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Bacillus subtillis for preventing and controlling cole sclerotium and antibioticsubstance separation

A technology for bacillus subtilis and rape sclerotinia, which is applied in the field of agricultural microbiology, can solve the problems of difficulty in separating and purifying single-molecule antibiotics, difficulty in structural identification, and unsatisfactory effect of separation and purification, and achieves strong inhibition of rape sclerotinia bacterium the effect of the ability

Inactive Publication Date: 2008-09-24
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the difference in amino acid composition and fatty acid side chain length, various isomers are formed. These lipopeptides have similar structures and properties. It is very difficult to separate and purify pure single-molecule antibiotics from bacterial fermentation broth, which gives further research Structural identification poses considerable difficulties
Acid precipitation is the most commonly used method for separating and purifying lipopeptides, but many impurities are also precipitated after acid precipitation, and the effect of separation and purification is not ideal (Mukherjee S et al., Towards commercial production of microbial surfactants.TRENDS in Biotechnology, 2006 , 24(11):509-515)

Method used

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  • Bacillus subtillis for preventing and controlling cole sclerotium and antibioticsubstance separation
  • Bacillus subtillis for preventing and controlling cole sclerotium and antibioticsubstance separation
  • Bacillus subtillis for preventing and controlling cole sclerotium and antibioticsubstance separation

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1: Isolation, screening and identification of rapeseed sclerotinia antagonistic strain X-01

[0029]Step 1: Isolation of bacterial strains (dilution plate method, refer to the method introduced by Zhao Bin et al., Microbiology Experiment (First Edition). Beijing: Science Press, 2002).

[0030] Take 10 g of soil samples from the rapeseed base of Huazhong Agricultural University in Wuhan City, Hubei Province, add them to a triangular flask containing 100 ml of sterile water and glass beads, shake on a shaker at 150 rpm for 30 min, and obtain a soil suspension. Heat the soil suspension at 80°C for 30 minutes to enrich the bacillus, take 1ml of the enriched culture solution and add it to 9ml of sterile water, and follow the steps of 10 -2 , 10 -3 , 10 -4 , 10 -5 Dilution, take 0.1ml of different gradient dilutions and spread evenly on beef extract peptone medium (beef extract 5g, peptone 10g, NaCl 5g, agar 15g, distilled water to 1L, pH7.2), invert the plate, 30 ...

Embodiment 2

[0038] Example 2: Purification and structural identification of lipopeptide antibacterial active substances of X-01 strain

[0039] Pick a single colony of the X-01 strain and culture it in beef extract liquid medium (see above for ingredients) at 30°C and vibrate at 150rpm for 16 hours to obtain seed liquid, then inoculate 1ml of the seed liquid into 100ml fermentation medium (see After) in a 500ml Erlenmeyer flask. The components of the fermentation medium used are: tryptone 10g, yeast extract 2.5g, glucose 5g, K 2 HPO42.5g, MgSO4 7H 2 O 0.2g, dilute to 1000ml with distilled water, pH7.2. The culture temperature is 30°C; the rotation speed of the shaker is 150rpm; the culture time is 48h. Centrifuge the X-01 fermentation broth on a centrifuge with a relative centrifugal force of 10,000×g for 15 minutes to remove the bacteria, and get the supernatant through an ultrafiltration tube (Amicon ultra-15 devices, Millipore, USA) with a molecular weight cut-off (MWCO) of 100KDa. ...

Embodiment 3

[0041] Example 3: In vitro control experiment of purified lipopeptide substances on Sclerotinia sclerotiorum

[0042] The lipopeptide antibiotic iturin A purified from the X-01 strain fermentation broth was formulated into 100 μg / ml, 10 μg / ml and 1 μg / ml solutions respectively with sterile water and sprayed on rape leaves (the rape variety is Westar, five leaf stage) on. The method is to place rapeseed leaves in a large glass plate (20 cm in diameter) lined with filter paper (soaked with sterile water), 3 leaves per plate, 3 repetitions per treatment, and a sclerotinia in the center of each leaf. The silk block (Let-27) was cultured at 28°C and light / dark 14h / 10h for 96h, and then the diameter of the lesion was measured for each treatment. Water and carbendazim (purchased in the market) were used as negative and positive controls respectively. Calculate the inhibition rate according to the calculation formula: inhibition rate (%)=(clear water control lesion diameter-treatment...

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Abstract

The invention belongs to the technical field of agromicrobiology, in particular relates to separation and screening of an antagonistic bacterial strain X-01 which can inhibit sclerotinia sclerotiorum,and separation and application of antibacterial active substance. The bacterial strain X-01 is preserved in China Center for Type Culture Collection with the preservation number of CCTCC No. M208067. The bacterial strain X-01 is identified as bacillus subtilis through morphologic observation, biophysical and biochemical study, and 16S rDNA autoploidy study. The bacterial strain can secrete a lipopeptid antibiotic, iturin A. The molecular weight of the lipopeptid antibiotic is 1042.9Da. The invention further discloses a method for preparing the lipopeptid antibiotic, iturin A, by the bacterial strain X-01. According to the biological control test in vitro, the lipopeptid antibiotic, iturin A, has significant inhibitory effect on the sclerotinia sclerotiorum. When the concentration of the lipopeptid antibiotic iturin A is 100 Mu g / ml, the effect to control the sclerotinia sclerotiorum can reach 100 percent; when the concentration is 10 Mu g / ml, the effect to control the sclerotinia sclerotiorum can reach 40.7 percent.

Description

technical field [0001] The invention belongs to the technical field of agricultural microbiology, and in particular relates to the isolation and screening of a strain of Bacillus subtilis for preventing and treating Sclerotinia sclerotiorum, and the isolation and application of antibacterial active substances. Background technique [0002] Rapeseed sclerotinia is a fungal disease caused by Sclerotinia sclerotiorum, which ranks first among the three major diseases of rapeseed. The annual yield reduction of mild disease is 10%-20%, and the annual production reduction of severe disease is about 50%. No immune material has been found in the screening of rapeseed varieties resistant to Sclerotinia sclerotiorum, and there is currently a lack of highly effective disease-resistant varieties (Shuangsang et al., Progress in Research on Sclerotinia and disease-resistant breeding, CROP RESEARCH, 2006, 5: 552- 556). For a long time, most of its control has been chemically controlled by ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P21/04A01N63/02A01N37/18A01P3/00C12R1/125
Inventor 喻子牛张吉斌何进孙明刘子铎郑世学李明顺徐爱章
Owner HUAZHONG AGRI UNIV
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