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Method of determining antagonistic activity of fengycin lipopeptide against fusarium

A fusarium and lipopeptide technology is applied in the field of measuring the resistance of fengycin-like lipopeptides to Fusarium pigmentosa, which can solve the problems of long time, cumbersome methods, inefficiency, accuracy and simplicity, and achieves high efficiency. , the effect of simple operation

Inactive Publication Date: 2013-06-19
SHENYANG INST OF APPL ECOLOGY CHINESE ACAD OF SCI
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Problems solved by technology

[0004] However, in actual work, multiple experiments have shown that the above two methods are not suitable for rapid, efficient and accurate detection of the effect of antibiotic resistance on Fusarium
There are two reasons: first, Fusarium grows slowly and produces few spores. Generally, it is necessary to prepare a special spore-forming medium for shaking flasks for at least a week to produce sufficient spores, which takes a long time; In the next step of the resistance test, re-cultivation, observation of the phenomenon, the method is cumbersome and very labor-intensive, so it is not suitable for method A; second, the extremely slow growth rate of Fusarium makes it take at least a week to cover the plate, and some Even up to half a month, such a long action time has caused some antibiotics to lose their effect, showing that the mycelium spreads over the bacteriostatic zone produced by the initial antibiotics, and the bacteriostatic zone gradually shrinks or even disappears after long-term cultivation (such as image 3 ), if the diameter of the inhibition zone is still used to measure the antibacterial force of antibiotics at this time, it is obviously not feasible, so it is not applicable to method B
[0005] Although some scientific research workers adopt the improved method B (hereinafter referred to as method C), when the culture time is about 3 days (at this time, it can be observed that the mycelium grows around the antibiotic to an arc such as Figure 4 ), the antibacterial force can be judged by measuring the distance from the outer edge of the Oxford cup or filter paper to the outer edge of the bacteria. Although this method is feasible, the error is large, the accuracy is not high, and the measurement is difficult
Therefore, at present, there is no efficient, accurate and simple method for the determination of the antagonistic activity of filamentous fungi that grow slowly and are not easy to produce spores.

Method used

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  • Method of determining antagonistic activity of fengycin lipopeptide against fusarium
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  • Method of determining antagonistic activity of fengycin lipopeptide against fusarium

Examples

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Embodiment 1

[0028] Example 1 Determination of the minimum action concentration of C16fengycin A to cotton wilt (Fusarium oxysporumf.sp.vasinfectum)

[0029] 1. Initial culture of the target strain: select a PSA plate with a fresh cotton wilt strain, dig out a round bacterial block with a diameter of 8mm with a sterile puncher, place it in the center of the sterile PSA plate, and cultivate it at 28°C 48h, at this time the diameter of the bacterial block grows to about 2cm.

[0030] 2. Prepare C16fengycin A solution (a lipopeptide antibiotic with good effect on resistance to filamentous fungi) diluted with sterile PBS (pH=8.5) at half concentration from 2 mg / ml to 125 μg / ml (concentrations are respectively 2 mg / ml ml, 1mg / ml, 500μg / m, 250μg / m, 125μg / ml), 10μl of sample was loaded on each filter paper sheet, each concentration was repeated three times, and the filter paper sheets loaded with different concentrations of C16fengycinA were equidistantly attached to the cotton Around the bacter...

Embodiment 2

[0035] Example 2 Determination of the minimum action concentration of C16fengycin A on cucumber wilt (Fusarium.oxysporumf.sp.cucumerinum) and wheat scab (Fusarium graminearum)

[0036] The difference from Example 1 is that the indicator bacteria are Fusarium.oxysporum f.sp.cucumerinum or Fusarium graminearum, and the culture temperature and culture time: the initial culture is 30 ° C for 48 hours, and the loading The second period of culture condition after C16fengycin A is 10°C for 6 days.

[0037] The operation method is exactly the same as that in Example 1, and the data results obtained at last are shown in Table 2 and Table 3 respectively. Using Minitab 15 to fit the concentration of C16fengycin A and the diameter of the pigment circle, it was found that different concentrations of C16fengycin A promoted the square of the radius r of the pigment circle (r 2 ) and the logarithm (lgM) of the mass M of C16fengycin A on the filter paper sheet are still linearly related, as ...

Embodiment 3

[0038] Example 3 Determination of the minimum action concentration of the novel fengycin family on cotton fusarium wilt (Fusarium oxysporum f.sp.vasinfectum), cucumber wilt (Fusarium.oxysporum f.sp.cucumerinum) and wheat scab (Fusarium graminearum)

[0039] The difference from Example 1 is that the fengycin antibiotic is a novel fengycin family antibiotic, and the difference from fengycin A is that the sixth amino acid in the lipopeptide ring is substituted by Abu for Ala, and the indicator bacterium is cotton wilt (Fusarium oxysporum f.sp .vasinfectum), Cucumber Fusarium wilt (Fusarium.oxysporum f.sp.cucumerinum) or Wheat Scab (Fusarium graminearum);

[0040] The operation method is exactly the same as that of Example 1, and the corresponding relationship between the finally obtained pigment circle and concentration is consistent with that of Example 1.

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Abstract

The invention relates to a method for determining an antagonistic activity of the lipopeptid antibiotic, fengycin, against fusarium, in particular to a method for determining an antagonistic activity of fengycin lipopeptide against pigment-producing fusarium. The method is realized by replacing an inhibition zone with a pigment ring as a determining standard, operating the plate bacteriostasis experiment (cylinder plate method, drilling method or filtering paper method), and adopting temperature segmentation method to culture indicator bacterium. The invention has the advantages of high efficiency, simple operation, and reliable result, solving the difficulty in determining the antagonistic activity of the antibiotic against fusarium.

Description

technical field [0001] The invention relates to a method for measuring the resistance of fengycin, a lipopeptide antibiotic, to Fusarium; specifically, it is a method for measuring the resistance of fengycin lipopeptide to Fusarium chromogenum Background technique [0002] Fusarium (also known as Neurospora) is a worldwide soil-borne disease fungus that causes plant wilt, causing large-scale production reduction or even extinction in diseased fields. Therefore, as an important target bacterium, Fusarium is widely used in screening agricultural antibiotics and measuring the strength of antibiotic resistance. [0003] There are two traditional methods for measuring the resistance of antibiotics to fungi: A. Mix the bacterial suspension or spore suspension of the fungus to be tested in the culture medium and pour the antibiotic solution into an Oxford cup or put it on a filter paper. Place the slice on the culture medium containing bacteria, and measure the diameter of the inh...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/18
Inventor 胡江春陈丽丽王书锦
Owner SHENYANG INST OF APPL ECOLOGY CHINESE ACAD OF SCI
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