阅读说明：本技术 一种防治辣椒疫霉的复合生防制剂及其防治方法 (A kind of compound biological prevention and control agent and its control method for preventing and treating Phytophthora capsici ) 是由 袁志辉 张斌 刘小文 何福林 张祖姣 于 2019-07-19 设计创作，主要内容包括：本发明属于植物病害的生物防治领域,具体涉及一种防治辣椒疫霉的复合生防制剂及其制备和使用方法。本发明制备的复合生防制剂包括如下成分：银杏内生解淀粉酶芽孢杆菌、银杏外种皮提取物、银杏内生解淀粉酶芽孢杆菌代谢物及蒸馏水。在具有广谱抗菌活性的银杏内生真菌淀粉芽孢杆菌菌液1.5份、银杏外种皮提取物1份、银杏内生解淀粉酶芽孢杆菌代谢物0.5份及蒸馏水7份的重量份的情况下,复合生防制剂对辣椒疫霉防治率最高。本发明制备的复合生防制剂速效性好；将三种作用机制完全不同的生防制剂成分进行复配,可增加了靶位点,使辣椒疫霉菌不易产生抗药性；成本较低且安全环保。(The invention belongs to the field of biological control of plant disease, and in particular to a kind of compound biological prevention and control agent and its preparation and application for preventing and treating Phytophthora capsici.Compound biological prevention and control agent prepared by the present invention includes following ingredient: gingko endogenous solution amylase bacillus, extraction fromginkgoseed coat, gingko endogenous solution amylase bacillus metabolin and distilled water.In the case where 1.5 parts of the gingko endogenous fungus bacillus amyloliquefaciens bacterium solution with broad spectrum antibiotic activity, 1 part of extraction fromginkgoseed coat, gingko endogenous 0.5 part of amylase bacillus metabolin of solution and 7 parts of distilled water of parts by weight, compound biological prevention and control agent is to Phytophthora capsici control rate highest.Compound biological prevention and control agent quick-acting prepared by the present invention is good；The entirely different biological prevention and control agent ingredient of three kinds of mechanism of action is compounded, target site can be increased, phytophthora blight of pepper is made to be not likely to produce drug resistance；Cost is relatively low and safety and environmental protection.)

1. a kind of compound biological prevention and control agent for preventing and treating Phytophthora capsici, which is characterized in that be made of the raw material of following parts by weight: in ginkgo Raw bacillus amyloliquefaciens 0.8-2.0 parts, 0.6-1.5 parts of extraction fromginkgoseed coat, gingko endogenous bacillus amyloliquefaciens generation Thank 0.3-1.0 parts of object and 5.0-8.0 parts of distilled water.

2. a kind of compound biological prevention and control agent for preventing and treating Phytophthora capsici according to claim 1, which is characterized in that by following weight The raw material composition of part: 1.5 parts of gingko endogenous bacillus amyloliquefaciens, 0.6 part of extraction fromginkgoseed coat, gingko endogenous solution starch 0.4 part of bacillus metabolin and 7 parts of distilled water.

3. a kind of compound biological prevention and control agent for preventing and treating Phytophthora capsici according to claim 1 or 2, which is characterized in that the silver The deposit number of raw solution amylase bacillus is CCTCC AB 2012028 in apricot, and the deposit number of the pseudomonas aeruginosa is CCTCC AB 205067。

4. a kind of compound biological prevention and control agent for preventing and treating Phytophthora capsici according to claim 1, which is characterized in that the compound life The preparation of anti-preparation the following steps are included:

S1- actication of culture: gingko endogenous bacillus amyloliquefaciens are cultivated in seed culture medium to strain density OD600 value it is 0.6-0.8 obtains bacterium solution；

S2- expands culture: in the gingko endogenous fungus bacillus amyloliquefaciens of 100 parts by weight inoculation of medium 2-5 parts by weight Bacterial strain is cultivated at 37 DEG C, until the viable count of gingko endogenous solution amylase bacillus is 2-20 × 10 in culture medium⁷A/gram；

S3- expands culture: the gingko endogenous solution amylase bacillus after inclined-plane culture is activated is connected to PDA solid plate 25- 35 DEG C culture 72-120 hours, then be forwarded to activation 48-72 hours of 25-35 DEG C of PDA liquid medium, 3-10% connects by volume Kind of amount, in liquid culture medium of transferring, pH to 6-9 at 25-35 DEG C, is cultivated 72 hours to 120 hours；It is pressed into container Proportion addition distilled water, seals a period of time at room temperature；

S4- matches bacteriostat: culture, aeration quantity 0.16-0.20m/min, when microorganism bacterium number is are aerated under the conditions of 25-30 DEG C 1×10⁸-1×10¹⁰When a/mL, the bacterium solution of strain is collected using storage tank；

S5- metabolin extracts: extracting gingko endogenous fungus solution starch gemma bar by solvent extraction, precipitating, absorption or chromatography Metabolin in bacterium bacterium solution is simultaneously freeze-dried it；

The preparation of S6- extraction fromginkgoseed coat: the gingko episperm that ethyl alcohol was impregnated is placed in boil to mention in pot and routinely add water to cook It 2-3 times and filters respectively, filtrate is merged and is concentrated under reduced pressure, concentrate is obtained, concentrate is transferred in container, container is placed in On the pedestal of magnetic stirring apparatus, ethyl alcohol is slowly added in concentrate, concentration of alcohol is made to reach 60-80 (V/V), ethyl alcohol is being added While start magnetic stirrer, sediment is washed with high concentration ethanol again after standing and is freeze-dried afterwards for several times；

The compound biological prevention and control agent preparation of S7-: at room temperature, extraction fromginkgoseed coat, silver are successively added in the bacterium solution according to the ratio Raw solution amylase bacillus metabolin, concussion are uniform in apricot.

5. a kind of compound biological prevention and control agent for preventing and treating Phytophthora capsici according to claim 1, which is characterized in that in step S1 In, the formula of the seed culture medium are as follows: beef extract 3g, peptone 10g, sodium chloride 5g, agar 15-20g, distilled water 1000mL, in 121 DEG C of sterilizing 30min, the pH value of the seed culture medium is 7.0-7.2.

6. a kind of compound biological prevention and control agent for preventing and treating Phytophthora capsici according to claim 1, which is characterized in that in step S2 In, the formula of the 100 parts by weight culture medium are as follows: glucose 30-35g/L, yeast extract 30g/L, manganese sulfate 0.05g/L, Calcium chloride 0.042g/L, ammonium sulfate 1.2g/L, magnesium sulfate 3g/L, phosphoric acid 0.4g/L, potassium phosphate 0.6g/L, 100 parts by weight The pH of culture medium is 7.0-7.2.

7. a kind of preparation method of compound biological prevention and control agent for preventing and treating Phytophthora capsici according to claim 1, which is characterized in that In step s 4, the storage tank is closed, saves under conditions of being placed in environment temperature lower than 20 DEG C.

8. a kind of control method for the compound biological prevention and control agent for preventing and treating Phytophthora capsici, which is characterized in that before Hot Pepper Seedling transplanting, will weigh Benefit requires the 1 compound biological prevention and control agent for impregnating Hot Pepper Seedling root, is then cultivated, the use of the compound biological prevention and control agent Amount is 1.0-1.5g/ plants of capsicums, soaking time 30min.

Technical field

The invention belongs to the field of biological control of plant disease, and in particular to a kind of compound biological and ecological methods to prevent plant disease, pests, and erosion system for preventing and treating Phytophthora capsici Agent and preparation method thereof and application method.

Background technique

Phytoph-thora capsici leonian is caused by Mastigomycotina fungi Phytophthora capsici.Germ is mainly with egg spore, thick Huan's spore in disease Overwintering wherein soil invalid body bacterial bearing rate is high in residuum or soil and seed, is main dip dyeing source.When condition is suitable for, after overwintering Germ is splashed through rainwater or irrigation water passes on basal part of stem or plant near the ground, causes to fall ill.Therefore, phytoph-thora capsici leonian becomes hair The destructive disease that disease cycle is short, epidemic rate is swift and violent.High temperature, high humidity, rainy days are more, rainfall is big, are conducive to disease.

Control method specifically includes that cultural control, chemical prevention and biological control at present.Cultural control mainly includes closing The reason rotation of crops and scientic planting.Rationally the rotation of crops is tillage control measure very important in agricultural production, reasonable cropping system and cloth Office, it is highly beneficial to the growth of capsicum.Chemical prevention includes soil medication and field medication；But chemical medication be easy to produce it is residual It stays and causes environmental pollution, and the generation of Antagonistic Fungi is so that the more tired chemopreventive effects the poorer.Biological control mainly uses antagonism Microorganism, environment friendly is strong, will not generate pollution.The single microbial inoculum of the prior art is more common, such as streptomycete, withered grass bud Spore bacillus etc., but single bacterial strain often has the defects of control efficiency is low, drug effect is slow, current multiple scientific research institutions into The research of the compound biological prevention and control agent of row.Develop it is a kind of using compound biological prevention and control agent prevention and treatment phytoph-thora capsici leonian method be that we need to solve Certainly the technical issues of.

Summary of the invention

In view of this, the object of the present invention is to provide a kind of microbial bacterial agent and its control method for preventing and treating Phytophthora capsici, To solve the problems such as chemical pesticide is existing at high cost, safety is poor, seriously polluted in terms of preventing and treating Phytophthora capsici.

To achieve the goals above, the present invention adopts the following technical scheme:

In a first aspect, the present invention provides a kind of compound biological prevention and control agent for preventing and treating Phytophthora capsici, by the raw material of following parts by weight Composition: gingko endogenous 0.8-2.0 parts of solution amylase bacillus, 0.6-1.5 parts of extraction fromginkgoseed coat, gingko endogenous Xie Dian 0.3-1.0 parts and 5.0-10.0 parts of distilled water of powder enzyme bacillus metabolin.

Further, it is made of the raw material of following parts by weight: the gingko endogenous fungus starch bud with broad spectrum antibiotic activity 1.5 parts of spore bacillus bacterium solution, 1 part of extraction fromginkgoseed coat, 0.5 part of gingko endogenous solution amylase bacillus metabolin and distillation 7 parts of water.

Further, the deposit number of solution amylase bacillus of the gingko endogenous fungus with broad spectrum antibiotic activity is CCTCC AB 2012028。

Second aspect, the present invention also provides a kind of preparation methods of above-mentioned compound biological prevention and control agent, including following preparation step:

S1- actication of culture: by gingko endogenous solution amylase bacillus (Bacillus amyloliquefaciens) In Culture to strain density OD600 value is 0.6-0.8 in seed culture medium, obtains bacterium solution；

S2- expands culture: in the gingko endogenous fungus starch gemma bar of 100 parts by weight inoculation of medium 2-5 parts by weight The bacterial strain of bacterium is cultivated at 37 DEG C, until the viable count of gingko endogenous solution amylase bacillus is 2-20 × 10 in culture medium⁷ A/gram；

S3- expands culture: the gingko endogenous solution amylase bacillus after inclined-plane culture is activated is connected to PDA solid plate 25-35 DEG C culture 72-120 hours, then be forwarded to activation 48-72 hours of 25-35 DEG C of PDA liquid medium, by volume 3- 10% inoculum concentration, in liquid culture medium of transferring, pH to 6-9 at 25-35 DEG C, is cultivated 72 hours to 120 hours；Xiang Rong Distilled water is added in device according to the ratio, at room temperature sealing a period of time；

S4- matches bacteriostat: culture, aeration quantity 0.16-0.20m/min, when each microorganism are aerated under the conditions of 25-30 DEG C Bacterium number is 1 × 10⁸-1×10¹⁰When a/mL, the bacterium solution of strain is collected using storage tank；

S5- metabolin extracts: extracting gingko endogenous fungus solution starch bud by solvent extraction, precipitating, absorption or chromatography Metabolin in spore bacillus bacterium solution is simultaneously freeze-dried it；

The preparation of S6- extraction fromginkgoseed coat: the gingko episperm impregnated by ethyl alcohol is placed in boil mention in pot routinely plus Water is decocted 2-3 times and is filtered respectively, and filtrate is merged and is concentrated under reduced pressure, concentrate is obtained, concentrate is transferred in container, will hold Device is placed on the pedestal of magnetic stirring apparatus, and ethyl alcohol is slowly added in concentrate, so that concentration of alcohol is reached 60-80 (V/V), is being added Magnetic stirrer is started while entering ethyl alcohol, is washed to freeze afterwards for several times with high concentration ethanol again by sediment after standing and be done It is dry；

The compound biological prevention and control agent preparation of S7-: at room temperature, gingko episperm is successively added in the bacterium solution according to the ratio and is extracted Object, gingko endogenous solution amylase bacillus metabolin, concussion are uniform；

Further, in step sl, the formula of the seed culture medium are as follows: beef extract 3g, peptone 10g, sodium chloride 5g, agar 15-20g, distilled water 1000mL, in 121 DEG C of sterilizing 30min, the pH value of the seed culture medium is 7.0-7.2.

Further, in step s 2, the formula of the 100 parts by weight culture medium are as follows: glucose 30-35g/L, yeast mention Take object 30g/L, manganese sulfate 0.05g/L, calcium chloride 0.042g/L, ammonium sulfate 1.2g/L, magnesium sulfate 3g/L, phosphoric acid 0.4g/L, phosphorus Sour potassium 0.6g/L, the pH of the 100 parts by weight culture medium are 7.0-7.2.

Further, in step s 4, the storage tank is closed, saves under conditions of being placed in environment temperature lower than 20 DEG C.

The third aspect, the present invention also provides the application method of above-mentioned compound biological prevention and control agent, the application methods are as follows: in capsicum Before seedling transplanting, the biological prevention and control agent is used to be soaked in Hot Pepper Seedling root, is then cultivated.

Further, the usage amount of the compound biological prevention and control agent is 1.0-1.5g/ plants of capsicums.

Compared with prior art, the invention has the following beneficial effects:

Preparation method provided by the invention, advantage are gingko endogenous solution amylase bacillus and its metabolin and ginkgo Both exosper extracts action target is different, and gingko endogenous solution amylase bacillus and its metabolin effect are to inhibit The expression of the normal growth and the enhancing anti-ospc gene of capsicum of germ mycelia, and extraction fromginkgoseed coat effect is to pass through phenolic acid Strong oxidizing property effect directly kill Phytophthora capsici.The two compounding can enhance the control efficiency to Phytophthora capsici, and can subtract Few Phytophthora capsici makes that capsicum is not likely to produce drug resistance and production cost is low to the resistance of preparation, is suitble to promote and the big rule of industrialization Mould production.

Application method provided by the invention, advantage are that only impregnating compound biological prevention and control agent in Hot Pepper Seedling root can save Cost, and the otherness generated with the effect for impregnating entire plant is little.

Biological agent provided by the invention, advantage are, without environmental pollution brought by chemical prevention and medicament residue The problems such as, be conducive to the No-harmful apple orchard of crop, peasant can not have to or reduce other prevention and treatment phytophthora root rots and improve resistance Measure, this can not only reduce the heavy burdens for peasant planting, but also be conducive to improve product quality and yield, increase agricultural incomes.

Specific embodiment

To make the object, technical solutions and advantages of the present invention clearer, below in conjunction with the embodiment of the present invention, to this hair Technical solution in bright embodiment is clearly and completely described, it is clear that described embodiment is that a part of the invention is implemented Example, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not making creativeness Every other embodiment obtained, shall fall within the protection scope of the present invention under the premise of labour.

Embodiments of the present invention will be further described below.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

In following embodiments, field experiment paddy field near Yongzhou City Dao County is carried out；It is Acid Paddy Soils for examination soil； It is phytophthora blight of pepper (Phytophthora capsici) It263 for examination pathogen, is stored in Agriculture in Hunan academy of sciences plant guarantor Protect research institute；Capsicum variety is Hunan green pepper No. 5, is purchased from Changsha Xin Wannong Seed company；Bacillus amyloliquefaciens used (Metarhizium anisopliae) bacterial strain CCTCCAB2012028 is purchased from China typical culture collection center.

The present invention is further elaborated by the following examples, but not as a limitation of the invention.