CN105028483A - Compound bactericide containing bacillus subtilis BAB-1 and boscalid and application thereof - Google Patents

Abstract

The invention relates to a compound fungicide containing bacillus subtilis BAB-1 and boscalid and its application in preventing and treating tomato gray mold and leaf mold. The compound fungicide raw material contains active components, fillers and/or auxiliary agents; the active components are composed of Bacillus subtilis BAB-1 and boscalid. After compounding Bacillus subtilis BAB-1 and boscalid, the compound fungicide of the present invention forms a complementary effect, which can not only exert the high-efficiency and rapid bacteriostatic effect of boscalid, but also be effective against botrytis cinerea. and leaf mold, and can play the role of Bacillus subtilis BAB-1 in inducing tomato resistance and protecting against gray mold and leaf mold; it can form a stable, balanced, and biologically diverse ecosystem , to achieve the purpose of improving drug efficacy, reducing the use of chemical pesticides, and lastingly controlling the prevalence of tomato gray mold and leaf mold.

Description

Compound fungicide containing Bacillus subtilis BAB-1 and boscalid and its application

technical field

The invention relates to a compound fungicide and its application, in particular to a compound fungicide containing bacillus subtilis BAB-1 and boscalid and its application in preventing and treating tomato gray mold and leaf mold.

Background technique

Botrytis cinerea is a fungal disease caused by Botrytis cinerea, which can infect more than 200 plants such as tomatoes, strawberries, cucumbers, gourds, chrysanthemums, apples, zucchini and grapes, causing gray mold the occurrence of disease. Botrytis cinerea is a worldwide important disease, especially on fruits and vegetables in facilities.

Leaf mold is another fungal disease in tomato production, which mainly damages leaves, and can also damage stems, flowers, and fruits in severe cases. Most of the diseased spots start from the lower leaves and gradually spread upwards. In severe cases, the whole leaves can dry up and curl, causing premature senescence of the plants and affecting the yield and quality of tomatoes.

Since there are no materials and varieties resistant to Botrytis cinerea in germplasm resources, chemical control is mainly used in production, supplemented by biological and ecological control. At present, chemical control is an important means to control Botrytis cinerea, but Botrytis cinerea has the characteristics of rapid reproduction, large genetic variation and high fitness, and continuous use of the same agent is easy to produce drug resistance. In terms of production, chemical fungicides commonly used in the prevention and treatment of Botrytis cinerea, such as carbendazim of the benzimidazole class, procymidone of the dicarboximide class, and pyrimethanil of the anilinopyrimidine class, are all due to the resistance of Botrytis cinerea. The control effect is greatly reduced. If the amount of pesticides is increased arbitrarily in order to improve the control effect, the result is that not only the occurrence of Botrytis cinerea is not effectively controlled, but the risk of pesticide residues in fruits and vegetables is increased, and the pollution of pesticides to the environment is increased.

With the increase of people's attention to food safety, biological control has increasingly attracted people's attention due to its characteristics of human and animal safety, good environmental compatibility, and less resistance to bacteria. Especially Bacillus subtilis, because it can produce a variety of antibacterial substances and has obvious control effect, many excellent Bacillus subtilis strains have been used in production practice at home and abroad. Biopesticides are of great significance to reduce chemical pesticide pollution in farmland and ensure the quality and safety of agricultural products. However, biological control also has some shortcomings, such as unstable field control effect and slow field efficacy, which seriously affect its popularization and use.

In order to make up for the deficiencies of biological control and chemical control, carry out technical research on the synergistic control of tomato gray mold by biocontrol microorganisms and low-toxicity chemical agents. The purpose of plant disease resistance and long-lasting control of the prevalence of Botrytis cinerea.

Boscalid is a new type of nicotinamide fungicide developed by BASF in Germany. It is mainly used to prevent gray mold, powdery mildew, various rots, brown rots and root rots. Boscalid is a succinate coenzyme Q reductase inhibitor in the mitochondrial respiratory chain, which has a strong inhibitory ability to spore germination and has no cross-resistance with other fungicides. In addition, the toxicity of boscalid is also very low, and the acute oral LD ₅₀ for male and female rats and mice is greater than 5000 mg/kg. The acute percutaneous LD ₅₀ of both male and female rats was >2000mg/kg. Acute inhalation LD ₅₀ of male and female mice >6.7mg/kg. No irritation to rabbit eyes and skin test. No sensitization in guinea pig test. It has no effect on bees, birds, earthworms, silkworms, etc. Bees and birds can be fed after the spray liquid is dry. At the same time, it has no effect on various natural enemies.

In view of this, the present invention is proposed.

Contents of the invention

The object of the present invention is to provide a method for treating tomato gray mold and leaf mold in view of problems such as large-scale use of chemical pesticides in current production to prevent and control the resistance of pathogenic bacteria caused by gray mold and leaf mold, seriously pollute the environment, cause pesticide residues to exceed the standard, and affect tomato quality. A compound fungicide containing bacillus subtilis BAB-1 and boscalid which has good control effect on mildew and leaf mold and its application. After compounding Bacillus subtilis BAB-1 and boscalid, the compound fungicide of the present invention forms a complementary effect, which can not only exert the high-efficiency and rapid bacteriostatic effect of boscalid, but also be effective against botrytis cinerea. and leaf mold, and can play the role of Bacillus subtilis BAB-1 in inducing tomato resistance and protecting against gray mold and leaf mold. Through the use of this compound fungicide, a large number of beneficial microorganisms are introduced into the tomato planting environment, and the living environment of pathogenic bacteria is deteriorated. Combined with agricultural measures such as cultivation management, a stable, balanced, and biologically diverse ecosystem is formed to achieve improved efficacy. , Reduce the use of chemical pesticides, and control the prevalence of tomato gray mold and leaf mold for a long time.

The Bacillus subtilis BAB-1 of the present invention is obtained through screening by the Institute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences. The Bacillus subtilis BAB-1 was preserved in the General Microorganism Center of the China Microbiological Culture Collection Management Committee on July 6, 2007, and the preservation number is CGMCCNo.2099; the Latin scientific name is: Bacillus subtilis BAB-1 (hereinafter referred to as BAB-1 ). The strain has the characteristics of high efficiency, wide antibacterial spectrum, simple use, low cost, no environmental pollution, etc., and can be used for the control of tomato gray mold and leaf mold. Bacillus subtilis BAB-1 is naturally resistant to the chemical agent boscalid, and this strain can grow on LB medium with a content of 1000 μg/mL of boscalid. Therefore, Bacillus subtilis BAB-1 and boscalid can be compounded into a fungicide to achieve long-term coexistence of the two.

The technical scheme for realizing the present invention is as follows: a compound fungicide containing Bacillus subtilis BAB-1 and boscalid, the active ingredient of which is composed of Bacillus subtilis BAB-1 and boscalid.

Preferably, the Bacillus subtilis BAB-1 is its bacterial body and/or its metabolites.

Preferably, the Bacillus subtilis BAB-1 is its fermentation broth or its freeze-dried bacterial powder; more preferably, the total number of viable bacteria in the fermentation broth is 7×10 ⁹ to 1×10 ¹⁰ CFU/mL; further preferably, the The total number of viable bacteria in the freeze-dried bacterial powder was 1.8×10 ¹⁰ -2.4×10 ¹⁰ CFU/g. Preferably, the weight ratio of boscalid to Bacillus subtilis BAB-1 in the active ingredient is 1-99:99-1; more preferably the weight ratio is 1-9:9-1; the preferred weight ratio is 1 ~4:4~1.

Specifically, a compound fungicide containing Bacillus subtilis BAB-1 and boscalid, its raw materials contain the following components in parts by weight: 10-80 parts of the above-mentioned active ingredients, 1-8 parts of dispersants, 1-8 parts of wetting agent, 10-80 parts of filler; preferably the raw materials contain the following components: 40-70 parts of active ingredient, 3-7 parts of dispersant, 3-7 parts of wetting agent, 20-50 parts of filler; More preferably, the raw material contains the following components: 50-60 parts of active ingredient, 4-6 parts of dispersant, 4-6 parts of wetting agent, and 30-40 parts of filler.

Preferably, the boscalid is the original drug of boscalid with a mass concentration of 98%.

Preferably, the dispersant is sodium lignosulfonate, sodium carboxymethyl cellulose, desugared sodium lignosulfonate, sulfurous acid pulp waste liquor, alkylphenol polyoxyethylene ether, ethylene glycol monobutyl One or more of base ether, polyoxyethyl alkyl ether, nonylphenol polyoxyethylene ether, sodium dodecylbenzenesulfonate.

Preferably, the wetting agent is sodium dibutylnaphthalene sulfonate (pull open powder BX), wetting agent wgwinD30, fatty alcohol polyoxyethylene ether, sodium dodecylsulfonate, dodecylbenzenesulfonate A mixture of one or more of acid sodium salt, alkylphenol polyoxyethylene formaldehyde condensate sulfonate.

Preferably, the filler is a mixture of one or more of white carbon black, kaolin, light calcium, diatomaceous earth, light calcium carbonate, and bentonite.

Specifically, a compound fungicide containing Bacillus subtilis BAB-1 and boscalid contains the following components in parts by weight: Boscalid original drug with a mass concentration of 98%: 20.41 parts; Bacillus subtilis Bacillus BAB-1 freeze-dried bacteria powder (the total number of viable bacteria is 2.0×10 ¹⁰ CFU/g): 40 parts; dispersant carboxymethylcellulose sodium salt: 3 parts; wetting agent: sodium dodecylsulfonate: 4.5 parts; filler light calcium carbonate: 32.09 parts. With this formula, 20% boscalid·8 billion Bacillus subtilis BAB-1 wettable powder can be prepared as follows.

The parts by weight in the present invention can be μg, mg, g, kg and other well-known weight units in the art, and can also be multiples thereof, such as 1/10, 1/100, 10 times, 100 times, etc.

The compound fungicide containing Bacillus subtilis BAB-1 and boscalid according to the present invention can be prepared into a liquid preparation or a wettable powder according to the prior art.

The present invention also provides the preparation method of the compound fungicide containing Bacillus subtilis BAB-1 and boscalid, which includes: mixing and stirring the various components according to the above ratio, and then performing primary airflow milling (particle fineness reaches 80 mesh); after mixing and stirring, carry out secondary jet milling (particle fineness reaches 325 mesh), that is.

The Bacillus subtilis BAB-1 freeze-dried bacterial powder of the present invention can be prepared from the Bacillus subtilis BAB-1 fermentation liquid according to the conventional method in the prior art. The Bacillus subtilis BAB-1 fermentation liquid can be prepared by the method described in the prior art such as Chinese patent CN200710121275.0; or by the following method.

A preparation method for Bacillus subtilis BAB-1 fermentation broth, comprising the following steps: first activate the BAB-1 bacterial classification in an LB plate, pick a single bacterium colony and streak it into a Klei flask, and after 3 days, use sterile water to Wash out the bacterial lawn and inoculate it into a seed tank (such as a 1-ton tank) as a seed liquid. After culturing at 31°C for 12 hours, inoculate the seed liquid into a fermenter (such as a 5-ton tank) for fermentation, and place it in the tank for 48 hours to obtain Bacillus subtilis BAB- 1 fermentation broth.

After putting the Bacillus subtilis BAB-1 fermentation broth into the tank, centrifuge and freeze-dry to obtain the Bacillus subtilis BAB-1 freeze-dried bacteria powder.

The present invention also includes the application of the compound fungicide containing Bacillus subtilis BAB-1 and boscalid in the prevention and treatment of botrytis cinerea and leaf mold; the plants include but are not limited to fruits, vegetables and flowers, More than 200 kinds of plants such as tomato, strawberry, cucumber, zucchini, grape, lettuce, lettuce, cabbage, eggplant, pepper, beans, gourd, chrysanthemum, apple, etc.; preferably tomato. The compound fungicide containing Bacillus subtilis BAB-1 and boscalid of the present invention has remarkable effect on the control of tomato gray mold and leaf mold, and the control effects can reach 88.6% and 87.5% respectively.

The method for using the compound fungicide containing Bacillus subtilis BAB-1 and boscalid according to the present invention comprises: diluting the compound fungicide containing Bacillus subtilis BAB-1 and boscalid The stems and leaves of the above-mentioned plants (such as tomato) are sprayed to prevent gray mold and leaf mold; it is preferable to prevent and control during the growth period of the plant (such as tomato).

Generally, when the compound fungicide containing Bacillus subtilis BAB-1 and boscalid is used, its active ingredient content is at least 300 μg/mL; preferably 500 μg/mL˜600 μg/mL.

Generally, the compound fungicide containing Bacillus subtilis BAB-1 and boscalid is diluted with water before use.

The advantage of the present invention is that by using Bacillus subtilis BAB-1 which is safe for humans and animals and has a strong inhibitory effect on Botrytis cinerea and leaf mold, it is compounded with the low-toxic chemical agent boscalid in a certain proportion, Complementary advantages are achieved, the amount of chemical fungicides is reduced, and while the control effect is improved, the ecological environment is improved and the residual amount of chemical agents in vegetables is reduced.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Unless otherwise specified, the raw material boscalid in the compound fungicide containing Bacillus subtilis BAB-1 and boscalid described in the following examples is the original drug with a mass concentration of 98% boscalid, manufactured by Jiangyin Suli Provided by Chemical Co., Ltd.; raw material Bacillus subtilis BAB-1 is Bacillus subtilis (Bacillus subtilis) BAB-1 freeze-dried bacteria powder (the total number of live bacteria is 2.0×10 ¹⁰ CFU/g), provided by the Institute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences supply.

Example 1

This embodiment provides a compound fungicide containing Bacillus subtilis BAB-1 and boscalid, which contains the following components: active ingredient 60kg; dispersant: alkylphenol polyoxyethylene ether 5Kg, ethylene glycol monobutyl Base ether 1Kg; wetting agent: dodecylbenzenesulfonic acid sodium salt 4Kg, fatty alcohol polyoxyethylene ether 3Kg; filler: white carbon black 5Kg, kaolin 25Kg; the active ingredient is composed of 98% boscalid medicine and Bacillus subtilis BAB-1 freeze-dried bacteria powder (the total number of viable bacteria is 2.0×10 ¹⁰ CFU/g), and the weight ratio of the two is 4:1.

Example 2

This example provides a compound fungicide containing Bacillus subtilis BAB-1 and boscalid, which differs from Example 1 only in that the weight ratio of the active ingredient boscalid to Bacillus subtilis BAB-1 is 2 : 1.

Example 3

This example provides a compound fungicide containing Bacillus subtilis BAB-1 and boscalid, which differs from Example 1 only in that the weight ratio of the active ingredient boscalid to Bacillus subtilis BAB-1 is 1 : 1.

Example 4

This example provides a compound fungicide containing Bacillus subtilis BAB-1 and boscalid, which differs from Example 1 only in that the weight ratio of the active ingredient boscalid to Bacillus subtilis BAB-1 is 1 : 2.

Example 5

This example provides a compound fungicide containing Bacillus subtilis BAB-1 and boscalid, which differs from Example 1 only in that the weight ratio of the active ingredient boscalid to Bacillus subtilis BAB-1 is 1 : 4.

Example 6

This example provides a compound fungicide containing Bacillus subtilis BAB-1 and boscalid, which differs from Example 1 only in that the weight ratio of the active ingredient boscalid to Bacillus subtilis BAB-1 is 1 : 99.

Example 7

This example provides a compound fungicide containing Bacillus subtilis BAB-1 and boscalid, which differs from Example 1 only in that the weight ratio of the active ingredient boscalid to Bacillus subtilis BAB-1 is 99 : 1.

Example 8

This embodiment provides a compound fungicide containing Bacillus subtilis BAB-1 and boscalid, which contains the following components in terms of 100kg: Boscalid with a mass concentration of 98%: 20.41kg; Bacillus subtilis Bacillus BAB-1 freeze-dried bacteria powder (the total number of viable bacteria is 2.0×10 ¹⁰ CFU/g): 40kg; dispersing agent carboxymethylcellulose sodium salt: 3kg; wetting agent sodium dodecylsulfonate: 4.5kg; Filler light calcium carbonate: 32.09kg.

Example 9

This example provides a preparation method for a compound fungicide formulated as described in Example 1, which specifically includes: mixing and stirring various components according to the proportion, performing primary jet milling, and the particle fineness reaches 80 mesh; mixing and stirring Afterwards, it is crushed in two airflow stages by a jet mill, and the particle fineness reaches 325 meshes, and it is obtained.

The preparation method of the compound bactericide described in embodiment 2-8 is the same as embodiment 9.

Experimental example 1

Toxicity test of Bacillus subtilis BAB-1 and boscalid against Botrytis cinerea

1 Experimental materials

The pathogen strain for testing is Botrytis cinerea, provided by the Institute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences, using PDA medium (potatoes 200g, glucose 20g, agar 10g, distilled water 1000mL) at 25 °C ± 1 °C. Cultivate for 3d and reserve for later use. The original drug of boscalid with a mass concentration of 98% was provided by Jiangyin Suli Chemical Co., Ltd. Bacillus subtilis (Bacillus subtilis) BAB-1 fermentation broth (the total number of viable bacteria is 8 billion CFU/mL) was provided by the Institute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences. The compound fungicide containing Bacillus subtilis BAB-1 and boscalid prepared in Example 1-5, the weight ratio of its active ingredient boscalid and Bacillus subtilis BAB-1 is 4:1, 2 respectively :1, 1:1, 1:2, 1:4.

2 Experimental methods

2.1 Dissolve the original drug of boscalid with a mass concentration of 98% in an appropriate amount of acetone to prepare a mother solution with a concentration of 10,000 μg/mL, then serially dilute it with sterile water, and mix it with PDA medium at a volume ratio of 1:9, respectively. Prepare drug-containing plates with concentrations of 10, 5, 1, 0.5, 0.1, and 0.05 μg/mL.

2.2 Dilute the Bacillus subtilis BAB-1 fermentation broth with sterile water, mix it with the PDA medium at a volume ratio of 1:9, and prepare Bacillus subtilis concentrations of 10, 5, 1, 0.5, 0.1 and 0.05 μg respectively /mL drug-containing plate.

2.3 The compound fungicide containing Bacillus subtilis BAB-1 and boscalid prepared in Examples 1-5 was diluted with sterile water, and mixed with PDA medium respectively to make concentrations of 10, 5, 1, 0.5, 0.1, 0.05 μg/mL drug-containing plates. The concentration here refers to the concentration of the active ingredients (ie Bacillus subtilis BAB-1 and boscalid) in the compound fungicide.

2.4 On the PDA plate of Botrytis cinerea that has been pre-cultivated for 3 days, use a puncher to punch a 5mm-diameter bacteria cake, and inoculate the bacteria cake face down to the center of the drug-containing plate prepared in the above steps 2.1-2.3. ; and use the PDA plate without the drug as the blank control, and operate in the same way; after constant temperature cultivation at 25°C±1°C for 3 days, the diameter of the colony is measured by the cross method.

3 result calculation

Use EXCEL to calculate the inhibition rate of each treatment, according to the concentration and inhibition rate of each treatment, utilize _DPSv6 . % treatment concentration in μg/mL). Then write out the toxicity regression equation: y=bx+a, where y represents the probability value of the treatment inhibition rate; x represents the logarithmic value of the concentration of the treatment.

Inhibition rate (%) = (net growth of control colonies - diameter of net growth of treated colonies) / net growth of control colonies × 100%

Calculate the synergistic coefficients of different compound drugs with reference to Wadley (1967) formula:

SR= _EC50 (th)/ _EC50 (ob)

In the above formula, a and b respectively represent the percentages of two fungicides of boscalid (A) and Bacillus subtilis (B) in the mixture; EC ₅₀ (A) and EC ₅₀ (B) represent the percentage of A and B respectively. The actual observed EC ₅₀ value, EC ₅₀ (th) represents the theoretical EC ₅₀ value after the two fungicides A and B are mixed according to a:b, and EC ₅₀ (ob) is the two fungicides A and B mixed according to a:b The actual observed _EC50 value. SR>1.5 indicates synergy; SR<0.5 indicates antagonism; SR between 0.5 and 1.5 indicates additive effect.

4 Experimental results

See Table 1. The results show that the compound fungicide containing Bacillus subtilis BAB-1 and boscalid described in Examples 1-5, boscalid, Bacillus subtilis BAB-1 and boscalid have good inhibitory effect on tomato Botrytis cinerea respectively and the synergistic coefficient of the compound bactericide described in embodiment 1-5 is all greater than 1.5 to tomato botrytis cinerea, shows that the compound sterilization that contains Bacillus subtilis BAB-1 and boscalid described in embodiment 1-5 Boscalid in the agent has a synergistic effect with Bacillus subtilis BAB-1.

Table 1 Combined toxicity of different ratios of compound fungicides containing Bacillus subtilis BAB-1 and boscalid against Botrytis cinerea

potion Virulence regression equation _EC50 relevant synergy

(μg/mL) coefficient coefficient Boscalid (A) y=0.4591x+4.9027 1.6291 0.9975 - Bacillus subtilis BAB-1(B) y=1.3081x+6.9959 0.0298 0.839 - Example 1 (A:B=4:1) y=0.6651x+5.6944 0.0903 0.9552 1.54 Example 2 (A:B=2:1) y=0.6430x+5.8558 0.0467 0.9384 1.85 Example 3 (A:B=1:1) y=1.3254x+6.9616 0.0331 0.8406 1.77 Embodiment 4 (A:B=1:2) y=1.5315x+7.5243 0.0225 0.845 1.97 Example 5 (A:B=1:4) y=1.3223x+7.1449 0.0239 0.86 1.55

"A:B=4:1" in Table 1 represents the compound fungicide containing Bacillus subtilis BAB-1 and boscalid described in Example 1, wherein the weight ratio of boscalid to Bacillus subtilis BAB-1 It is 4:1. Others are similar.

Experimental example 2

Determination of synergistic effect of compound fungicides containing Bacillus subtilis BAB-1 and boscalid against tomato leaf mold

1 Experimental materials

Experimental agent: a compound fungicide containing Bacillus subtilis BAB-1 and boscalid prepared according to Example 8.

Control agent 1: Boscalid water-dispersible granules with a mass concentration of 50% (provided by BASF Europe, batch number 140820000).

Control drug 2: Bacillus subtilis BAB-1 fermentation liquid (provided by the Institute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences, the total number of viable bacteria is 8 billion CFU/mL).

The pathogen strain for testing is tomato leaf mold fungus (Fulviafulva (Cooke) Cif.), provided by the Institute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences, using PDA medium (potatoes 200g, glucose 20g, agar 10g, distilled water 1000mL) at 24 ° C ± Pre-cultivate at 1°C for 20 days, and set aside after sporulation.

2 Experimental methods

2.1 Dissolve the control drug 1 in an appropriate amount of acetone to make a mother solution with a concentration of 10000 μg/mL, then serially dilute it with sterile water, mix it with PDA medium at a volume ratio of 1:9, and make a concentration of 10, 5 , 1, 0.5, 0.1, 0.05 μg/mL drug-containing plates.

2.2 Dilute the Bacillus subtilis BAB-1 fermentation broth with sterile water, mix it with the PDA medium at a volume ratio of 1:9, and prepare Bacillus subtilis concentrations of 10, 5, 1, 0.5, 0.1, and 0.05 μg respectively /mL drug-containing plate.

2.3 Dilute the compound fungicide containing Bacillus subtilis BAB-1 and boscalid prepared according to Example 8 with sterile water, and mix it with PDA medium in a volume ratio of 1:9 to make a concentration of 10 , 5, 1, 0.5, 0.1, 0.05 μg/mL drug-containing plates. The concentration here refers to the concentration of the active ingredients (ie Bacillus subtilis BAB-1 and boscalid) in the compound fungicide.

2.4 Add 10 mL of 0.5% Tween water to the PDA plate of tomato leaf mold that has been pre-cultured for 20 days, scrape off the leaf mold spores with a stick, and adjust the spore suspension to ¹ ×105 by using a microscope /mL, use a pipette to inject 0.1mL of spore suspension into the drug-containing plate prepared in the above steps 2.1-2.3, and then spread it evenly with a stick. And use the PDA plate without the drug as the blank control, and operate in the same way; after 20 hours of constant temperature cultivation at 24°C±1°C, investigate the total number of spores and the number of germination under a low-power microscope, and investigate about 100 spores per treatment .

3 result calculation

Calculate the spore germination rate and inhibition rate according to the formula, and calculate the virulence regression equation, correlation coefficient, _EC50 (the treatment concentration when effectively inhibiting the growth of pathogenic bacteria by 50%, in μg/mL) and the synergistic coefficient by the method of Experimental Example 1.

Germination rate (%) = germination number/total survey number × 100%

Inhibition rate (%)=(control germination rate-treatment germination rate)/control germination rate×100%

4 Experimental results

See Table 2. The results show that the compound fungicide containing Bacillus subtilis BAB-1 and boscalid described in Example 8, boscalid, Bacillus subtilis BAB-1 and embodiment 8 have good inhibitory effect on tomato leaf mold respectively; And the synergistic coefficient of the compound fungicide described in embodiment 8 to tomato leaf mold is 1.90, shows that in the compound fungicide containing Bacillus subtilis BAB-1 and boscalid described in embodiment 8, boscalid It has synergistic effect with Bacillus subtilis BAB-1.

Table 2 Combined toxicity of compound fungicides containing Bacillus subtilis BAB-1 and boscalid against tomato leaf mold

"A:B=1:2" in Table 2 represents the compound fungicide containing Bacillus subtilis BAB-1 and boscalid described in Example 8, wherein the weight ratio of boscalid to Bacillus subtilis BAB-1 It is 1:2.

Experimental example 3

Field control effect of compound fungicide containing Bacillus subtilis BAB-1 and boscalid against Botrytis cinerea

The experimental location is Baitapu Village, Gaolin Village Town, Xushui County, Baoding City, Hebei Province. The previous stubble in the experimental field was cucumbers, and tomatoes had been planted for more than 15 years, and botrytis cinerea occurred over the years. The tomato variety to be tested was Dongsheng, and the outside of the test greenhouse was covered with thatch to keep warm, and rolled up during the day. When the temperature in the shed is too low, use a fire to heat up. It was planted on January 18, 2014, and 3,000 tomato seedlings were planted per mu.

1 Experimental materials

Experimental agent: a compound fungicide containing Bacillus subtilis BAB-1 and boscalid prepared according to the method of Example 8. Contrast agent 1: 50% mass concentration boscalid water-dispersible granules (provided by BASF Europe, batch number 140820000. Contrast agent 2: Bacillus subtilis BAB-1 dry powder (provided by Institute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences, total number of viable bacteria For 20 billion CFU/g).Contrast agent 3: concentration is 400g/L pyrimethanil suspension concentrate (provided by Bayer Crop Science Company of Germany, batch number 20131127).Contrast agent 4: mass concentration 10% difenoconazole water dispersible granule (provided by Syngenta (Suzhou) Crop Protection Co., Ltd., batch number SJ14BWG004). Blank control: water.

2 Experimental methods

There are 7 treatments in the experiment: after diluting the experimental drug with water, spray it according to the dosage of 750g/hm ² , 1125g/hm ² and 1500g/hm ² respectively; control drug 1 (50% boscalid water-dispersible granule ) was diluted with water, and sprayed at a dosage of 720g/hm ² ; after the contrast agent 2 (20 billion CFU/mL dry powder of Bacillus subtilis BAB-1) was diluted with water, it was sprayed at 3600g/hm ² ; the contrast agent 3 ( Concentration 400g/L pyrimethanil suspension concentrate) after diluting with water, according to preparation dosage 1500mL/hm ² spray medicine; Amount of 600g/hm ² spray medicine; water as a blank control. The experimental plots were arranged in random blocks, with 4 repetitions for each treatment, and the plot area was 13m ² . The first drug was administered on March 1, the second and third drugs were administered on March 10 and March 17 respectively, and the drugs were shared 3 times during the test. The "Guardian" brand knapsack manual sprayer is used for spraying, the spray is even and thoughtful, and the water consumption is 900L/hm ² .

Botrytis gray mold occurred sporadically before spraying, and the base number was regarded as zero. The incidence was investigated 7 days after the last spraying. Calculate the disease index and control effect. Randomly select 5 points in each plot for investigation, investigate 2 plants at each point, investigate all fruits of each plant, and record the number of diseased fruits. The classification criteria for diseased fruits are:

Grade 0: no lesions;

Grade 1: Residual petal disease or stigma disease;

Grade 3: sepal rot or stigma disease spreading to the navel of the fruit;

Grade 5: There are infiltrated spots on the navel but no mold layer;

Grade 7: There is a mold layer on the navel but it has not spread to other parts;

Grade 9: The mold layer on the fruit navel extends to other parts.

Diseased fruit rate (%) = (number of diseased fruits/total number of investigated fruits) × 100

Disease index = ∑[(number of diseased fruits×relative degree of disease)/(total number of investigated fruits×highest level)]×100

Control effect (%)=[(control condition index-treatment condition index)/control condition index]×100

3 The experimental results are shown in Table 3.

Experimental example 4

Field control effect of compound fungicide containing Bacillus subtilis BAB-1 and boscalid against tomato leaf mold

The experimental location is Baitapu Village, Gaolin Village Town, Xushui County, Baoding City, Hebei Province. The previous stubble in the experimental field was cucumber, and tomato had been planted for more than 15 years, and leaf mold had occurred over the years. The tomato variety tested was Caesar. The outside of the test greenhouse was covered with grass thatch for heat preservation and rolled up during the day. When the temperature in the shed is too low, use a fire to heat up. It was planted on January 18, 2014, and 3,000 tomato seedlings were planted per mu.

1 Experimental materials

Same as experimental example 3.

2 Experimental methods

Experimental medicament, control medicament and dosage are the same as Experimental Example 3.

The first drug was administered on March 4, the second and third drugs were administered on March 14 and March 21 respectively, and the drug was shared 3 times during the test. The "Guardian" brand knapsack manual sprayer is used for spraying, the spray is even and thoughtful, and the water consumption is 900L/hm ² .

The base number of sporadic occurrence of leaf mold before spraying was regarded as zero, and the incidence was investigated 7 days after the last spraying. Calculate the disease index and control effect. Randomly select 3 points in each plot for investigation, and investigate 5 plants at each point. Each plant is divided into upper, middle and lower parts to take a representative compound leaf, and the area of disease spots on each small leaf on the compound leaf accounts for the entire leaf area. Percentage grading, the grading method is:

Grade 0: no lesions;

Grade 1: Lesion area accounts for less than 5% of the entire leaf area;

Grade 3: The lesion area accounts for 5% to 10% of the entire leaf area;

Grade 5: Lesion area accounts for 11% to 20% of the entire leaf area;

Grade 7: The lesion area accounts for 21% to 50% of the entire leaf area;

Grade 9: The lesion area accounts for more than 51% of the entire leaf area.

Disease index = ∑[(number of diseased fruits×relative degree of disease)/(total number of investigated fruits×highest level)]×100

Control effect (%)=[(control condition index-treatment condition index)/control condition index]×100

3 The experimental results are shown in Table 3.

As shown in table 3, field experiment result shows: the control effect of control agent 1 (single agent 50% boscalid water-dispersible granule, mu dosage 48g, diluted 1250 times) to tomato botrytis cinerea and leaf mold is respectively 83.6% and 82.3%. Contrast medicament 2 (20,000,000,000 bacillus subtilis dry powders of single dose, mu dosage 240g, diluted 250 times) is respectively 76.7% and 77.8% to the control effect of tomato botrytis cinerea and leaf mold; experimental medicine (embodiment 8 contains The compound fungicide of Bacillus subtilis BAB-1 and boscalid, 100g per mu, diluted 600 times) has the best control effect on tomato gray mold and leaf mold, which are 88.6% and 87.5% respectively; 3 (400g/L pyrimethanil suspension concentrate, 100mL per mu, diluted 600 times) has a control effect of 62.3% on tomato gray mold.

Experimental medicine (embodiment 8 contains the compound bactericide of Bacillus subtilis BAB-1 and boscalid) when the dosage per mu is 100 grams (diluted 600 times), wherein the consumption of chemical bactericide boscalid is about its unit The dosage of Bacillus subtilis BAB-1 in the described compound fungicide is about 16.7% of its single agent Bacillus subtilis BAB-1, but the control effect on Botrytis cinerea is lower than that of single agent Boscalid is equivalent, but the control effect on leaf mold is better than its single agent boscalid.

When the experimental medicine (the compound fungicide containing Bacillus subtilis BAB-1 and boscalid) in the mu dosage of 50 grams (diluted 1200 times) in Example 8, the consumption of the chemical fungicide boscalid was about its unit 41.7% of the agent boscalid; the consumption of Bacillus subtilis BAB-1 in the compound fungicide is about 8.3% of its single agent Bacillus subtilis BAB-1, and the compound fungicide is effective against Botrytis cinerea of tomato and leaf mold were 78.7% and 78.0%, respectively, which were equivalent to the control effects of two single doses of control agent 1 and control agent 1 on gray mold and leaf mold; the control effect on gray mold was significantly higher than that of Conventional medicament 400g/L pyrimethanil suspension concentrate (100mL per mu, 600 times diluted); the control effect on leaf mold is the same as that of conventional medicament 10% difenoconazole water dispersible granules (40 grams per mu, 1500 times diluted ) has no significant difference.

Table 3 Field control effects on tomato gray mold and leaf mold

The above embodiments are not specific limitations to the present invention, as long as the compound bacterial agent is used in the disease prevention and control of tomato and other crops according to the scope defined in the claims, under the inspiration of this patent and in combination with the basic common sense in this field, All belong to the protection scope of the present invention.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (10)

1. A compound fungicide containing Bacillus subtilis BAB-1 and boscalid, the active ingredient of which is composed of boscalid and Bacillus subtilis BAB-1. 2. The compound fungicide according to claim 1, characterized in that, the Bacillus subtilis BAB-1 is its bacterium and/or its metabolites. 3. The compound fungicide according to claim 1, characterized in that, said Bacillus subtilis BAB-1 is its fermented liquid or its freeze-dried bacteria powder; preferably the total number of viable bacteria in said fermented liquid is 7×10 ⁹ to 1×10 ¹⁰ CFU/mL; preferably, the total number of viable bacteria in the freeze-dried bacterial powder is 1.8×10 ¹⁰ to 2.4×10 ¹⁰ CFU/g. 4. The compound fungicide according to claim 3, characterized in that, the weight ratio of boscalid and Bacillus subtilis BAB-1 in the active ingredient is 1～99:99～1; further preferred weight ratio It is 1～9:9～1; the preferred weight ratio is 1～4:4～1. 5. The compound fungicide according to any one of claims 1-4, characterized in that, in parts by weight, its raw materials contain the following components: 10 to 80 parts of active ingredients, 1 to 8 parts of dispersants, and 1-8 parts of wetting agent, 10-80 parts of filler; preferably the raw materials contain the following components: 40-70 parts of active ingredient, 3-7 parts of dispersant, 3-7 parts of wetting agent, 20-50 parts of filler; more Preferably, the raw materials contain the following components: 50-60 parts of active ingredients, 4-6 parts of dispersants, 4-6 parts of wetting agents, and 30-40 parts of fillers. 6. compound fungicide according to claim 5, is characterized in that, described dispersant is sodium lignosulfonate, sodium carboxymethyl cellulose, desugared sodium lignosulfonate, sulfurous acid pulp waste One or more of liquid, alkylphenol polyoxyethylene ether, ethylene glycol monobutyl ether, polyoxyethyl alkyl ether, nonylphenol polyoxyethylene ether, sodium dodecylbenzenesulfonate Mixture; the wetting agent is sodium dibutylnaphthalene sulfonate, wetting agent wgwinD30, fatty alcohol polyoxyethylene ether, sodium dodecylsulfonate, sodium dodecylbenzenesulfonate, alkylphenol A mixture of one or more of polyoxyethylene formaldehyde condensate sulfonate; the filler is one or more of white carbon black, kaolin, light calcium, diatomaceous earth, light calcium carbonate, bentonite mixture. 7. The compound fungicide according to claim 1, characterized in that, in parts by weight, it contains the following components: Boscalid original drug with a mass concentration of 98%: 20.41 parts; the total number of viable bacteria is 2.0×10 ¹⁰ CFU/g Bacillus subtilis BAB-1 freeze-dried bacterial powder: 40 parts; Dispersant sodium carboxymethyl cellulose: 3 parts; Wetting agent: Sodium dodecylsulfonate: 4.5 parts; Light filler Calcium carbonate: 32.09 parts. 8. The preparation method of the compound bactericide described in any one of claims 1-7, is characterized in that, comprises the following steps: mix and stir various components according to the proportioning, carry out primary pulverization, particle fineness reaches 80 mesh; after mixing and stirring, carry out secondary crushing through a jet mill, and the particle fineness reaches 325 mesh. 9. The application of the compound fungicide described in any one of claims 1-7 in the control of botrytis cinerea and leaf mold. 10. The application according to claim 9, wherein the plants include but not limited to fruits, vegetables and flowers; preferably tomato.