermis, and the short-stem bacterium GXW15-4 is sucked into the plant pests by mouth, causing the plant scales within 24 hours. Hymenoptera pest death.

The present invention is directed to a biological pesticide produced by the short-lived bacillus GXW15-4, in view of the fact that plant major pests have certain resistance to chemical pesticides and people are generally concerned about the use of chemical pesticides. Its beneficial effects are: safe use, no pollution to the environment, no toxic effects on plants themselves, natural enemies of pests, mammals and aquatic animals. DRAWINGS

Figure 1 is a flow chart of the preparation process of short-lived bacillus pesticides. detailed description

Empedobacter brevis) W\^, is a plant pest killed by rickets The plant pests and pathogens were extracted and screened. The strain was deposited at the China Center for Type Culture Collection (CCTCC) on December 11, 2002, under the accession number CCTCC NO: M202046.

The extraction and culture methods of B. brevis strain GXW15-4 are as follows:

Step 1. Find the corpse of the lepidopteran pest from the leaves of the field plant, store it and bring it back indoors. Step 2: Dissect the dead body of the infected insect and observe it under the microscope;

Step 3: According to the individual morphological characteristics of the strain of B. oxysporum GXW15-4, the strain of B. brevis strain GXW15-4 is distinguished and extracted;

Step 4, the GXW15-4 strain was inoculated into a beef soup medium test tube, LB slanted surface culture for 24 to 36 hours, temperature 24~30 ° C, light 3000~5000Lx, pH 7~7. 2, obtained short stability Bacillus GXW15-4 strain. According to the test and identification report of the Institute of Microbiology of the Chinese Academy of Sciences ([2009] Micro-Detection No. 175), the 16SrR A gene sequence of B. oxysporum GXW15-4 is shown in the sequence listing.

According to the test and appraisal report of the Institute of Microbiology, Chinese Academy of Sciences ([2009] Micro-Detection No. 175), the physical and chemical test results of the suspension culture of GXW15-4 are shown in Table 1:

Physicochemical test results of short-lived bacillus suspension (BIOLIG GN2)

Test item Result Test item Result Test item Result Test item Result Cell morphology Rod-shaped Gram stain Negative Oxidase + Contact enzyme +

Color

Control - D - honey disaccharide - P-phenylethanol - L-histidine -

Acid

a - cyclodextrin + methyl grape - itaconic acid + hydroxy guanidine +

Glycosylate

Dextrin + D-alloxone - a-oxobutyl + L-leucine +

Sugar

Glycogen + D-raffinose - D-oxopentane + L-uranium ―

Diacid

Tween40 + L - rhamnose - D-oxopentane + L-phenylalanine +

Acid

Tween80 + D-sorbitol - D, L-lactic acid - L-valine +

N-acetyl half - sucrose - malonic acid - L - pyroglutamine -

Lactosamine

N-acetylgluco D-trehalose - propionic acid + D-serine -

Glucosamine

Ribitol - pine disaccharide - quinic acid - L - serine + L-Arab - Xylitol-D-hexose acid-L-threonine+

Sugar

D-arabitol pyruvate methyl + azelaic acid ― D, L -carnitine ―

― ester

D-fiber dibutyl succinate - + succinic acid ―

Sugar - methyl ester

i -erythritol - acetic acid + bromobutane + uric acid

Acid

D-Fructose - Shun-Akang - Succinyl Amine - Inosine +

Acid

L-fructose - citric acid - glucuronyl - uridine +

Amine

D-galactose formic acid ― L-alanyl + thymidine +

― Amine

Gentian disaccharide - D - galactose - D-alanine phenylethylamine

Acid lactone

a - D-grape + D-galactose - L - alanine + putrescine -

Uronic acid

Inositol alcohol - D - gluconic acid - L-alanyl + one

Alcohol

a - D-lactose - b - methyl gluconate L-asparagine + 2, 3. butyl 2

Glycoside amine alcohol

Lactulose - D-glucuron I L-aspartame + glycerol +

Acid

Maltose + a-hydroxybutanol L-glutamic acid + D, L-glycerol ―

Acid phosphate

D-mannitol ― b-hydroxybutane + glycyl day + D- glucose +

Acid towine - 1-phosphate

D-mannose ― g -hydroxybutyl + glycyl valley + D - glucose +

Acidic acid-6-phosphobacillus GXW15-4 has strong regenerative capacity and can be propagated in large quantities under factory conditions. The main substrate of the fermentation is soybean powder, soy milk powder, silkworm cocoon powder, etc. The main matrix material is Renewable resources, and abundant domestic and international markets, can meet the needs of industrial production. The final product of the present invention is a short-lived bacillus pesticide, including a short-lived bacillus suspension and a short-lived bacillus parent drug.

The suspension of short-lived bacillus is a milky white suspension containing 10 billion spores/ml of B. oxysporum GXW15-4 in vivo; the product of the above-mentioned short-lived bacterium GXW15-4 is directly packaged into a suspension of short-lived bacillus As shown in Figure 1, the specific preparation steps are as follows - Step 1: The GXW15-4 strain is inoculated into a medium test tube, cultured for 24 to 36 hours, temperature 24 to 30 ° C, light 3000~5000 Lx, pH Value 7~7, 2; medium is not limited, here is selected The weight percentage is 97% of beef soup, 1.5% of sucrose, 1.5% of agar, or LB medium.

Step 2: According to the characteristics of the colony of GXW15-4, the cultured short-lived bacterium GXW15-4 was selected by microscopic observation and inoculated into the culture flask. The medium is not limited. The weight percentage is beef soup. 98. 5% and sucrose 1.5% of the medium; the first-stage fermentation is carried out by a shaker culture method, and the other culture conditions are the same as the first one except the medium;

Step 3: Inoculating the first-stage fermented B. oxysporum GXW15-4 into a secondary fermenter with a volume gradient of 10 times for secondary fermentation, using the sterilized soy flour (or beans) in percentage by weight Milk powder) 4. 5%, corn flour 4.5%, silkworm cocoon powder 3%, sucrose 1.5%, calcium powder 1%, salt 0.5%, water 85%, other culture conditions are the same as step one, fermentation formation 5%。 After the sterilized soy flour (or soy milk powder) 4. 5% by weight of the third-stage fermentation apparatus. 4, 5% of corn flour, 3% of silkworm pupa, 5% of sucrose, 1% of calcium powder, 5% of salt, 85% of water, and other culture conditions are the same as in the first step, fermenting to form a suspension of Bacillus brevis. After being qualified for product quality inspection, it is packaged to obtain a suspension of short-lived bacillus.

The suspension of short-lived bacillus can be directly diluted into an aqueous solution of 1-1.3 billion spores/ml and sprayed evenly on the positive and negative epidermis of the stems and leaves of the plant. After the lepidopteran pests are sucked into the body, the lesions die within 24 hours.

The short-lived bacillus parent drug is a pale yellow powder having a life of 30 billion spores/mg of B. oxysporum GXW15-4. The powdery product formed by the high-speed centrifugal dehydration and the third-stage freeze-drying of the first-stage fermentation product or the second-stage fermentation product of the above-mentioned B. oxysporum GXW15-4 is as shown in Fig. 1, and the specific preparation steps are as follows:

Steps 1 to 3 are the same as the short-lived suspension method, wherein the secondary fermentation medium is sterilized soybean powder (or soy milk powder) 4.5%, corn flour 4.5%, silkworm powder 3 %, sucrose 1. 5%, calcium powder 1%, salt 0.5%, water 85%; Step 4: injecting the first-stage fermentation product or the secondary fermentation product into a centrifugal drying machine, and performing conventional centrifugal dewatering to a pickable and movable state;

Step 5: The product after centrifugal dehydration is subjected to three-stage freeze-drying treatment with a freeze dryer, and the temperature is controlled at _5 ° C, - 15 ° C, -75 ° C, respectively, to prepare a powder having a moisture content of less than 5%. After being inspected and packaged, the short-acting bacillus parent drug is obtained.

The short-stabilizing bacillus can be further processed to form short-acting bacillus pesticides of other dosage forms for the control of lepidopteran pests, and can also be used to treat other plant diseases and insect pests after compounding with chemical pesticides.

The virulence and insecticidal effects of the short-lived bacillus suspension prepared in this example are as follows:

1. As determined by Nanjing Agricultural University, the suspension of short-lived bacillus has significant insecticidal activity against the larvae of Plutella xylostella. Tested by the Nanjing Institute of Environmental Sciences of the Ministry of Environmental Protection, the normal use of short-lived bacillus suspension in the field is low risk for zebrafish, large cockroaches, chlorella, bees and cockroaches.

2. Tested by Jiangsu Provincial Center for Disease Control and Prevention, the suspension of short-lived bacillus is slightly toxic to male and female rats, and is not irritating to rabbit skin, and is a weakly sensitizing substance. Tested by the Occupational Health and Poison Control Institute of the Chinese Center for Disease Control and Prevention, the suspension of short-lived bacillus is not pathogenic to the human body.

3. The results of the test report issued by the four institutions of the Agricultural College of China Agricultural University, the Institute of Plant Protection of Henan Academy of Agricultural Sciences, the Plant Protection Institute of Shandong Academy of Agricultural Sciences, and the Jiangsu Provincial Pesticide Control Institute indicate: The mortality rate of Plutella xylostella and Spodoptera litura larvae increased day by day within 7 days after drug administration. On the 7th day after the drug, the lethal rate of 10 billion spores/ml of Bacillus sinensis suspension 800 times solution and 900 times solution against Plutella xylostella and Spodoptera litura larvae was higher than 16000 IU/mg Bacillus thuringiensis WP 1000 times; The lethal rate of the 100-fold solution of the billion spores/ml of the short-lived bacillus suspension against the larvae of Plutella xylostella and Spodoptera litura was slightly lower than that of 16000 IU/mg of Bacillus thuringiensis WP 1000 times.

4. The Anhui Provincial Plant Protection Station and the Jiangsu Plant Protection Station respectively published a demonstration test report on the insecticidal effects of vegetable pests in 2010. 10 billion spores/ml short-lived bacillus suspension agent 600 times solution, 800 times solution, 1000 times solution The average lethal rate of P. xylostella larvae was 81.78%, 77.17%, 70.88%, respectively, better than 16000 IU/mg Bacillus thuringiensis WP 500 times (see Table 2); The average lethal rate of 10 billion spores/ml of Bacillus sinensis suspension 600 times solution and 800 times solution against larvae of Spodoptera litura larvae was 85.11%, 83.30%, both higher than 16000 IU. /mg Bacillus thuringiensis wettable powder 500 times solution; 10 billion spores / ml short-lived bacillus suspension agent 1000 times liquid efficacy and 16000 IU / mg Bacillus thuringiensis wettable powder 500 times liquid phase imitation (see Table 3). Summary of the insecticidal effects of demonstration tests on Plutella xylostella in two places (%)

3 days after treatment, 5 days after treatment, 7 days after treatment

10 billion spores / ralsporin suspension 600 times solution 86. 2 82. 55 81. 78

10 billion spores/ml short-lived bacillus suspension 800 times liquid 83. 87 77. 85 77. 17

10 billion spores/ml short-lived bacillus suspension 1000 times liquid 77. 63 72. 31 70. 88

16000 IU/mg Bacillus thuringiensis wettable powder 500 times liquid 78. 18 73. 18 70. 44

Table 3 Summary of the insecticidal effects of the demonstration test on Spodoptera litura (%)

3 days after treatment, 5 days after treatment, 7 days after treatment

10 billion spores/ml short-lived bacillus suspension 600 times liquid 89. 22 91. 54 85. 11

10 billion spores Ail short-lived bacillus suspension 800 times liquid 85. 67 88. 63 83. 30

10 billion spores/ml short-lived bacillus suspension 1000 times liquid 79. 35 82. 96 77. 03

16000 IU/mg Bacillus thuringiensis wettable powder 500 times liquid 82. 67 85. 26 77. 05

In addition to the above implementations, this product may have other embodiments. Any technical solution formed by equivalent replacement or equivalent transformation falls within the scope of protection required by the invention of this product.

Claims

1. Empedobacter brevis GXW15_4 (CCTCC NO. M202046) is a plant pest pathogen that extracts and screens the dead bodies of plant pests that have died of rickets.

A pesticide comprising the B. oxysporum according to claim 1, wherein the agricultural drug is a suspension agent containing a milky white suspension of a living body of 10 billion spores/ml of B. oxysporum GXW15-4.

A pesticide comprising the B. oxysporum according to claim 1, wherein the agrochemical is a parent drug, and has a pale yellow powder having a living body of 30 billion spores/mg of B. oxysporum GXW15-4.

The method according to claim 2, comprising the following steps - Step 1: inoculating the GXW15-4 strain into a test tube, culturing for 24 to 36 hours, temperature 24 to 30 ° C, illumination 3000 ~5000Lx, pH 7~7. 2;

Step 2: inoculation of the cultured B. oxysporum GXW15-4 into a medium culture culture flask, and performing a first-stage fermentation by a shaker culture method, and the fermentation culture conditions are the same as those in the first step;

Step 3: inoculating the first-stage fermented Bacillus brevis strain GXW15-4 into a secondary fermenter with a volume gradient of 10 times for secondary fermentation, and the fermentation culture conditions are the same as those in the first step, and fermenting to form a short-lived bacillus stock;

Step 4: Inoculate the short-lived Bacillus subtilis to a three-stage fermentation device with a volume gradient of 10 times. The fermentation culture conditions are the same as in the first step, and the suspension is formed into a short suspension of Bacillus licheniformis. After the product quality inspection, the package is packaged to obtain Bacillus brevis. Suspending agent.

The method according to claim 3, comprising the steps of: Step 1: inoculating the GXW15-4 strain into a test tube, culturing for 24 to 36 hours, temperature 24 to 30 ° C, illumination 3000 〜5000Lx, pH 7~7. 2;

Step 2: inoculation of the cultured B. oxysporum GXW15-4 into the medium culture culture flask, and performing the first-stage fermentation by a shaker culture method, and the fermentation culture conditions are the same as those in the first step;

Step 3: Inoculating the first-stage fermented B. oxysporum GXW15-4 into a secondary fermenter with a volume gradient of 10 times for secondary fermentation, and the fermentation culture conditions are the same as those in the first step, and fermenting to form a short-lived Bacillus subsp.

Step 5: The product after centrifugal dehydration is freeze-dried by a freeze dryer to prepare a powder having a moisture content of less than 5%, and after being inspected and packaged, the short-acting Bacillus parent drug is obtained.

The method for preparing a short-lived bacillus pesticide according to claim 5, wherein: the freeze-drying treatment in the step 5 is divided into three stages, and the temperature is controlled at -5 ± 2 ° C, - 15 ± 2 ° C, respectively. , -75

± 2 °C.

The method for preparing a short-lived bacillus pesticide according to claims 4 and 5, characterized in that - the secondary or tertiary culture medium in the fermentation process is sterilized soybean powder or soybean milk powder by weight percentage. 5%, corn flour 4. 5%, silkworm pupa powder 3%, sucrose 1. 5%, calcium powder 1%, salt 0.5%, water 85%.