CN110079470B

CN110079470B - Pseudomonas with antibacterial activity

Info

Publication number: CN110079470B
Application number: CN201910160005.3A
Authority: CN
Inventors: 李文; 王佳莹; 何月秋; 刘峰; 王志龙
Original assignee: Ningbo City College of Vocational Technology
Current assignee: Ningbo City College of Vocational Technology
Priority date: 2019-03-04
Filing date: 2019-03-04
Publication date: 2020-11-03
Anticipated expiration: 2039-03-04
Also published as: CN110079470A

Abstract

The invention discloses a pseudomonas with bacteriostatic activity, belonging to pseudomonas family: (a)Pseudomonadaceae) Pseudomonas morganii (M. Morgans.) (Pseudomonas mosselii) The preservation number is CCTCC NO: m2019081. The pseudomonas morganii LWB10 has strong bacteriostatic activity, and the metabolite thereof also has bacteriostatic activity, can be applied to prevention and treatment of agrobacterium tumefaciens and preparation of agrobacterium tumefaciens antibacterial drugs, and can be fermented to produce the metabolite thereof, thereby providing a powerful basis for large-scale popularization and application of biocontrol bacteria.

Description

Pseudomonas with antibacterial activity

Technical Field

The invention relates to pseudomonas, in particular to pseudomonas with bacteriostatic activity.

Background

Agrobacterium tumefaciens (A. tumefaciens) (B)Agrobacterium tumefaciens) Is an important plant pathogenic bacterium, has a very wide host range, and can infect more than 1193 plants of 138 families, such as peach, cherry, plum, apricot, pear, apple and the like. After the bacterium invades from the wound of a plant, a tumor is formed on a root and a branch, the transportation of water and nutrient substances by the root system is blocked, the growth of the root is influenced, and the nutrient deficiency, the tree vigor weakness and even the dead tree are caused. In a cherry blossom breeding base in Ningbo areas, the root cancer is an important disease on the cherry blossom, and great economic loss is caused to the breeding of the cherry blossom.

For many years, the inspection and quarantine work of the cherry seedling root cancer is not kept up, and the root cancer occurs in large area in various places. With the increase of cultivation time, the occurrence degree of the root cancer in the same nursery can be enhanced year by year; because the circulation of each oriental cherry seedling is frequent, the root cancer is also propagated along with the circulation of the seedling, and the disease is more serious. Early-stage research shows that the average incidence of the main culture area of the oriental cherry in Ningbo city reaches 30%, and the incidence of the plant in the retransmission block is even as high as 92% (Wangzhiling et al, 2014). In production, before the root cancer disease occurs, the method of dipping the root with lime sulphur mixture or irrigating the root with streptomycin sulfate is adopted, and the like, so that the root cancer disease is prevented to a certain extent. However, after the disease occurs, the method has poor control effect. Through soil fumigation, the number of agrobacterium tumefaciens in a diseased land can be reduced, and the probability of pathogen infection is reduced. However, fumigation of soil kills not only the pathogenic bacteria but also some other antagonistic microorganisms in the soil, which may lead to recurrence of root cancer (Yakabe et al, 2010).

In the control of fruit tree diseases, research reports have been made on the use of antagonistic microorganisms in the control of fruit tree diseases. The strain A102 of streptomyces lydicus is separated from the Liu Gung and the like, the strain can synthesize an active product, when the concentration of the active product is more than 30mg/L, the strain has stronger inhibition activity on botrytis cinerea, penicillium spinulosum and monilinia persicae, and the inhibition rate can reach 100%. Bacillus AiL3 separated from mangrove can synthesize antibacterial protein, and inhibit the growth of Cerbera Manghas Linnaeus by influencing thallus metabolism. In the aspect of biological control of fruit tree root canceration, Australian scientists isolated an agrobacterium rhizogenes K84 strain and modified strains K1026 and WJK84-1 thereof can be successfully used for controlling the root canceration of stone fruit trees such as peach trees and the like (Kerr, 1980; Copping, 2001; Wangguan and the like, 2004). Two strains of Alcaligenes faecalis 51-A and 51-B of peach rhizosphere are obtained by separating high buds and the like, and the two strains have obvious inhibition effect on Agrobacterium tumefaciens ATCC 23308T. The inoculation experiment shows that the prevention effect on the root cancer can reach over 86 percent by infiltrating the antagonistic bacterium suspension on the indication plant tomato in advance. However, the above antagonistic bacteria and metabolites thereof having antibacterial action have not been applied to production on a large scale at present. Therefore, the screening and obtaining of new biocontrol bacteria resources have important practical significance for the prevention and control of Ningbo local root cancer.

Disclosure of Invention

In order to solve the technical problems, the invention provides a strain of pseudomonas which has strong bacteriostatic activity, can obviously inhibit agrobacterium tumefaciens and provides a basis for biological prevention and development of new antibacterial drugs.

The technical scheme of the invention is to provide pseudomonas with bacteriostatic activity, which belongs to pseudomonas (Pseudomonas sp.)Pseudomonadaceae) Pseudomonas morganii (M. Morgans.) (Pseudomonas mosselii) Named as:Pseudomonas mosseliiLWB 10. The culture is preserved in China center for type culture Collection, the preservation address is Wuhan university in Wuhan city, the preservation date is 2019, 1 month and 24 days, and the preservation number is CCTCC NO: m2019081, hereinafter abbreviated as Pseudomonas morganii LWB 10.

Pseudomonas morganii LWB10 of the present invention is a gram-negative bacterium, an aerobic bacterium, having a cell size of (0.5-0.8) μm x (1.5-1.8) μm with terminal plexus flagella, as shown in FIG. 1.

The length of the 16S rDNA sequence of the pseudomonas LWB10 is 1407bp, and the sequence is shown as SEQ ID NO: 1 is shown.

The invention has the advantages and beneficial effects that: the pseudomonas morganii LWB10 has strong bacteriostatic activity, and the metabolite thereof also has bacteriostatic activity, can be applied to prevention and treatment of agrobacterium tumefaciens and preparation of agrobacterium tumefaciens antibacterial drugs, and can be fermented to produce the metabolite thereof, thereby providing a powerful basis for large-scale popularization and application of biocontrol bacteria.

Drawings

FIG. 1 is an electron micrograph of Pseudomonas LWB10 of the present invention.

FIG. 2 is a photograph showing the inhibition zone of Pseudomonas LWB10 of the present invention against Agrobacterium tumefaciens C58.

FIG. 3 is a phylogenetic tree of Pseudomonas LWB10 of the present invention.

Detailed Description

The present invention will be further described with reference to the following embodiments.

The invention provides pseudomonas LWB10, belonging to the family of Pseudomonas (A)Pseudomonadaceae) Pseudomonas morganii (M. Morgans.) (Pseudomonas mosselii) The culture is preserved in China Center for Type Culture Collection (CCTCC), the preservation address is Wuhan university in Wuhan, the preservation date is 2019, 1 month and 24 days, and the preservation number is CCTCC NO: m2019081 which is named asPseudomonas mosseliiLWB10。

Example 1

The pseudomonas morganii LWB10 gram-negative and aerobic bacteria are provided. Positive in catalase test, oxidaseThe test is negative, and the result of the motility test is that the test has motility. The strain is cultured in YEB medium (containing 5g peptone, 5g sucrose, 1g yeast extract, 5g beef extract, 10g agar powder, pH 7.0-7.2, and autoclaving at 121 deg.C for 20 min), and sterilized by adding 1M MgSO₄2ml of the extract is obtained; or LB and KB culture medium added with beef extract and peptone) can grow well, can grow on Macconk culture medium, can grow normally on a blood plate, forms gray-white flat wet colonies, and has smooth colonies, neat edges and no hemolysis. The optimum growth temperature was 28 ℃ and growth at 4 ℃ and 42 ℃ was not possible. The culture at 22-28 ℃ can produce a large amount OF yellow pigment, and the test OF glucose oxidation and fermentation (OF test) is positive, the test OF hydrolyzed DNA is negative, the test OF arginine double hydrolase is positive, the test OF nitrate reduction is negative, the test OF acetamide hydrolysis is negative, and the test OF xylose, mannitol and maltose acid production is positive.

Example 2

The separation process of the pseudomonas morganii LWB10 of the invention is as follows: selecting oriental cherry plants infected with Agrobacterium tumefaciens (in this example, collected in Nibo city, Zhejiang province, 37150in campus, Nibo city occupational technology institute, State region, excavating downwards along rhizosphere, taking about 5g of soil 10-20cm away from surface soil, soaking in sterile 50ml centrifuge tube with 10ml of sterile distilled water, mixing uniformly on vortex mixer, fully releasing microorganisms in soil, standing for 10min, sucking small amount of upper liquid in super clean bench for gradient dilution, setting 10min¹-10⁵Five different gradients, each 50ul of liquid was pipetted evenly onto a 9cm YEB solid plate and incubated overnight at 28 ℃. And on the next day, selecting single colonies on a culture dish without fungal pollution, transferring the single colonies into a liquid YEB culture medium for culture, and obtaining different soil bacteria single colonies for the following antagonistic effect identification process.

And (3) identifying the antagonistic effect of the soil bacteria on the agrobacterium tumefaciens: a large amount of antagonistic bacteria of the agrobacterium tumefaciens in the soil are screened by observing whether the inhibition zone is generated, and the experiment operation of the inhibition zone is as follows. 15 ml of YEB solid culture medium is poured into a sterile culture dish and dried for standby. To 10ml at a temperature of about 60 DEG C1 ml of Agrobacterium tumefaciens bacterial liquid with OD600 value of 1.0 is added into the YEB culture medium and mixed evenly. And (4) quickly pouring the culture medium mixed with the bacterial liquid into the culture dish prepared above, uncovering and drying. A0.6 cm diameter disc of sterile filter paper was carefully placed on the surface of the medium with tweezers, and a 10. mu. lOD600 drop of a 1.0 value of a different soil bacteria was pipetted into the center of the filter paper, followed by sterile ddH₂O as blank control. After the liquid permeates the culture medium, the culture medium is carefully moved to 28 ℃ for overnight culture, the generation of a bacteriostatic circle is observed on the next day and is photographed and recorded, after 2 days of culture, a bacterial liquid with the bacteriostatic circle diameter larger than 2cm is selected as a candidate antagonistic bacterium for further research, and the bacteriostatic circle after 2 days of co-culture of the screened bacterial strain LWB10 and the agrobacterium tumefaciens C58 is shown in figure 2.

Example 3

The strain LWB10 obtained by screening is subjected to molecular identification according to the following steps: extracting bacterial genome DNA by a wall breaking method, and carrying out PCR amplification on the genomic DNA by using international bacterial identification universal primers F8 (5'-AGAGTTTGATCCTGGCTCAG-3')/R1492 (5'-ACGGCTACCTTGTTACGACTT-3'). And then verifying the size of the amplified product by agarose gel electrophoresis, tapping and recovering, recovering the product, carrying out TA cloning, connecting the product to a pMD-18-T vector, transforming escherichia coli TG1 competent cells, carrying out overnight culture, selecting positive clones by antibiotic screening and PCR amplification, carrying out amplification culture on the positive clones, and then committing Shanghai biological engineering technical service company to carry out sequencing.

The length of the 16S rDNA sequence of the strain LWB10 is 1407bp, and the sequence is shown as SEQ ID NO: 1, the sequencing results were subjected to homologous alignment in GenBank, and then a phylogenetic tree was constructed using software, as shown in FIG. 3, to determine the species relationship of the strains, from which it was found that LWB10 of the present invention is an independent branch in Pseudomonas. The homology analysis result shows that the strain has the highest homology with the 16S rDNA sequence of the pseudomonas morganii, the similarity is 99 percent, the strain is the pseudomonas morganii through mass spectrometry and database comparison, and in addition, the strain LWB10 is determined to belong to the pseudomonas morganii by combining with the thallus morphological characteristics, the growth conditions, the physiological and biochemical identification results and the mass spectrum identification result analysis in the example 1Medicine (Ke (C)Pseudomonadaceae) Pseudomonas morganii (M. Morgans.) (Pseudomonas mosselii）。

Example 4

Preparation of pseudomonas morganii LWB10 fermentation product: the isolated Pseudomonas morganii LWB10 was inoculated into YEB liquid medium and cultured on a shaker at 28 ℃ and 250rpm for 3 days. After 3 days, the broth was centrifuged at 10,000 rpm for 10min to collect the supernatant, which was then fractionated with an equal volume of ethyl acetate and the organic phase was re-extracted with 1/10 volumes of sterile distilled water. The collected organic phase was then concentrated on a rotary evaporator and after fractional concentration the remaining 2ml of solution was completely blown dry on a nitrogen blower. And finally, adding 200 ul of chromatographic pure grade dimethyl sulfoxide (DMSO) into a blow-dried test tube, and performing vortex oscillation until the DMSO is completely dissolved to obtain the purified fermentation product of the pseudomonas LWB 10.

And (3) verifying the antibacterial activity of the fermentation product: the method for verifying the bacteriostatic activity of the fermentation product is the same as the process for identifying the antagonistic effect in example 2, except that the bacterial liquid on the filter paper sheet is changed into the extracted fermentation product, the fermentation product is cultured for 24 hours at 28 ℃, the bacteriostatic activity of the fermentation product on different strains of agrobacterium tumefaciens is observed, the size of a bacteriostatic zone is determined, and the result is shown in table 1:

TABLE 1 statistics of the bacteriostatic effect of Pseudomonas bacteria LWB10 of the present invention on Agrobacterium tumefaciens

Test strains

C58

GV3101

LAB4404

EHA105

C58C1

NT1RE

LWB10

+++

In Table 1, + represents the diameter of the zone of inhibition <10mm, + represents the diameter of the zone of inhibition 10mm-20mm, and, + + + represents the diameter of the zone of inhibition >30 mm. The Agrobacterium tumefaciens strains listed in the tables are all disclosed in the literature.

The bacteriostatic effect shows that the pseudomonas morganii LWB10 has strong bacteriostatic activity on agrobacterium tumefaciens, and metabolites of the pseudomonas morganii LWB10 also contain a large amount of bacteriostatic active substances, so that the pseudomonas morganii LWB10 has deep development and application values as biocontrol bacteria of agrobacterium tumefaciens.

Materials, reagents and experimental equipment related to the embodiment of the invention are all commercial products meeting the field of plant protection unless specified otherwise.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, modifications and decorations can be made without departing from the core technology of the present invention, and these modifications and decorations shall also fall within the protection scope of the present invention. Any changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Sequence listing

<110> Ningbo city profession and technology college

<120> pseudomonas with bacteriostatic activity

<160>1

<170>SIPOSequenceListing 1.0

<210>2

<211>1407

<212>DNA

<213> Pseudomonas morganii (Pseudomonas moselii)

<400>2

acatgcagtc gagcggatga cgggagcttg ctccttgatt cagcggcgga cgggtgagta 60

atgcctagga atctgcctgg tagtggggga caacgtttcg aaaggaacgc taataccgca 120

tacgtcctac gggagaaagc aggggacctt cgggccttgc gctatcagat gagcctaggt 180

cggattagct agtaggtgag gtaatggctc acctaggcga cgatccgtaa ctggtctgag 240

aggatgatca gtcacactgg aactgagaca cggtccagac tcctacggga ggcagcagtg 300

gggaatattg gacaatgggc gaaagcctga tccagccatg ccgcgtgtgt gaagaaggtc 360

ttcggattgt aaagcacttt aagttgggag gaagggcagt aagttaatac cttgctgttt 420

tgacgttacc gacagaataa gcaccggcta actctgtgcc agcagccgcg gtaatacaga 480

gggtgcaagc gttaatcgga attactgggc gtaaagcgcg cgtaggtggt tcgttaagtt 540

ggatgtgaaa gccccgggct caacctggga actgcatcca aaactggcga gctagagtat 600

ggtagagggt ggtggaattt cctgtgtagc ggtgaaatgc gtagatatag gaaggaacac 660

cagtggcgaa ggcgaccacc tggactgata ctgacactga ggtgcgaaag cgtggggagc 720

aaacaggatt agataccctg gtagtccacg ccgtaaacga tgtcaactag ccgttggaat 780

ccttgagatt ttagtggcgc agctaacgca ttaagttgac cgcctgggga gtacggccgc 840

aaggttaaaa ctcaaatgaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 900

ttcgaagcaa cgcgaagaac cttaccaggc cttgacatgc agagaacttt ccagagatgg 960

attggtgcct tcgggaactc tgacacaggt gctgcatggc tgtcgtcagc tcgtgtcgtg 1020

agatgttggg ttaagtcccg taacgagcgc aacccttgtc cttagttacc agcacgtcat 1080

ggtgggcact ctaaggagac tgccggtgac aaaccggagg aaggtgggga tgacgtcaag 1140

tcatcatggc ccttacggcc tgggctacac acgtgctaca atggtcggta cagagggttg 1200

ccaagccgcg aggtggagct aatctcacaa aaccgatcgt agtccggatc gcagtctgca 1260

actcgactgc gtgaagtcgg aatcgctagt aatcgcaaat cagaatgttg cggtgaatac 1320

gttcccgggc cttgtacaca ccgcccgtca caccatggga gtgggttgca ccagaagtag 1380

ctagtctaac ctcggaggac ggtacca 1407

Claims

1. The pseudomonas with antibacterial activity is characterized in that the preservation number is CCTCC NO: m2019081 belonging to Pseudomonas morganii (M2019081)Pseudomonas mosselii）。

2. The process for producing a fermentation product of Pseudomonas as claimed in claim 1, wherein the steps comprise:

(1) inoculating pseudomonas into YEB liquid culture medium, and shake culturing at 28 deg.C for 3 days;

(2) then centrifuging the bacterial liquid and collecting the supernatant;

(3) extracting the supernatant with an equal volume of organic solvent;

(4) and concentrating and drying the organic phase obtained by extraction.

3. The process for producing a fermentation product of Pseudomonas as claimed in claim 2, wherein the dried product obtained in the step (4) is reconstituted with dimethyl sulfoxide to further purify the fermentation product; the rotating speed of the shaking table in the step (1) is 200-250 rpm; the centrifugation parameter in the step (2) is 8000-12000 rpm for 5-15 min.

4. The process for the preparation of a fermentation product of Pseudomonas according to claim 2, wherein the organic solvent in step (3) is ethyl acetate and is extracted in multiple steps, and further comprising the step of re-extracting the organic phase with 1/10 volumes of sterile distilled water after the ethyl acetate extraction.

5. The use of the pseudomonads of claim 1 for controlling agrobacterium tumefaciens and for preparing an agrobacterium tumefaciens antibacterial agent.

6. The fermentation product obtained by the preparation method of any one of claims 2 to 4 is applied to prevention and treatment of Agrobacterium tumefaciens and preparation of an Agrobacterium tumefaciens antibacterial drug.