Bacillus methylotrophicus AR3, bacillus subtilis AR4 and bacillus amyloliquefaciens AR10 and application thereof

A technology of Bacillus subtilis and methylotrophy, which is applied in the application field of three strains of Bacillus subtilis and R. solanacearum bacteria agent, can solve the problems of large pollution, low efficiency of chemical fungicides, weak pertinence, etc., and achieve effective results Good, fruitful, targeted effects

Active Publication Date: 2015-11-11
武汉华恩绿农生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to provide three biocontrol bacillus strains in order to overcome the defects of existing chemical fungicides such as low efficiency, weak perti

Method used

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  • Bacillus methylotrophicus AR3, bacillus subtilis AR4 and bacillus amyloliquefaciens AR10 and application thereof
  • Bacillus methylotrophicus AR3, bacillus subtilis AR4 and bacillus amyloliquefaciens AR10 and application thereof
  • Bacillus methylotrophicus AR3, bacillus subtilis AR4 and bacillus amyloliquefaciens AR10 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1 prepares three kinds of bacillus AR3, AR4, AR10

[0026] 1. Screening of Bacteria

[0027] Soil samples were taken from healthy tobacco fields in Xuan'en County and brought back to the laboratory for analysis. Weigh 5 grams of soil sample and place it in a sterilized 150mL Erlenmeyer flask containing 15-20 small glass beads, add 45mL of sterile water, shake the Erlenmeyer flask for 20min, and let stand for 30min to obtain the supernatant which is 10 -1 Dilute the gradient soil drench. Then use a 1mL sterile pipette to draw 10 -1 Transfer 1mL of the diluent into a test tube filled with 9mL of sterile water, blow and inhale 3 times, let the bacteria solution mix evenly, and serve as 10 -2 Diluent, take 100μl concentration as 10 -2 ~10 -4 The soil immersion solution was evenly spread on the plates with a coating stick, and after standing for 20 minutes, the culture plates were turned over and incubated at 37°C for 24 hours. Select the plate that is most ...

Embodiment 2 3

[0032] Embodiment 2 Three strains of biological control bacteria activity verification

[0033] Pick a single colony from the three preserved strains and put them in 5 mL of LB medium, culture them on a shaker at 37°C and 180 rpm / min for 72 hours, take 1 mL of fermentation broth in 1.5 mL centrifuge tubes, centrifuge at 12000 r / min for 5 minutes, and collect the supernatant , use a syringe to draw the supernatant and sterilize it with a filter. Prepare a PDA plate, take 100 μl of R. solanacearum cultured for 24 hours and evenly spread it on the PDA plate with a spreader, and then punch holes (5 mm in diameter) on the plate with a sterilized hole puncher. Inject 50 μl of the fermented liquid after filter sterilization into the round hole, and use an equal amount of sterile water as a control, and finally cover the round hole with a sterilized filter paper. Place the plate in a 37°C incubator and incubate for 48 hours, observe and record the antibacterial results, such as fig...

Embodiment 3

[0034] The identification of embodiment 3 biocontrol bacteria

[0035] Pick a single colony from the three preserved strains into 5mL LB medium, culture at 37°C, 180rpm / min on a shaker for 5-6h, take out 1mL of the cultured bacterial solution into a 1.5mL centrifuge tube, centrifuge at 12000r / min for 5min, discard Collect the bacteria in the supernatant, add 400 μl of deionized water to the centrifuge tube, cook in boiling water for 10 minutes, centrifuge at 12000 r / min for 5 minutes, and take the supernatant as a template.

[0036] The total volume of the PCR system is 50 μl: template DNA 2 μl, forward and reverse primers 2 μl each, 2×EasyTaqPCR superMix 25 μl, ddH 2 O19 μl.

[0037] Use the prepared supernatant as a template for PCR amplification. The reaction was carried out on a Thermo-Hybaid PCR instrument, and the program was 30 cycles of denaturation at 94°C for 3 min, denaturation at 94°C for 45 s, renaturation at 59°C for 45 s, extension at 72°C for 45 s, and extens...

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Abstract

The invention provides three biocontrol bacilli namely bacillus methylotrophicus AR3, bacillus subtilis AR4 and bacillus amyloliquefaciens AR10 and application thereof to preparation of a crop ralstonia solanacearum resistant bacteria agent. According to the invention, three biocontrol bacilli namely bacillus methylotrophicus AR3, bacillus subtilis AR4 and bacillus amyloliquefaciens AR10 are obtained through soil sample taking, screening, analysis and cultivation in a healthy tobacco field in the Xuan'en county, results of plenty of field experiments prove that when being used separately or used in a mixed manner, the three biocontrol bacilli have the good effect of preventing and controlling crop ralstonia solanacearum, are highly targeted and low in pollution, and can be stored for a long term and conveniently used in the field after being prepared into the bacteria agent, so that the achievement of the bacilli in the field of biocontrol bacterium research is enriched and guiding significance on later related research work is realized.

Description

technical field [0001] The present invention specifically relates to three strains of biocontrol bacillus, methylotrophic bacillus AR3, subtilis AR4, amyloliquefaciens AR10, and their application in the preparation of bacterial wilt bacteria agents resistant to tobacco, tomato, pepper, and ginger. The invention belongs to the field of agricultural microbiology technology and biological control technology. Background technique [0002] Crop bacterial wilt is an important disease caused by Ralstonia solanacearum that seriously endangers agricultural production. It is a major disease that restricts agricultural production and occurs commonly in many provinces in my country. Bacterial wilt is a vascular bundle disease. When the disease occurs, the roots, stems, and leaves of the crops will all suffer from the disease, but it is mainly concentrated in the roots and stems. blight. [0003] In recent years, in order to prevent and control plant diseases, the abuse of chemical age...

Claims

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Application Information

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IPC IPC(8): C12N1/20A01N63/00A01P1/00A01G7/06A01G13/00C12R1/07C12R1/125
Inventor 祁高富蹇小平
Owner 武汉华恩绿农生物科技有限公司
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