CN109439603B

CN109439603B - A kind of bacillus thuringiensis cultural method and its fluid nutrient medium

Info

Publication number: CN109439603B
Application number: CN201910025388.3A
Authority: CN
Inventors: 汪少斌
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-01-11
Filing date: 2019-01-11
Publication date: 2019-11-12
Anticipated expiration: 2039-01-11
Also published as: CN109439603A

Abstract

The present invention discloses the fluid nutrient medium and cultivation and fermentation method of a kind of bacillus thuringiensis, achieves significant virulence due to addition taurine in the fluid nutrient medium and is promoted；And taurine and salicylic combination are even more to obtain apparent synergistic effect, and bacillus thuringiensis fermentation liquid virulence is promoted to and reaches surprising 96.7% to 3 age diamondback moth lethalities.The fluid nutrient medium of bacillus thuringiensis of the invention is at low cost, cultural method is simple, and stability is preferable, convenient for long-distance transport and long-term preservation, has good popularization and application foreground.

Description

A kind of bacillus thuringiensis cultural method and its fluid nutrient medium

Technical field

The present invention relates to a kind of microbial culture method and its fluid nutrient mediums, and in particular to a kind of Dipel liquid Fermentation medium.

Background technique

Insect pest is to limit a stable, high-yielding important factor of crops, accounts for total output by the grain that pest is annexed every year 1/4 or so.For a long time, people solve pest problem by chemical pesticide；But not only the use of pesticide could not solve very well Pest problem but causes a series of serious hazard analysis and HACCPs, such as person poultry poisoning, environmental pollution simultaneously.Therefore, chemical insecticide Application by environmental protection organization and society immense pressure.Along with the awakening and constantly enhancing of people's environmental consciousness, biological agriculture Medicine gradually starts to be taken seriously, and receives more and more attention.Biological control is with pollution-free, insecticide efficiency is high, is not likely to produce The advantages that resistance, it is considered to be ideal means of prevention.Biological control is also increasingly by relevant department and scientific research institution Attention, wherein what is attracted people's attention is to utilize the research application in terms of entomopathogenic bacterium mosquito control.Among these, Su Yunjin Bacillus is universally acknowledged is most widely used and successful one of microbial insecticide.

Bacillus thuringiensis (Bacillus thuringiensis, abbreviation Bt) is a kind of Gram-positive reaction (G+) bacterium forms one or more companion cells with protein properties in thallus while key property is to be formed gemma Crystal.And the parasporal crystal is to Lepidoptera, Diptera, coleoptera, Hymenoptera, Homoptera insect and animals and plants parasitic nematode etc. There is stronger toxic action, and therefore to nontarget organism safety, Bt insecticide has single-minded, efficiently and excellent to safety of human and livestock etc. Point.Dipel commercial preparation is up to more than 100 kinds at present, be most widely used in the world, dosage is maximum, effect is best Microbial insecticide, thus paid close attention to by people.Bt Israel subclass (B.thuringiensis subsp.israelensis, Abbreviation Bti) have to a variety of mosquitoes high-efficiency activated, successfully exploitation is biological insecticides for a variety of Diptera elder brothers such as mosquito and buffalo gnat The prevention and treatment of worm played an important role in the control of mosquitos matchmaker's disease such as malaria, dengue fever, yellow fever.However production at present That there are still insecticidal ranges is narrow for the Bti bacterium of application, effective component is degradable, vulnerable to such environmental effects the disadvantages of, also have Bt successively The relevant report of certain drug resistance is formed in laboratory conditions.

Summary of the invention

The object of the present invention is to provide a kind of efficient bacillus thuringiensis liquid culture medium and its preparation methods.Micro- life Before the commercialization of object insecticide, the selection of culture medium is of crucial importance, and efficient fermentation medium can give full play to insecticidal activity bacterial strain Virulence and pathogenicity rate.The present invention is to provide a kind of Su Yunjin bud for comprehensively utilizing carbon source, nitrogen source and metal ion and being configured to Spore bacillus cultural method and its fluid nutrient medium, especially taurine and salicylic addition, even more to Su Yun bacillus Insecticidal effect brings great promotion, achieves unexpected technical effect.Culture medium of the invention is by Su Yunjin gemma Bacillus fermentation liquid is to the virulence of 3 age diamondback moths, and the lethality from 60% or so is increased to 95% or more, and insecticidal effect is promoted bright It is aobvious.

Applicant has found that taurine is added in bacillus thuringiensis liquid culture medium to be greatly improved under study for action Fermentation liquid is to the lethality of insect pest (such as diamondback moth), and salicylic addition is even more that can further cooperate with to reinforce this killing Power, the fermentation liquid being prepared reach 95% or more to the lethality of 3 age diamondback moths.

Fluid nutrient medium of the invention is composed of the following components: glucose, urea, dipotassium hydrogen phosphate, anhydrous sodium acetate, sulphur Sour magnesium, iron chloride, taurine, salicylic acid and sterile water.

The raw material proportioning of the fluid nutrient medium is (w/v): glucose 4-9%, urea 0.1-1%, dipotassium hydrogen phosphate 0.1-2%, anhydrous sodium acetate 0.1-2.5%, magnesium sulfate 0.01-0.1%, iron chloride 0.01-0.1%, taurine 0.1-2%, Salicylic acid 0.1-2%, remaining is sterile water.

Preferably, the proportion is (w/v): glucose 5-7%, urea 0.1-0.5%, dipotassium hydrogen phosphate 0.5-1%, nothing Water sodium acetate 1-2%, magnesium sulfate 0.01-0.02%, iron chloride 0.01-0.03%, taurine 0.2-1%, salicylic acid 0.2- 1%, remaining is sterile water.

It is highly preferred that the proportion is (w/v): glucose 5%, urea 0.1%, dipotassium hydrogen phosphate 0.5%, anhydrous acetic acid Sodium 1%, magnesium sulfate 0.01%, iron chloride 0.01%, taurine 0.5%, salicylic acid 0.5%, remaining is sterile water.

Beneficial effects of the present invention: 1, fluid nutrient medium of the invention is at low cost, and composition is simple, overcomes traditional Su Yun Golden bacillus culture medium is at high cost, should not be mass produced, the disadvantage that production operation is excessively complicated, convenient for long-distance transport and length Phase saves.2, the bacillus thuringiensis fermentation liquid obtained using bacillus thuringiensis of the invention to 3 age diamondback moths have compared with High virulence, insecticidal effect are significant.The present invention also has superior insecticidal properties to various insects such as ladybug, wheat cecidomyiias；Tool There is good popularization and application foreground.

Specific embodiment

Embodiment 1: the preparation of culture medium

Prepare four groups of culture mediums, respectively control medium, culture medium 1 (addition taurine), (the addition bigcatkin willow of culture medium 2 Acid) and culture medium 3 (addition taurine and salicylic acid), it is following (w/v) specifically to match when preparation method:

Control medium: glucose 5%, urea 0.1%, dipotassium hydrogen phosphate 0.5%, anhydrous sodium acetate 1%, magnesium sulfate 0.01%, iron chloride 0.01%, remaining is sterile water；Its preparation method are as follows: precision weighs glucose 50g, urea 1g, phosphoric acid hydrogen Dipotassium 5g, anhydrous sodium acetate 10g, magnesium sulfate 0.1g, iron chloride 0.1g, are separately added into volumetric flask, are settled to using sterile water 1L is mixed.

Culture medium 1: glucose 5%, urea 0.1%, dipotassium hydrogen phosphate 0.5%, anhydrous sodium acetate 1%, magnesium sulfate 0.01%, iron chloride 0.01%, taurine 1%, remaining is sterile water；Its preparation method are as follows: precision weighs glucose 50g, urine Plain 1g, dipotassium hydrogen phosphate 5g, anhydrous sodium acetate 10g, magnesium sulfate 0.1g, iron chloride 0.1g, taurine 10g, are separately added into capacity In bottle, it is settled to 1L using sterile water, is mixed.

Culture medium 2: glucose 5%, urea 0.1%, dipotassium hydrogen phosphate 0.5%, anhydrous sodium acetate 1%, magnesium sulfate 0.01%, iron chloride 0.01%, salicylic acid 1%, remaining is sterile water；Its preparation method are as follows: precision weighs glucose 50g, urine Plain 1g, dipotassium hydrogen phosphate 5g, anhydrous sodium acetate 10g, magnesium sulfate 0.1g, iron chloride 0.1g, salicylic acid 10g, are separately added into capacity In bottle, it is settled to 1L using sterile water, is mixed.

Culture medium 3: glucose 5%, urea 0.1%, dipotassium hydrogen phosphate 0.5%, anhydrous sodium acetate 1%, magnesium sulfate 0.01%, iron chloride 0.01%, taurine 0.5%, salicylic acid 0.5%, remaining is sterile water；Its preparation method are as follows: precision claims Take glucose 50g, urea 1g, dipotassium hydrogen phosphate 5g, anhydrous sodium acetate 10g, magnesium sulfate 0.1g, iron chloride 0.1g, taurine 5g, Salicylic acid 5g, is separately added into volumetric flask, is settled to 1L using sterile water, mixes.

The fermented and cultured and virulence determination of 2: Su Yun bacillus of embodiment

By the above culture medium prescription, every group of culture medium 3 250ml triangular flasks of setting, every 250ml triangular flask 40ml loading amount, It is 7.0 with sodium hydroxide tune Medium's PH Value, after high pressure sterilization, every shaking flask is inoculated with bacillus thuringiensis H14 bacterial strain 3.5%, Temperature is 31 DEG C, cultivates 72h, extracts 5ml fermentation liquid from every bottle of culture medium and is diluted to 100ml with sterile water and is made and ferments for examination Liquid；Pakchoi blade is impregnated for examination fermentation liquid using described, takes out, is added in foster worm bottle after drying, with sterile water logging after 5min The leaf of bubble is blank control, and every bottle of access 20 is in the same size, hungry 3 instar larvae of diamondback moth for 24 hours, 23 DEG C of constant temperature have been raised It supports, lethality is counted after 48h, test result is shown in Table 1.

Table 1:

It is as shown in table 1: the bacillus thuringiensis fermentation that bacillus thuringiensis cultivation and fermentation method of the invention obtains Liquid has higher virulence to 3 age diamondback moths；Wherein, the addition of taurine can significantly improve the virulence of the fermentation liquid of culture； Salicylic addition acts on limited although can also generate certain raising to the virulence of fermentation liquid；And taurine and salicylic acid While be added, but achieve apparent virulence and promoted, enable bacillus thuringiensis fermentation liquid average to the 48h of 3 age diamondback moths Lethality reaches 96.7% (even up to 100%), and insecticidal effect is brilliant, achieves unexpected technical effect.In addition, this Invention has also carried out insecticidal effect test to the various insects such as ladybug insect, equally has significant insecticidal effect.

Claims

1. a kind of fluid nutrient medium of bacillus thuringiensis, which is characterized in that the Liquid Culture of the bacillus thuringiensis Base comprises the following raw materials by weight percent: glucose 4-9%, urea 0.1-1%, dipotassium hydrogen phosphate 0.1-2%, anhydrous second Sour sodium 0.1-2.5%, magnesium sulfate 0.01-0.1%, iron chloride 0.01-0.1%, taurine 0.1-2%, salicylic acid 0.1-2%, Remaining is sterile water.

2. the fluid nutrient medium of bacillus thuringiensis according to claim 1, which is characterized in that the Su Yunjin gemma The fluid nutrient medium of bacillus comprises the following raw materials by weight percent: glucose 5-7%, urea 0.1-0.5%, phosphoric acid hydrogen two Potassium 0.5-1%, anhydrous sodium acetate 1-2%, magnesium sulfate 0.01-0.02%, iron chloride 0.01-0.03%, taurine 0.2-1%, Salicylic acid 0.2-1%, remaining is sterile water.

3. the fluid nutrient medium of bacillus thuringiensis according to claim 2, which is characterized in that the Su Yunjin gemma The fluid nutrient medium of bacillus comprises the following raw materials by weight percent: glucose 5%, urea 0.1%, dipotassium hydrogen phosphate 0.5%, anhydrous sodium acetate 1%, magnesium sulfate 0.01%, iron chloride 0.01%, taurine 0.5%, salicylic acid 0.5%, remaining is nothing Bacterium water.

4. the preparation method of culture medium described in claim 1-3, it is characterised in that: formed according to the raw material, precision weighs respectively Raw material components are placed in volumetric flask, are then settled to 1L using sterile water, are mixed.

5. the described in any item thuringiensis liquid culture mediums of claims 1 to 3 are in culture thuringiensis bacterium Purposes in kind.

6. a kind of cultivation and fermentation method of bacillus thuringiensis, it is characterised in that: will be trained described in any one of claim 1-3 Base is supported, every 250ml triangular flask 40ml loading amount is 7.0, after sterilizing with sodium hydroxide tune Medium's PH Value, every bottle of inoculation Su Yunjin Bacillus H14 bacterial strain 3.5%；31 DEG C of temperature, shake culture 72h, the fermentation liquid of collection.