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Bacillus pumilus as well as culture method and application thereof

A technology of bacillus pumilus and its cultivation method, which is applied in the field of preparation of bacillus and its antibacterial active substances, to achieve a good control effect and the effect of inhibiting the growth of mycelium

Inactive Publication Date: 2011-01-12
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, Bacillus is an antagonistic microorganism. At present, it is basically mixed with Jinggangmycin and other pesticides to make microbial compound pesticides. The biological control of other antagonistic microorganisms for sheath blight is still in the experimental research stage and needs to be further developed.

Method used

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  • Bacillus pumilus as well as culture method and application thereof
  • Bacillus pumilus as well as culture method and application thereof
  • Bacillus pumilus as well as culture method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Obtainment of Bacillus pumilus 223 strain:

[0034] (1) Isolation and purification of bacteria:

[0035] Single spore separation method: Weigh 1 g of rice samples from different regions, put them into 100 ml of sterile water, and shake them for 20 minutes to obtain a bacterial suspension. Pipette 1 ml respectively, dilute 1000-fold, and spread 30 μl on LB plate, invert and incubate at 30°C after slightly drying. After 48 hours, according to the characteristics of the colony morphology and color on the plate, representative colonies were selected and streaked and purified on the LB plate. The purified bacterial isolates were inoculated into the slant medium, and stored at 4°C for later use. Finally, 238 strains were isolated from rice plants.

[0036] The formula of LB medium is: peptone 10g, yeast extract 5g, NaCl 10g, agar 18g, add water to 1000ml, adjust pH to 7.0, sterilize at 121°C for 20min.

[0037] (2) Screening of antagonistic bacteria:

[0038] T...

Embodiment 2

[0047] Embodiment 2, a kind of preparation method of antibacterial active substance, carry out the following steps successively:

[0048] 1), take Bacillus pumilus CCTCC NO: M2010075 (Bacillus pumilus 223CCTCC NO: M2010075) stored at -70°C, streak on PDA medium, and culture at 35°C for 24h.

[0049] The preparation method of PDA culture medium is as follows: add 800ml of water to 200g of potatoes and boil for 30min, filter with two layers of gauze to obtain a filtrate; add 20g of glucose and 18g of agar to the obtained filtrate, add water to 1000ml, natural pH, sterilize at 121°C for 20min, PDA medium was obtained.

[0050] 2), dip the single colony that grows on the PDA plate of step 1) gained with an inoculation loop, insert into a 500ml conical flask containing 100ml SMA liquid medium, and cultivate at 35°C, 200r / min for 30h; Bacterial fluid.

[0051] SMA liquid medium is: (NH 4 ) 2 SO 4 2.0g, KH 2 PO 4 6.0g, K 2 HPO 4 14.0g, NaCitrate 3H 2 O1.0g, MgSO 4 ·7H ...

Embodiment 3

[0056] Example 3. Effect of Bacillus pumilus CCTCC NO: M2010075 (Bacillus pumilus 223CCTCC NO: M2010075) on the growth of Rhizoctonia solani under in vitro conditions:

[0057] Confrontation culture method (Bell D K., et al. 1982): Take the sclerotia of Rhizoctonia solani cultured at a constant temperature of 28 °C and connect it to one side of the PDA solid medium, and connect it to the other side of the Bacillus microbacterium CCTCC. NO: M2010075. The cells were cultured at a constant temperature of 28°C, and the antibacterial status was observed after 24 hours. The experiment was repeated three times.

[0058] The confrontation culture showed that Bacillus pumilus CCTCC NO: M2010075 had obvious antagonistic effect on Rhizoctonia solani, and the edge structure of the hyphae of R. The edges are looser ( figure 1 ). Under the microscope, the inhibited mycelial structure of R. figure 2 ).

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Abstract

The invention discloses bacillus pumilus with a collection name of Bacillus pumilus 223, a collection unit of Chinese Type Culture Collection Center, a collection address of Wuhan University in Wuhan China, a collection date of April 8, 2010 and a collection number of CCTCC NO: M2010075. The invention also discloses a culture method of the bacillus pumilus and a method for preparing an antibiotic active substance by using the bacillus pumilus, comprising the following steps of: (1), carrying out streak culture on bacterial strains on a PDA (Potato Dextrose Agar) culture medium; (2), culturing a single colony in an SMA (Standard Methods Agar) fluid culture medium; (3), culturing bacterial liquid after being coated on an SMA solid culture medium; (4), soaking an obtained substance of the step (3) by using phosphate buffer and centrifuging; and (5) adding (NH4)2SO4 into supernatant liquid, centrifuging after standing and redissolving obtained precipitates by using the phosphate buffer. In the invention, the bacillus pumilus and the antibiotic active substance can be used for preventing and treating rice sheath blight diseases.

Description

technical field [0001] The invention belongs to the technical field of biological pesticides, and relates to a new strain for preventing and treating rice sheath blight, in particular to a Bacillus sp. Background technique [0002] Rice is the second largest food crop in the world after wheat. More than 50% of the world's population eat rice as the staple food. Therefore, the development of rice production is of great significance to ensure the stable production and supply of global food. Rice sheath blight occurs in a wide area, with strong prevalence and high frequency, and its losses often exceed those of rice blast, which has become a serious obstacle to stable and high yield of rice. How to effectively control the occurrence and damage of rice sheath blight, strengthen the prevention and control of the disease and ensure stable and high yield of rice has attracted widespread attention. [0003] With the aggravation of the rice sheath blight disease, a lot of work has b...

Claims

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Application Information

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IPC IPC(8): C12N1/20A01N63/02A01P3/00C12R1/07
Inventor 陈卫良姚岚程丹丹
Owner ZHEJIANG UNIV
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