CN116042728A - Pseudomonas aeruginosa extract with antagonism and application thereof - Google Patents
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Abstract
The invention relates to the technical field of microorganisms, in particular to a pseudomonas aeruginosa extract with antagonism and application thereof, the pseudomonas aeruginosa CJ9 strain extract prepared by the invention has stronger effect of inhibiting pathogenic bacteria of Vorticella canker, namely xanthomonas, and the antibacterial components of fermentation liquor are found to have high temperature resistant effect, and the active components of the pseudomonas aeruginosa CJ9 strain still have stronger antibacterial activity after being treated in water bath at 90 ℃ for 30 minutes; the components extracted by different solvents have different antibacterial effects on different bacteria, and the research discovers that the pseudomonas aeruginosa CJ9 fermentation broth: ethyl acetate was 2:1 (v: v) and vortex violent shaking extraction for 5min, the extract obtained by extraction has stronger effect of inhibiting xanthomonas, and the research result can provide a more efficient way for preventing and controlling the citrus canker.
Description
[ field of technology ]
The invention relates to the technical field of microorganisms, in particular to a pseudomonas aeruginosa extract with antagonism and application thereof.
[ background Art ]
The citrus tangerines (Orah) are the hybrid of the Tanpul oranges and the single-western kumquats, have the advantages of strong tree vigor, beautiful fruit appearance, excellent quality, late maturing, high sugar, early bearing, high yield and the like, are introduced in 2012 to Guangxi nan ning planting, are expanded in outbreak after 2014 because of excellent performance, and account for about 10 thousand ha by half of the total national area of Guangxi citrus tangerines at the bottom of 2018. The citrus tangerines are late-maturing citrus varieties with the largest planting area and influence in China at present, and are the most main citrus varieties after the sugar oranges in Guangxi. However, the citrus reticulata is sensitive to ulcer diseases, and the ulcer diseases become the most serious diseases in the citrus reticulata planting area, and are important factors for restricting the further development of the citrus reticulata industry. The citrus canker is an important bacterial quarantine disease which is harmful to citrus industry worldwide, and is mainly characterized in that leaf spots or wood embolism appear at the damaged part of the citrus in the new-tip extraction period and the young-fruit period of the citrus, so that fallen leaves, dried tips and fallen fruits are seriously caused to influence the yield and quality of the citrus, and the disease spread speed is high and the prevention and control difficulty is high.
The Vorticella is divided into 5 bacterial systems such as A, B, C, D and E according to geographic distribution and pathogenicity difference to host plants, and the bacterial system A is also called Asian species, belongs to the citrus subspecies of xanthomonas citri, and is the most pathogenic and most extensive bacterial system. Although the occurrence of ulcer disease has been 100 years old, no effective method can be completely cured at present, and the production mainly depends on chemical agents for prevention and treatment. The epidemic areas in China are mainly treated by chemical control, and mainly comprise copper preparations, streptomycin and other medicaments according to medicament classification. However, long-term application of chemical agents can cause problems such as drug residues and environmental pollution, and cause enhancement of pathogen resistance (Behlaiu et al 2011), so that green prevention and control of bacteria by using biocontrol microorganisms are particularly important. The biocontrol microorganisms mainly include Bacillus subtilis, pseudomonas fluorescens, eubacterium herbicola, pseudomonas syringae, and the like (Das, 2003). Yao Tingshan et al (2014) have stronger antagonism on the Uygorskite bacteria by antagonistic actinomyces A16 screened from soil, and the diameter of a bacteriostasis ring is 26.00mm; liu Bing and the like (2015) improve 20.7 percent of antibacterial effect after being mixed with agricultural streptomycin by the bacillus subtilis. Therefore, more and more researchers at home and abroad are researching about the prevention and treatment of the Wobbe ulcer disease by microorganisms.
Although the biological activity organic copper compound of pseudomonas aeruginosa can inhibit the growth of xanthomonas citrifolia, the biological activity organic copper compound is not studied, but the extracellular substance component secreted by pseudomonas aeruginosa is complex, and the components extracted by different solvents have different antibacterial effects on different bacteria, therefore, further intensive research on fermentation broth obtained by extracellular substance secreted by pseudomonas aeruginosa is needed, and an extraction method is adopted to ferment and extract an extract with higher effect of antagonizing xanthomonas citrifolia so as to provide a more reliable way for preventing and controlling the xanthomonas citrifolia.
[ invention ]
In view of the above, it is necessary to make intensive studies on the extract of pseudomonas aeruginosa by which technical means the fermentation broth secreted to the extracellular space of pseudomonas aeruginosa is extracted, thereby obtaining an extract with high antibacterial effect.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the pseudomonas aeruginosa extracting solution with antagonism is prepared by the following steps:
(1) Activating strains: inoculating pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ to LB liquid medium, and culturing at 37 ℃ at 220rpm/min overnight to obtain pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ9 culture;
(2) Preparing seed liquid: inoculating the pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ9 culture in LB liquid medium to culture at 37 ℃ for 220rpm/min for 8 hours to obtain pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ seed liquid;
(3) Preparing a fermentation liquid: inoculating the obtained pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ seed solution into an LB culture medium for culturing at 37 ℃ with 220rpm/min inoculum size of 2%, and fermenting for 48 hours to obtain pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ fermentation broth;
(4) Preparing an extracting solution: the obtained pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ fermentation broth is prepared according to the following steps: ethyl acetate volume=3-1:1, vortex shaking and extracting for 1-5min, then centrifuging at 10000rpm for 1min to separate ethyl acetate from fermentation broth, sucking out the colorless ethyl acetate at the uppermost layer, and obtaining pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ extract.
Further described, the pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ accession No. GDMCC No. (62903).
Further, the formula of the LB liquid medium is as follows: 10g/L peptone, 5g/L yeast powder, 10g/LNaCl.
The invention also provides application of the pseudomonas aeruginosa extract with antagonism in antagonism of xanthomonas aeruginosa.
The invention also provides application of the pseudomonas aeruginosa extract with antagonism in antagonism of xanthomonas citrina.
The invention also provides application of the pseudomonas aeruginosa extract with antagonism in preparing microbial agents for preventing and treating citrus canker.
The invention has the following beneficial effects:
in the invention, extracellular substances secreted by pseudomonas aeruginosa are found in fermentation broth in the research process, and when the antibacterial effect of pseudomonas aeruginosa CJ9 strain is researched, the supernatant fermented by pseudomonas aeruginosa CJ9 strain is found to have stronger effect of inhibiting xanthomonas, antibacterial components of the fermentation broth have high temperature resistant effect, and active components of pseudomonas aeruginosa CJ9 strain still have stronger antibacterial activity after being treated in water bath at 90 ℃ for 30 minutes; the components extracted by different solvents have different antibacterial effects on different bacteria, and the research discovers that the pseudomonas aeruginosa CJ9 fermentation broth: ethyl acetate was 2:1 (v: v) and vortex violent shaking extraction for 5min, the extract obtained by extraction has stronger effect of inhibiting xanthomonas, and the research result can provide a more efficient way for preventing and controlling the citrus canker.
[ description of the drawings ]
FIG. 1 is a morphology of CJ9 strain of the invention on a plate;
FIG. 2 is a graph showing the comparison of the effect of different components of the fermentation broth of the CJ9 strain of the present invention on antagonizing pathogenic bacteria of Vortichthys (Xanthomonas sp); remarks: f1, fermenting liquid stock solution; f2, fermenting supernatant (sterilized by 0.22 μm filter membrane); f3, bacterial cell resuspension; f4, bacterial cell cytoblast;
FIG. 3 is a graph showing comparison of heat resistance of active ingredients of fermentation broth of CJ9 strain of the present invention; wherein, CK: control (broth stock); 50: carrying out water bath treatment on the fermentation liquor at 50 ℃ for 30 minutes; 60: carrying out water bath treatment on the fermentation liquor at 60 ℃ for 30 minutes; 70: carrying out water bath treatment on the fermentation liquor at 70 ℃ for 30 minutes; 80: carrying out water bath treatment on the fermentation liquor at 80 ℃ for 30 minutes; 90: carrying out water bath treatment on the fermentation liquor at 90 ℃ for 30 minutes; 100: carrying out water bath treatment on the fermentation liquor at the temperature of 100 ℃ for 30 minutes;
FIG. 4 is a graph showing the comparison of the effect of the extract of the fermentation broth of different organic solvent extraction strain CJ9 on antagonizing the pathogenic bacteria of Vorticella (Xanthomonas).
FIG. 5 is a graph showing the comparison of the effect of the organic solvent extract of the strain CJ9 on antagonizing pathogenic bacteria of Vorticella (Xanthomonas campestris) in different proportions;
fig. 6 is a graph showing the comparison of the effect of the extract of the strain CJ9 on antagonizing pathogenic bacteria of Vorticella (Xanthomonas).
[ detailed description ] of the invention
All of the features disclosed in this specification, or all of the steps in a method or process disclosed, may be combined in any combination, except for mutually exclusive features and/or steps.
Any feature disclosed in this specification (including any accompanying claims, abstract) may be provided with respect to each feature disclosed herein, unless otherwise indicated, as an example of a generic series of equivalent or similar features.
Example 1:
screening of pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ:
pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ with accession number GDMCC No.62903 deposited in: the collection of microbial strains, guangdong province, address: the collection date is 2022, 10 months and 19 days, which is the institute of microbiology, national institute of sciences, guangdong province, building 5, 100, mitsui, guangzhou City.
The strain was isolated from the soil of the Guangxi Sakya fruit orchard in 2022.
The separation method comprises the following steps: a flat bacteriostasis ring method.
The isolated strains were morphologically classified and identified by molecular biology, as follows:
1. morphological classification of strains
Morphological classification of strains:
as shown in FIG. 1, the strain CJ9 forms large and flat colonies after culturing on LB agar plates, is in a ground glass shape, is irregular, stretched, umbrella-shaped at the edge, has metallic luster and ginger flavor, generates a grayish green, blue green or yellow green pigment, and is diffused in a culture medium.
2. Molecular biological identification
Sequencing and verifying the strain: and amplifying the gene sequence of the strain by adopting a universal primer, sequencing the purified PCR product, wherein the sequenced sequence is shown in a sequence table. And inputting the CJ9 sequence obtained by sequencing into a NCBI database for sequence alignment, and identifying the CJ9 strain as pseudomonas aeruginosa (Pseudomonas aeruginosa) through sequence alignment.
Example 2:
preparation of pseudomonas aeruginosa fermentation broth with antagonism:
(1) Activating strains: inoculating pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ to LB liquid medium, and culturing at 37 ℃ at 220rpm/min overnight to obtain pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ9 culture; the formula of the LB culture medium is as follows: 10g/L peptone, 5g/L yeast powder, 10g/LNaCl;
(2) Preparing seed liquid: inoculating the pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ9 culture in LB liquid medium to culture at 37 ℃ for 220rpm/min for 8 hours to obtain pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ seed liquid;
(3) Preparing a fermentation liquid: and inoculating the obtained pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ seed solution into an LB culture medium for culturing at 37 ℃ and 220rpm/min, wherein the inoculum size is 2%, and fermenting for 48 hours to obtain pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ fermentation broth.
1. Antagonizing bacteriostasis experiments of pathogenic bacteria of Vorticella (Xanthomonas):
the pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ9 broth was subjected to 4 treatments as follows:
f1 fermentation liquor stock solution
F2 fermentation broth supernatant (sterilized by 0.22 μm filter membrane)
F3 bacterial cell resuspension
F4 bacterial cell cytoblast
Respectively taking the above 4 100ul liquids for carrying out antibacterial experiments for antagonizing pathogenic bacteria (xanthomonas) of the Vortilli ulcer disease;
(1) Preparation of Xanthomonas indicator plate:
symmetrically placing 4 oxford cups on an LB solid culture medium plate, uniformly mixing Xanthomonas and LB agar culture medium which is melted and cooled to 50 ℃ according to the ratio of 1:100, pouring about 25ml into the oxford cup placing plate, removing the oxford cups by using sterile forceps after the culture medium is solidified, and preparing an indication plate;
(2) Loading:
100ul of each of the above 4 treated liquids was dropped into the oxford cup well of the indicator plate.
(3) Culturing:
after loading was completed, the Xanthomonas indicator plate was incubated at 30℃for 24 hours.
(4) Experimental result processing:
after the cultivation is finished, the flat plate is photographed and observed, the flat plate indication result is shown in figure 2, the antibacterial effect of the fermentation stock solution is strongest, and the fermentation supernatant and the living bacteria resuspended in sterile water also have stronger antibacterial effect. The bacterial cell cytoblast can also inhibit Xanthomonas, but has weaker antibacterial effect, and further shows that the antibacterial active substance is produced by the living Pseudomonas aeruginosa and secreted extracellularly, and the antibacterial substance stored in the cells is less.
2. Heat resistance test of antibacterial active substances of Pseudomonas aeruginosa:
(1) Preparation of Xanthomonas indicator plate:
symmetrically placing 4 oxford cups on an LB solid culture medium plate, uniformly mixing Xanthomonas and LB agar culture medium which is melted and cooled to 50 ℃ according to the ratio of 1:100, pouring about 25ml into the oxford cup placing plate, removing the oxford cups by using sterile forceps after the culture medium is solidified, and preparing an indication plate;
(2) And (3) heating fermentation liquor:
200ul of fermentation broth is respectively taken for the following treatment:
CK: control (stock solution of fermentation broth)
50: the fermentation broth was subjected to a water bath at 50℃for 30 minutes
60: the fermentation broth was subjected to a water bath at 60℃for 30 minutes
70: the fermentation broth was subjected to a water bath at 70℃for 30 minutes
80: the fermentation broth was subjected to a water bath at 80℃for 30 minutes
90: the fermentation broth was subjected to a water bath at 90℃for 30 minutes
100: the fermentation broth was subjected to a water bath at 100deg.C for 30 minutes
100ul of each of the 7 treated liquids was dropped into the oxford cup well of the indicator plate.
(3) Loading:
the above 4 treated liquids were dripped into the oxford cup well of the indicator plate.
(4) Culturing:
after loading was completed, the Xanthomonas indicator plate was incubated at 30℃for 24 hours.
(5) Experimental result processing:
from the bacteriostasis experiment shown in fig. 3, after the fermentation broth is treated in a water bath at 50 ℃ for 30 minutes, the pseudomonas aeruginosa thallus still has activity, when the temperature is raised to 60 ℃, the thallus is deactivated, and the bacteriostasis substance can resist the treatment at a higher temperature, and the pseudomonas aeruginosa thallus still has stronger activity after being treated in a water bath at 90 ℃ for 30 minutes. There is a major loss of bacteriostatic activity from the 30-minute treatment in a water bath at 100 ℃.
Example 3:
preparation of pseudomonas aeruginosa extract with antagonism: the fermentation broth of the pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ obtained in example 2 was directly added into an organic solvent to extract for 5 minutes, so as to obtain an extract of the pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ.
The organic solvent is designed as follows:
a: acetic acid ethyl ester
B: diethyl ether
C: chloroform (chloroform)
D: petroleum ether
E: toluene (toluene)
Extracting the extract by the 5 different organic solvents to obtain an extract, and performing a xanthomonas bacteriostasis experiment:
(1) Preparation of Xanthomonas indicator plate:
symmetrically placing 4 oxford cups on an LB solid culture medium plate, uniformly mixing Xanthomonas and LB agar culture medium which is melted and cooled to 50 ℃ according to the ratio of 1:100, pouring about 25ml into the oxford cup placing plate, removing the oxford cups by using sterile forceps after the culture medium is solidified, and preparing an indication plate;
(2) Loading:
100ul of each of the above 4 treated liquids was dropped into the oxford cup well of the indicator plate.
(3) Culturing:
after loading was completed, the Xanthomonas indicator plate was incubated at 30℃for 24 hours.
(4) Experimental result processing:
after the cultivation is finished, the flat plate is photographed and observed, the indication result of the flat plate is shown in figure 4, the transparent inhibition zone of the extracting solution extracted by the ethyl acetate is the largest, and the transparent inhibition zones of the extracting solutions extracted by other solvents are smaller.
Example 4:
preparation of pseudomonas aeruginosa extract with antagonism: the fermentation broth of the pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ obtained in example 2 was directly added with ethyl acetate for extraction for 5 minutes to obtain an extract of the pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ.
Fermentation liquid: the ethyl acetate (v: v) was designed as follows:
fermentation liquid: ethyl acetate (v: v) =3:1
Fermentation liquid: ethyl acetate (v: v) =2:1
Fermentation liquid: ethyl acetate (v: v) =2:1
Fermentation liquid: ethyl acetate (v: v) =1:2
Fermentation liquid: ethyl acetate (v: v) =1:3
Extracting the extract with the above 5 different organic solvent ratios to obtain a xanthomonas bacteriostasis experiment:
(1) Preparation of Xanthomonas indicator plate:
symmetrically placing 4 oxford cups on an LB solid culture medium plate, uniformly mixing Xanthomonas and LB agar culture medium which is melted and cooled to 50 ℃ according to the ratio of 1:100, pouring about 25ml into the oxford cup placing plate, removing the oxford cups by using sterile forceps after the culture medium is solidified, and preparing an indication plate;
(2) Loading:
100ul of each of the above 4 treated liquids was dropped into the oxford cup well of the indicator plate.
(3) Culturing:
after loading was completed, the Xanthomonas indicator plate was incubated at 30℃for 24 hours.
(4) Experimental result processing:
the results of the measurement are shown in FIG. 5, when the fermentation broth: the volume ratio of the ethyl acetate is 2:1, the extract obtained by the method has the best antibacterial effect.
Example 5:
preparation of pseudomonas aeruginosa extract with antagonism: the pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ broth obtained in example 2 was directly processed as a broth: ethyl acetate volume=2:1 mixed vortex vibration extraction to obtain pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ9 extract.
The time design of vortex vibration extraction is as follows:
a: for 1 minute
B:2 minutes
C:3 minutes
D:4 minutes
E: for 5 minutes
The extract obtained through the extraction of the above 5 comparison times is subjected to a xanthomonas bacteriostasis experiment:
(1) Preparation of Xanthomonas indicator plate:
symmetrically placing 4 oxford cups on an LB solid culture medium plate, uniformly mixing Xanthomonas and LB agar culture medium which is melted and cooled to 50 ℃ according to the ratio of 1:100, pouring about 25ml into the oxford cup placing plate, removing the oxford cups by using sterile forceps after the culture medium is solidified, and preparing an indication plate;
(2) Loading:
100ul of each of the above 4 treated liquids was dropped into the oxford cup well of the indicator plate.
(3) Culturing:
after loading was completed, the Xanthomonas indicator plate was incubated at 30℃for 24 hours.
(4) Experimental result processing:
the measurement result is shown in fig. 5, and the obtained extract has the best antibacterial effect after vortex violent vibration extraction for 5 min.
In conclusion, the extract of the pseudomonas aeruginosa CJ9 strain has obvious inhibition effect on xanthomonas, and the fermentation broth has high temperature resistant effect, and the thalli of the pseudomonas aeruginosa CJ9 strain still have stronger activity after being treated in a water bath at 90 ℃ for 30 minutes; the components of different parts extracted by different solvents have different antibacterial effects on different bacteria, and the research discovers that the pseudomonas aeruginosa CJ9 fermentation broth: ethyl acetate was 2:1 (v: v) and vortex violent shaking extraction for 5min, the extract obtained by extraction has stronger effect of inhibiting xanthomonas, and the research result can provide a more efficient way for preventing and controlling the citrus canker.
The above examples merely represent a few embodiments of the present invention, which are described in more detail and are not to be construed as limiting the scope of the present invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of the invention should be assessed as that of the appended claims.
Claims (6)
1. The pseudomonas aeruginosa extract with antagonism is characterized in that the extract is prepared by the following steps:
(1) Activating strains: inoculating pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ to LB liquid medium, and culturing at 37 ℃ at 220rpm/min overnight to obtain pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ9 culture;
(2) Preparing seed liquid: inoculating the pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ9 culture in LB liquid medium to culture at 37 ℃ for 220rpm/min for 8 hours to obtain pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ seed liquid;
(3) Preparing a fermentation liquid: inoculating the obtained pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ seed solution into an LB culture medium for culturing at 37 ℃ with 220rpm/min inoculum size of 2%, and fermenting for 48 hours to obtain pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ fermentation broth;
(4) Preparing an extracting solution: the obtained pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ fermentation broth is prepared according to the following steps: mixing ethyl acetate with volume=3-1:1, vortex shaking and extracting for 1-5min, centrifuging at 10000rpm for 1min to separate ethyl acetate from fermentation broth, and sucking out the colorless ethyl acetate at the uppermost layer to obtain Pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ extract.
2. The antagonistic pseudomonas aeruginosa extract according to claim 1, wherein pseudomonas aeruginosa strain Pseudomonas aeruginosa CJ accession No.62903.
3. The antagonistic pseudomonas aeruginosa extract according to claim 1, wherein the formula of the LB liquid medium is: 10g/L peptone, 5g/L yeast powder, 10g/LNaCl.
4. Use of the antagonistic pseudomonas aeruginosa extract according to claim 1 for antagonizing xanthomonas aeruginosa.
5. Use of the antagonistic pseudomonas aeruginosa extract according to claim 1 for antagonizing xanthomonas citri.
6. Use of the antagonistic pseudomonas aeruginosa extract according to claim 1 for preparing microbial agents for controlling citrus canker.
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