CN112280714A

CN112280714A - Pseudomonas aeruginosa 8-7 and application thereof

Info

Publication number: CN112280714A
Application number: CN202011210825.8A
Authority: CN
Inventors: 戴利铭; 蔡志英; 李岚岚; 施玉萍; 刘一贤; 熊延林; 杨耀文; 吴忠华; 苏海鹏
Original assignee: Yunnan Institute of Tropical Crops
Current assignee: Yunnan Institute of Tropical Crops
Priority date: 2020-11-03
Filing date: 2020-11-03
Publication date: 2021-01-29
Anticipated expiration: 2040-11-03
Also published as: CN112280714B

Abstract

The invention discloses pseudomonas aeruginosa 8-7 and application thereof, and belongs to the technical field of microorganisms. The invention discloses pseudomonas aeruginosa 8-7 with the preservation number of CGMCC No. 20694. The pseudomonas aeruginosa 8-7 disclosed by the invention has good antagonistic action on various pathogenic bacteria, especially pathogenic bacteria of fungi imperfecti; the pseudomonas aeruginosa 8-7 has strong bacteriostasis to the pathogenic bacteria of the rubber tree clavospora leaf spot and the pestalotiopsis microsporus, and the bacteriostasis rates are 79.33 percent and 79.67 percent respectively; is obviously superior to the prior known antagonistic pseudomonas aeruginosa strains. Compared with the existing chemical synthesis bactericide for prevention and treatment, the pseudomonas aeruginosa 8-7 comes from the natural environment, has the advantages of high efficiency, low toxicity, no environmental pollution, difficult generation of drug resistance and the like, and has great biocontrol potential in the prevention and treatment aspects of corynespora polystachya and pestalotiopsis.

Description

Pseudomonas aeruginosa 8-7 and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to pseudomonas aeruginosa 8-7 and application thereof.

Background

The Corynespora hevea deciduous pathogen is Corynespora polytrichoides (Berk and cure.) Wei), belongs to deuteromycins (deuteromycinotina), Hyphomycetes (Hyphomycetes), Hyphomycetales (Hyphomycetales), darkling family (Dematiaceae), corynebacterium (corynebacterium) and is a plant pathogenic fungus with wide host range and worldwide distribution, and is also the earliest species found in the corynebacterium and the widest host range. Can infect 530 plants in 380 genera of Cucurbitaceae, Sapindaceae, Euphorbiaceae, Caricaceae, Malvaceae, Compositae, Solanaceae, Convolvulaceae, Amaranthaceae, Leguminosae, Araceae, Oleaceae and Meliaceae in tropical and subtropical regions.

Pestalotiopsis microspora (Pestalotiopsis microspora) is an anamorphic endophytic fungus of fungi imperfecti, and is a famous plant pathogen variety, such as leaf blight of Phoebe bournei, olive fruit rot, gray leaf spot of Douglas fir, leaf blight of oil palm seedlings, leaf blight of oil tea camellia, leaf blight of Jatropha curcas and the like. In a Yunnan macadamia nut planting base, diseases such as macadamia nut leaf blight, leaf fall and the like caused by pestalotiopsis microsporus are often found, so that the tree vigor gradually declines, and the yield of an orchard is reduced.

At present, chemical pesticides such as prochloraz emulsifiable solution, chlorothalonil wettable powder, benlate, chlorothalonil, mancozeb, thiophanate methyl and the like are mainly adopted for preventing and treating corynespora deciduous leaf disease and pestalotia trabeculosa leaf blight. The chemical pesticide is frequently used to cause the drug resistance of pathogenic bacteria, so that the control effect is weakened, and the long-term use causes serious pollution to soil and water resources, which affects the health of human beings; the research of the high-efficiency, environment-friendly and safe biological control method is imperative.

Pseudomonas spp is a kind of microorganism which is active in plant rhizosphere and leaf surface, the metabolite (especially antibiotic) produced by the Pseudomonas spp plays an important role in preventing and controlling crop diseases, and the antibiotic has the characteristics of high efficiency, low toxicity, easy degradation, no residue, environmental friendliness and the like. Among the numerous pseudomonas bacteria, pseudomonas aeruginosa (p. chrysogenis), pseudomonas fluorescens (p. fluorescens), and pseudomonas aeruginosa (p. aeruginosa) have been most isolated and studied extensively. The pseudomonas can prevent and treat tomato fusarium wilt, tobacco phytophthora blight, pepper phytophthora blight, sclerotinia rot of colza, cypress canker, bean anthracnose and the like. However, there are no international reports on the control of the pseudomonas aeruginosa preparation and the produced antibiotic thereof on the leaf spot of polyspora clavuligera and the leaf blight of pestalotiopsis.

Therefore, providing a pseudomonas aeruginosa strain 8-7 and application thereof is a problem which needs to be solved urgently by the technical personnel in the field.

Disclosure of Invention

In view of the above, the invention provides pseudomonas aeruginosa 8-7 and application thereof, which are used for inhibiting fungi imperfecti pathogenic bacteria: the Cladosporium polyspora and Pestalotiopsis microphylla are used for preventing and treating Cladosporium clavatum defoliating disease and Pestalotiopsis pseudopeyrodis leaf blight.

In order to achieve the purpose, the invention adopts the following technical scheme:

pseudomonas aeruginosa (Pseudomonas aeruginosa)8-7 with a preservation number of: CGMCC No.20694, which is deposited in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, is called CGMCC for short, and is deposited at the institute of microbiology, China academy of sciences, No.3, West Lu 1, North Cheng, south China, at the Beijing city, with the date of deposit being 09-21 days, 2020 years, and is classified and named as Pseudomonas aeruginosa.

Further, the application of the pseudomonas aeruginosa 8-7 in inhibiting fungi imperfecti.

Further, the fungi imperfecti pathogenic bacteria are polyspora and pestalotiopsis microsporus.

Further, the application of pseudomonas aeruginosa 8-7 in preventing and treating corynespora leaf drop disease.

Further, the application of the pseudomonas aeruginosa 8-7 in preventing and treating the bacterial leaf blight of the pestalotiopsis.

Further, the application of volatile substances generated by pseudomonas aeruginosa 8-7 in inhibiting fungi of fungi imperfecti is provided.

Further, the application of volatile substances generated by the pseudomonas aeruginosa 8-7 in inhibiting corynespora deciduous bacteria is provided.

According to the technical scheme, compared with the prior art, the invention discloses and provides the pseudomonas aeruginosa 8-7 and the application thereof, the pseudomonas aeruginosa 8-7 has good inhibition effect on the Corynespora cassiicola and the Pestalotiopsis microspora, the diameters of inhibition zones are 57.68 +/-0.66 mm and 66.25 +/-2.03 mm respectively, and the inhibition rates are 79.33% and 79.67% respectively; the results of the antibacterial spectrum test show that the strain 8-7 has broad-spectrum antibacterial activity, particularly has obvious inhibiting effect on fungi pathogenic bacteria of fungi of semi-known class, and the conjecture shows that the strain 8-7 has good application prospect in the field of biological control of fungi pathogenic bacteria of fungi of semi-known class. Volatile substances generated by pseudomonas aeruginosa 8-7 can inhibit pathogenic bacteria of fungi imperfecti.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a diagram showing the colony morphology of strains 8 to 7 of the present invention in LB medium;

FIG. 2 is a drawing showing the antagonistic action of the strain 8-7 of the present invention against Cladosporium clavatum deciduous bacteria;

wherein, A, control; b, antagonism of the strain 8-7;

FIG. 3 is a schematic diagram of a phylogenetic tree constructed in accordance with the present invention;

FIG. 4 is a graph showing the antagonistic effect of strains 8 to 7 of the present invention on Pestalotiopsis microspora (Pestalotiopsis microphylla); wherein, A, antagonistic action of the strain 8-7; CK, control.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Colletotrichum oxysporum DFMP1E (Colletotrichum acutum), Colletotrichum oxysporum MLZZP3(Colletotrichum acutum), Colletotrichum gloeosporioides (Colletotrichum gloeosporioides), Corynespora clava (Corynespora cassicola), Corynespora koshikoshikoensis (Photinus noxius), Petasites microphyllus (Pestalotiopsis microspora), Phytophthora rubber (Phytophthora heveae), Halisnobacter pentaerythraeum (Caloneriaceae), and Fusarium solani (Fusarium solani) were isolated from research center for the use of tropical microorganisms and plant protection in the scientific institute of Yunnan province crops. Bacteria Genomic DNA Kit: beijing kang is a century company; PCR primers: beijing Liu He Hua Dagen science and technology Co., Ltd; glucose, yeast extract, tryptone and other analytical reagents, bio-engineering, ltd; a PCR instrument: applied Biosystems, Inc.; electrophoresis apparatus, six biotech ltd, beijing; constant temperature incubator, Shanghai Boxun industries Co., Ltd.

EXAMPLE 1 isolation and screening of antagonistic bacteria

(1) Sample source

Collecting rhizosphere soil samples from rubber tree planting areas in Jinghong city, Xishuangbanna, Yunnan province, removing surface soil by a five-point sampling method, filling the soil with the depth of about 5-20cm into a sterile self-sealing bag, and taking the sterile self-sealing bag back to a laboratory for refrigeration at 4 ℃ for later use.

(2) Culture medium

LB liquid medium: 5g of yeast powder, 10g of tryptone, 10g of NaCl and 1L of water.

PDA medium (Potato dextrose agar): 200g of potatoes, 20g of glucose and 1000mL of water.

Gao's first medium: soluble starch 20g, KNO₃1 g、K₂HPO₄0.5 g、MgSO₄·7H₂O0.5g、NaCl 0.5g、FeSO₄·7H₂0.01g of O and 1000mL of water.

King's B: peptone 20g, K₂HPO₄1.5 g、MgSO₄1.5 g, 1000mL of water.

NA medium (nutriagar): 10g of tryptone, 3g of beef extract, 5g of NaCl and 1000mL of water.

Charles agar medium (Czapek): 2g of sodium nitrate, 1g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate, 0.5g of potassium chloride, 0.01g of ferric sulfate, 30g of sucrose, 15-20 g of agar and water with constant volume of 1000 mL. All media pH 7.2. + -. 0.2, solid media 20g agar/1000 mL, 1X 10⁵Sterilizing with Pa for 30 min.

(3) Isolation of antagonistic bacteria

And (4) screening the collected soil sample through a 100-mesh sieve. Weighing 1g of sieved soil sample, adding 1000mL of sterile water, oscillating at 25 ℃ and 180r/min for 30min, and sequentially diluting to 10^-3-10^-8g/L, and fully mixing. Adding 1mL of the diluted solution into LB culture medium cooled to 50 ℃, shaking up gently, culturing at 37 ℃ for 12h, picking different types of colonies, streaking and purifying on a new plate for culture.

(4) Antagonistic bacteria primary screen

Punching a plate of activated Corynespora deciduous (Corynespora cassiicola) by using a puncher with the diameter of 5mm, taking a bacterium block to inoculate in the center of a new PDA plate, inoculating different strains to be tested around the new PDA plate, culturing at the constant temperature of 28 ℃ for 5 days, selecting a bacterium colony with the effect of inhibiting the growth of pathogenic bacteria, streaking and storing, and numbering and recording.

(5) Antagonistic bacteria rescreening

And (3) measuring the antagonistic bacteria bacteriostasis efficiency by adopting a flat plate confronting method. A puncher with the diameter of 5mm is used for punching on the activated pathogenic bacteria flat plate, and pathogenic bacteria blocks are taken and placed in the center of a new PDA flat plate. Inoculating strains with antagonistic action at equal intervals around the strain block, repeating for 3 times with the culture dish inoculated with pathogenic bacteria as control, adjusting the temperature of the incubator to 28 deg.C, placing the plate in the incubator for 4-5 days, and taking out to measure the diameter of the pathogenic bacteria. And evaluating the antagonistic action of the antagonistic bacteria on the pathogenic bacteria by measuring the growth inhibition rate of the indicator bacteria. The antagonistic bacteria with the best inhibition effect is named as 8-7, and is mixed with 20% of glycerol and then stored at the temperature of-20 ℃. The colony morphology of the strain 8-7 on LB medium is shown in figure 1, and the antagonism to the Cladosporium clavatum deciduous bacteria is shown in figure 2.

EXAMPLE 2 identification of antagonistic bacteria

(1) And (3) colony morphology characteristics: inoculating antagonistic bacteria 8-7 on LB, PDA, Gao's first culture medium, NA, and King's B culture medium, culturing at 30 deg.C for 1d, observing colony morphology, and showing colony morphology on different culture medium in Table 1.

TABLE 1 cultural characteristics of strains 8-7

(2) Physiological and biochemical indexes: the physiological and biochemical indexes of the antagonistic bacteria, such as M.R test, V-P test, catalase test, starch hydrolysis, nitrate reduction, gelatin hydrolysis, denitrification, citrate utilization and the like, are measured by referring to the methods of 'common bacteria system identification manual' and 'Bergey bacteria identification manual'.

The results show that the antagonistic bacteria 8-7 screened by the invention can utilize D-glucose, citrate and methyl red, can reduce nitrate, is oxidase-positive, catalase-positive, and V-P test is negative, can not utilize starch, belongs to aerobic bacteria, can grow at 42 ℃, but can not grow at 4 ℃, and has the highest salt tolerance concentration of 5%.

(3)16Sr DNA Gene identification

8-7 strains are subjected to shaking culture in an LB culture medium at 30 ℃ and 180r/min until the logarithmic phase, and the strains are collected by centrifugation at 10000r/min for 5 min. Extracting thallus genome DNA by using the kit, and selecting primers 27F and 1492R for PCR amplification;

the specific primer sequences are as follows:

27F：5’-AGAGTTTGATCCTGGCTCAG-3’；SEQ ID NO.1；

1492R：5’-GGTTACCTTGTTACGACCCTT-3’；SEQ ID NO.2。

and (3) PCR reaction system: 2 × EsTaq Master Mix 25 μ L, genomic DNA 2 μ L, 27F and 1492R primers (10 μmol/L) each 1 μ L, plus ddH₂O to 50. mu.L. And (3) PCR reaction conditions: denaturation at 95 deg.C for 5 min; 30s at 95 ℃, 30s at 55 ℃ and 2min at 72 ℃ for 35 cycles; 10min at 72 ℃. The PCR product was purified and sequenced. And (3) comparing the sequencing result with a BLASTn program in a GenBank database, performing multiple sequence comparison and phylogenetic analysis through MEGA 7.0 software to determine the classification status of the strain, and constructing a phylogenetic tree by adopting a Neighbor-Joining method, wherein Bootstrap is set as 1000.

The species of the strain 8-7 is identified by 16Sr DNA identification, and the sequencing result is shown in SEQ ID NO. 3;

CAATTGATACCTCCGTGGTACCGTCCCCCTTGCGGTTAGACTAGCTACTTCTGGAGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGACATTCTGATTCACGATTACTAGCGATTCCGACTTCACGCAGTCGAGTTGCAGACTGCGATCCGGACTACGATCGGTTTTATGGGATTAGCTCCACCTCGCGGCTTGGCAACCCTTTGTACCGACCATTGTAGCACGTGTGTAGCCCTGGCCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCCTTAGAGTGCCCACCCGAGGTGCTGGTAACTAAGGACAAGGGTTGCGCTCGTTACGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTGTCTGAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTCAGCATGTCAAGGCCAGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTATCGCGTTAGCTGCGCCACTAAGATCTCAAGGATCCCAACGGCTAGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTCAGTGTCAGTATCAGTCCAGGTGGTCGCCTTCGCCACTGGTGTTCCTTCCTATATCTACGCATTTCACCGCTACACAGGAAATTCCACCACCCTCTACCGTACTCTAGCTCAGTAGTTTTGGATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCCAACTTGCTGAACCACCTACGCGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTTCGTATTACCGCGGCTGCTGGCACGAAGTTAGCCGGTGCTTATTCTGTTGGTAACGTCAAAACAGCAAGGTATTAACTTACTGCCCTTCCTCCCAACTTAAAGTGCTTTACAATCCGAAGACCTTCTTCACACACGCGGCATGGCTGGATCAGGCTTTCGCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGACTGATCATCCTCTCAGACCAGTTACGGATCGTCGCCTTGGTAGGCCTTTACCCCACCAACTAGCTAATCCGACCTAGGCTCATCTGATAGCGTGAGGTCCGAAGATCCCCCACTTTCTCCCTCAGGACGTATGCGGTATTAGCGCCCGTTTCCGGACGTTATCCCCCACTACCAGGCAGATTCCTAGGCATTACTCACCCGTCCGCCGCTGAATCCAGGAGCAAGCTCCCTTCATCCGCTCGACTCGCATGGTAGCATCGCTA；SEQ ID NO.3。

the phylogenetic tree constructed based on the 16Sr DNA sequencing results is shown in FIG. 3; the full length of the 16Sr DNA sequence of the strain is 1435bp, and the homology of the 16Sr DNA sequence of the Pseudomonas aeruginosa reaches 99 percent. The strain is judged to belong to the pseudomonas aeruginosa (P.aeruginosa) by combining the sequence analysis, morphological characteristics, culture characteristics and physical and chemical characteristics of the strain 8-7 and the common bacteria system identification manual, and is named as the pseudomonas aeruginosa (P.aeruginosa) 8-7.

(4) Determination of antagonistic Strain 8-7 antibiogram

Taking Colletotrichum oxysporum DFMP1E (Colletotrichum acutum), Colletotrichum oxysporum MLZZP3(Colletotrichum acutum), Colletotrichum gloeosporioides (Colletotrichum gloeosporioides), Corynespora deciduous fungus (Corynespora cassicola), brown root of rubber tree (Photinus noxius), Petasium microphyllum (Pestalotiopsis microspora), Phytophthora rubber (Phytophthora heae), Chinesium pentaseptate (Calonetiaceae), and Fusarium solani (Fusarium solani) as the pathogenic bacteria, simultaneously inoculating antagonistic bacteria and pathogenic bacteria on a flat plate, taking the treatment of inoculating only the indicator bacteria as a control, repeating three times, culturing at 28 ℃ for 5d, measuring the diameter of a circle, observing the antibacterial condition and taking a picture in an incubator; among them, the antagonistic action of the strain 8-7 against Pestalotiopsis microspora (Pestalotiopsis microphylla) is shown in FIG. 4. The growth inhibition ratios of the 8-7 strains against different pathogenic bacteria [ inhibition ratio (%) - (control group colony growth diameter-treatment group colony growth diameter)/(control group colony growth diameter-inoculated block diameter) × 100%) were measured, and the antibacterial broad spectrum of antagonistic bacteria was evaluated, and the results are shown in table 2.

TABLE 2 inhibition of 9 pathogenic fungi by strains 8-7

Note: different lower case english letters indicate that the same column data differed significantly at the P <0.05 level.

Table 2 shows that strains 8 to 7 have inhibitory effects on 9 pathogenic fungi tested, Colletotrichum acutum DFMP1E, Colletotrichum acutum MLZZP3, Colletotrichum gloeosporides, Corynespora casilicola, Phellinus noxius, Pestalotiopsis microspora, Phytophtora heveae, Calonectiapentaeta, and Fusarium solani, wherein the inhibitory effects on Corynespora casicicola and Pestalotiopsis microspora are the best, the inhibitory effects on 57.68 + -0.66 and 66.25 + -2.03 mm, respectively, and secondly, the inhibitory effects on both Calipentana and Phellinus noxius pathogens are the most weak. The result of an antibacterial spectrum test shows that the strain 8-7 has broad-spectrum antibacterial activity, particularly has obvious inhibiting effect on fungi pathogenic bacteria of fungi of semi-known, and the strain 8-7 is presumed to have good application prospect in the field of biocontrol of fungi pathogenic bacteria of fungi of semi-known.

EXAMPLE 3 Effect of volatile products of strains 8-7 on Cladosporium clavatum

Single colony of 8-7 strains is selected and inoculated in 50mL LB liquid culture medium, and cultured for 12h at 37 ℃ and 180 r/min. Taking 50 μ L of cultured fermentation broth, uniformly coating on LB plate, placing a piece of Corynespora clavuligerus with diameter of 5mm in the center of PDA culture medium, covering the latter on the former, sealing the contact position of the two dishes with sealing film, and repeating each treatment for 3 times by using blank LB plate as control. And (3) carrying out dark culture at 30 ℃, measuring the colony diameters of the control and treated corynespora deciduous leaf bacteria when the mycelium of the control group grows to 2/3 culture dishes, and calculating the inhibition rate.

The result shows that when the average colony diameter of the hypha of the control corynespora deciduous leaf bacteria reaches 67.34mm, the average colony diameter of the hypha of the corynespora deciduous leaf bacteria in the experimental group is 36.98mm, and the average inhibition rate reaches 48.70%, which indicates that 8-7 strains can produce volatile substances and the substances have good antagonistic action on the corynespora deciduous leaf bacteria.

EXAMPLE 4 Effect of exogenous Nutrition bacteriostatic Activity of volatile substances of strains 8-7

The method comprises the steps of taking the corynespora deciduous leaf bacteria as indicator bacteria, taking 6 different culture media LB, NA, Gauss, Kirschner B, Chachi and PDA as nutrient sources, and determining the inhibition rate of volatile substances on the bacteria according to the growth rate of hyphae by adopting a double-dish buckling method. Activating the strain 8-7 in an LB culture medium, selecting a single colony, inoculating the single colony in 50mL of LB liquid culture medium, and culturing at 37 ℃ and 180r/min for 12 h. 50 μ L of the cultured fermentation broth was applied evenly to the bottom of a petri dish (diameter 9cm) containing the 6 solid media described above. And (3) selecting pathogenic bacteria blocks (with the diameter of 5mm) to inoculate in a culture dish cover containing a PDA culture medium, then buckling the dish cover inoculated with the fungi with the bottom of a dish coated with the bacteria, sealing by using a sealing film, and culturing at constant temperature of 30 ℃. Using a culture medium without inoculating bacterial fermentation liquor as a control, when the mycelium of the control group grows to 2/3 culture dishes, measuring the colony diameter by adopting cross, calculating the result according to an inhibition rate formula, and repeating the treatment for 3 times, wherein the result is shown in table 3. Evaluating the action activity of volatile substances produced under 8-7 under different nutritional conditions by using the inhibition rate, and screening an optimal culture medium for producing antibacterial volatile substances.

As a result, the strains 8-7 can grow on LB, NA, Gauss, King's B and PDA, but cannot grow on the Chachien medium; and 8-7 of the strain can generate volatile substances on LB, NA, Gauss, King's B and PDA, the antagonistic effect displayed on an LB culture medium is strongest, and the bacteriostasis rate reaches 48.69%. Therefore, LB medium was selected as the optimal medium for volatile substances production by strains 8-7.

TABLE 3 inhibitory Effect of volatile substances of strains 8 to 7 on Cladosporium clavatum L.on different media

Example 5 GC-MS identification of volatile substances of strains 8-7

Preparing LB slant culture medium in 20mL sterilized headspace bottle, coating 50 μ L strain 8-7 seed solution on the slant culture medium, rapidly sealing with bottle cap with rubber pad, sealing the outer layer of the bottle cap with sealing film, culturing in 30 deg.C biochemical incubator for 48h, and repeating for 3 times each treatment with the un-inoculated slant culture medium as blank control. The volatiles in the headspace were extracted using a solid phase microextraction process. The extraction head used 50/30 μm DVB/CAR/PDMS. A chromatographic column: DB-5MS (30m 250 μm 0.25 μm); chromatographic conditions are as follows: a non-shunting mode; temperature rising procedure: maintaining at 50 deg.C for 2min, increasing to 100 deg.C at 4 deg.C/min, maintaining for 1min, increasing to 200 deg.C at 10 deg.C/min, maintaining for 1min, increasing to 250 deg.C at 25 deg.C, and maintaining for 5 min; the temperature of a gas phase injection port is 250 ℃; carrier gas: helium gas. Mass spectrum conditions: the ion source temperature is 230 ℃, the quadrupole rod temperature is 150 ℃, the ionization mode EI is adopted, the electron energy is 70eV, and the scanning mass range is 35-600 m/z.

The mass spectra of the resulting gas fractions were compared with the international standard database (Library of National Institute of standard and Technology, NIST) respectively for a matching rate of > 85%, and after identification of the volatile substance fraction, the volatile substance fraction was determined by removing the compound fractions present in both the control and treatment.

And (3) performing substance matching analysis on the detected volatile substances according to a NIST standard library, removing substances shared in LB culture medium treatment and strain 8-7+ LB culture medium treatment, and analyzing the volatile substances obtained by extraction and identification to obtain 35 volatile substances comprising amines, alcohols, alkenes, phenols, esters, pyrazines and the like, which are detailed in Table 4.

TABLE 4 volatile substance fractions of strains 8-7

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> scientific research institute for tropical crops in Yunnan province

<120> pseudomonas aeruginosa 8-7 strain and application thereof

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agagtttgat cctggctcag 20

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caattgatac ctccgtggta ccgtccccct tgcggttaga ctagctactt ctggagcaac 60

ccactcccat ggtgtgacgg gcggtgtgta caaggcccgg gaacgtattc accgtgacat 120

tctgattcac gattactagc gattccgact tcacgcagtc gagttgcaga ctgcgatccg 180

gactacgatc ggttttatgg gattagctcc acctcgcggc ttggcaaccc tttgtaccga 240

ccattgtagc acgtgtgtag ccctggccgt aagggccatg atgacttgac gtcatcccca 300

ccttcctccg gtttgtcacc ggcagtctcc ttagagtgcc cacccgaggt gctggtaact 360

aaggacaagg gttgcgctcg ttacgggact taacccaaca tctcacgaca cgagctgacg 420

acagccatgc agcacctgtg tctgagttcc cgaaggcacc aatccatctc tggaaagttc 480

tcagcatgtc aaggccaggt aaggttcttc gcgttgcttc gaattaaacc acatgctcca 540

ccgcttgtgc gggcccccgt caattcattt gagttttaac cttgcggccg tactccccag 600

gcggtcgact tatcgcgtta gctgcgccac taagatctca aggatcccaa cggctagtcg 660

acatcgttta cggcgtggac taccagggta tctaatcctg tttgctcccc acgctttcgc 720

acctcagtgt cagtatcagt ccaggtggtc gccttcgcca ctggtgttcc ttcctatatc 780

tacgcatttc accgctacac aggaaattcc accaccctct accgtactct agctcagtag 840

ttttggatgc agttcccagg ttgagcccgg ggatttcaca tccaacttgc tgaaccacct 900

acgcgcgctt tacgcccagt aattccgatt aacgcttgca cccttcgtat taccgcggct 960

gctggcacga agttagccgg tgcttattct gttggtaacg tcaaaacagc aaggtattaa 1020

cttactgccc ttcctcccaa cttaaagtgc tttacaatcc gaagaccttc ttcacacacg 1080

cggcatggct ggatcaggct ttcgcccatt gtccaatatt ccccactgct gcctcccgta 1140

ggagtctgga ccgtgtctca gttccagtgt gactgatcat cctctcagac cagttacgga 1200

tcgtcgcctt ggtaggcctt taccccacca actagctaat ccgacctagg ctcatctgat 1260

agcgtgaggt ccgaagatcc cccactttct ccctcaggac gtatgcggta ttagcgcccg 1320

tttccggacg ttatccccca ctaccaggca gattcctagg cattactcac ccgtccgccg 1380

ctgaatccag gagcaagctc ccttcatccg ctcgactcgc atggtagcat cgcta 1435

Claims

1. Pseudomonas aeruginosa (Pseudomonas aeruginosa)8-7, characterized in that the preservation number is as follows: CGMCC No. 20694.

2. The use of a strain of pseudomonas aeruginosa 8-7 as claimed in claim 1 for inhibiting fungi pathogenic in fungi imperfecti.

3. The application of the pseudomonas aeruginosa 8-7 in inhibiting fungi imperfecti according to claim 2, wherein the fungi imperfecti is selected from the group consisting of corynebacterium polyspora and pestalotiopsis microsporus.

4. The application of the pseudomonas aeruginosa 8-7 as claimed in claim 1 in preventing and treating corynespora leaf drop disease.

5. The application of the pseudomonas aeruginosa 8-7 as claimed in claim 1 in controlling the bacterial leaf blight of pestalotiopsis.

6. Use of the volatile substance produced by a strain of pseudomonas aeruginosa 8-7 according to claim 1 for inhibiting pathogenic bacteria of the fungi imperfecti.

7. Use of the volatile substance produced by a strain of pseudomonas aeruginosa 8-7 according to claim 1 for inhibiting corynespora deciduous bacteria.