CN115975886B - Novel pseudomonas and application thereof in crop bacterial disease control - Google Patents

Novel pseudomonas and application thereof in crop bacterial disease control Download PDF

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CN115975886B
CN115975886B CN202211689593.8A CN202211689593A CN115975886B CN 115975886 B CN115975886 B CN 115975886B CN 202211689593 A CN202211689593 A CN 202211689593A CN 115975886 B CN115975886 B CN 115975886B
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pseudomonas
canker
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pathogenic bacteria
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CN115975886A (en
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朱红惠
刘阳
段娇
姚青
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Institute of Microbiology of Guangdong Academy of Sciences
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Institute of Microbiology of Guangdong Academy of Sciences
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Abstract

The invention discloses a novel pseudomonas strain and application thereof in crop bacterial disease control. The strain is a potential new species of Pseudomonas and named Pseudomonas sp.F0096, and is deposited in the microorganism strain collection of Guangdong province at the month 2 of 2022, and the deposition number is GDMCC No:63027. The novel pseudomonas F0096 can inhibit the growth of pathogenic bacteria of bacterial diseases of various crops in a high-efficiency broad-spectrum manner, and can generate siderophores. Therefore, the novel pseudomonas F0096 has good application potential in the aspects of crop bacterial disease control, crop iron absorption promotion and the like.

Description

Novel pseudomonas and application thereof in crop bacterial disease control
Technical Field
The invention relates to the technical field of microorganisms, in particular to novel pseudomonas and application thereof in crop bacterial disease control.
Background
Crop bacterial diseases refer to diseases caused by bacterial pathogen infection. Bacterial diseases are easy to attack under high-temperature and high-humidity conditions, often cause plant rot, wilting, fading, spots and other symptoms, seriously affect the yield and quality of crops, and cause great loss to agricultural production. The loss caused by bacterial disease of the main crop worldwide accounts for about 20-40% of the total crop yield, with direct economic losses of billions of dollars per year, based on incomplete statistics. The Chinese is a large population country, and the demand of grains is very large. At present, bacterial diseases of crops have seriously affected the growth of crops in China and threaten the grain safety in China. In order to cope with increasingly serious bacterial diseases of crops, a large number of chemical pesticides are used. But create a series of problems due to the unreasonable use of long-term, high doses. The use of chemical pesticides causes a wide range of pesticide residue problems, and causes serious harm to the quality and safety of agricultural products. Meanwhile, the use of chemical pesticides also causes serious environmental pollution problems, pollutes the atmosphere, soil, water and the like, and threatens the health of human beings. In addition, the use of chemical pesticides also causes the generation of drug resistance and drug tolerance of crop pathogenic bacteria, and seriously reduces the action effect of the chemical pesticides. Therefore, it is of great importance to develop a new control method for bacterial diseases.
In recent years, biological control methods for controlling plant diseases by using biological interactions and bacteria, fungi and other organisms have the advantages of no residue, no pollution, environmental friendliness and the like, and development and utilization of the biological control methods are attracting attention of more researchers. Among biocontrol bacteria, pseudomonas is an important group, is widely distributed in nature, is fast in propagation, has strong colonization capability and simple nutrition requirement, and has the functions of inhibiting various plant diseases and promoting plant growth. Pseudomonas is also an important bacterium in rhizosphere and phyllosphere, and plays an important role in maintaining the microbial ecological balance of the rhizosphere and phyllosphere of crops. The most recently reported Pseudomonas bacteria with biocontrol effect include Pseudomonas fluorescens, pseudomonas aeruginosa and Pseudomonas aeruginosa. Pseudomonas fluorescens can produce a large amount of secondary metabolites inhibiting plant pathogenic bacteria, such as ampelopsis grossedentata, nitropyrrolin and 2, 4-diacetyl gamboge, so that the biological control effect is good. Pseudomonas aeruginosa can also secrete a plurality of active products antagonizing plant pathogenic bacteria, and can prevent and treat a plurality of plant fungi and bacterial diseases. Pseudomonas aeruginosa can produce phenazine-1 carboxylic acid and inhibit wheat take-all pathogen, cucumber anthracnose, rice sheath blight, pepper epidemic disease and the like. However, the pseudomonas with broad spectrum for preventing and treating bacterial diseases of crops such as citrus canker, bacterial wilt, tomato canker, fruit tree canker and the like is not reported at present.
Disclosure of Invention
It is a first object of the present invention to overcome the disadvantages and shortcomings of the prior art and to provide a potential novel species of Pseudomonas, pseudomonas sp.) F0096. The strain was deposited at the cantonese microbiological strain collection center (GDMCC), address: 30, building 5, post code of 30 # 100 college in Guangzhou city View area martyr: 510070, the preservation number is GDMCC No:63027, and the preservation date is 2022, 12 months and 2 days.
The morphological characteristics of the Pseudomonas F0096 are as follows: gram staining was negative, single colony was round, yellow, smooth, opaque, and irregular at the edges.
The second object of the invention is to provide the application of the novel pseudomonas F0096 in preparing medicines for preventing and treating citrus canker, bacterial wilt, tomato canker and fruit tree canker and/or microbial agents or fertilizer additives for promoting iron absorption of crops.
A third object of the present invention is to provide a microbial preparation for controlling canker of citrus, comprising the above Pseudomonas F0096 as an active ingredient.
The fourth object of the present invention is to provide a microbial preparation for controlling bacterial wilt, comprising the above Pseudomonas F0096 as an active ingredient.
A fifth object of the present invention is to provide a microbial preparation for controlling tomato canker, comprising the above Pseudomonas F0096 as an active ingredient.
The sixth object of the present invention is to provide a microbial preparation for controlling canker of fruit trees, which contains the above Pseudomonas F0096 as an active ingredient.
The seventh object of the present invention is to provide a microbial live bacteria preparation for promoting iron absorption of crops, which contains the above Pseudomonas F0096 as an active ingredient.
An eighth object of the present invention is to provide an active substance for inhibiting pathogenic bacteria of citrus canker, pathogenic bacteria of bacterial wilt, laurella of Solanaceae, pathogenic bacteria of tomato canker, corynebacterium michiganense, pathogenic bacteria of fruit tree canker, pseudomonas syringae, and a fermentation broth derived from the above Pseudomonas F0096, which is used for preparing an inhibitor, a pharmaceutical agent, etc. for preventing and treating citrus canker, bacterial wilt, tomato canker, and fruit tree canker.
The invention also provides a method for preventing and treating citrus canker, bacterial wilt, tomato canker and fruit tree canker, which is to spray pseudomonas F0096 to the area to be prevented and treated.
Compared with the prior art, the invention has the following advantages and effects:
The invention reports a potential new species in pseudomonas for the first time, has good control effect on citrus canker, bacterial wilt, tomato canker and fruit tree canker, and can generate siderophores to promote iron absorption of crops. The first discovery of the strain enriches the beneficial microorganism resources for biocontrol growth promotion in China, has the advantages of stable antibacterial effect, high efficiency and environmental friendliness, and has good application prospect in the aspects of preventing and controlling crop bacterial diseases, promoting crop growth and the like.
Pseudomonas sp.f0096, deposited at the cantonese collection of microorganisms and strains (GDMCC), address: 30, building 5, post code of 30 # 100 college in Guangzhou city View area martyr: 510070, the preservation number is GDMCCNo:63027, and the preservation date is 2022, 12 months and 2 days.
Drawings
FIG. 1 shows colony morphology of novel Pseudomonas F0096 on NA medium.
FIG. 2 is a novel P.sp.F 0096 system genome developmental tree.
FIG. 3 shows the growth curve of novel Pseudomonas F0096 in NA broth.
FIG. 4 shows the inhibitory effect of novel Pseudomonas F0096 on the pathogenic bacteria Xcc of citrus canker.
FIG. 5 shows the inhibitory effect of novel Pseudomonas F0096 on bacterial wilt-pathogenic bacteria Laurella of Solanaceae.
FIG. 6 shows the inhibitory effect of novel Pseudomonas F0096 on Corynebacterium michiganensis, the pathogenic bacteria of tomato canker.
FIG. 7 shows the inhibitory effect of novel Pseudomonas F0096 on Pseudomonas syringae, the pathogenic bacteria of fruit tree canker.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Example 1: isolation and characterization of novel Pseudomonas F0096
Healthy fruit trees in citrus orchards in the urban and urban mouth service area (N25 DEG 18 '33', E113 DEG 43 '48') of Shaoguan city, shaoguan, cheng county, guangdong are selected, fresh citrus leaves are cut out by using sterile scissors and placed in sterile sampling bags. Subsequently, the leaf samples were stored in a 4 ℃ sampling box. After bringing the sample back to the laboratory, 10g of the leaf was weighed in an ultra clean bench, added to a 500mL triangular flask containing 190mL of sterile PBS buffer (0.2 mol/L, ph=7.0), shaken well, and placed on a constant temperature shaker at 28 ℃, and shaken at 200rpm for 30min. Subsequently, the triangular flask was placed in an ultrasonic vibrator at 40KHz for 8s with 2s intervals and sonicated for 10min. Finally, 1mL of eluent is taken and added into a test tube filled with 9mL of PBS buffer, the eluent is diluted to 10 -1、10-2、10-3 and 10 -4 in sequence, 100 mu L of each of eluent stock solution, 10 -1、10-2、10-3 and 10 -4 diluted samples are respectively coated on TSA culture medium (Qingdao sea Bo, product number HB 0177), and the mixture is placed in a biochemical incubator at 28 ℃ for 7 days. By observation, depending on the size, color, degree of wetting, smoothness of colonies and whether halos are produced, different single colonies were picked from the medium onto new TSA medium and purified by streaking multiple times until pure culture was obtained. The purified strain was numbered and was mixed with 25% glycerol (v/v) and stored in an ultra-low temperature refrigerator, thereby obtaining strain F0096.
The bacterial strain F0096 has gram staining negative, the bacterial colony morphology of the bacterial strain when being cultured on the R2A culture medium for 4 days is shown in the attached figure 1, and the bacterial colony is round, has the diameter of 2-8 mm, is yellow, has smooth surface, is opaque and has irregular edges.
Example 2: 16S rRNA gene sequence analysis of novel Pseudomonas F0096
Genomic DNA of strain F0096 was extracted and purified using HiPure bacterial DNA extraction kit (cat No. D3146-03, guangzhou Mei-Biotechnology Co., ltd.). The bacterial 16S rRNA gene amplification universal primers 27F/1492R, namely 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were used to amplify the 16S rRNA gene sequence of strain F0096 using purified genomic DNA as template. After amplification, a proper amount of PCR products are taken for gel electrophoresis detection, and under the condition that the existence of a target band is determined, the rest PCR products are sent to Suzhou gold and other intelligent biotechnology limited company for sequencing, and the sequence is shown as SEQ ID NO.1, and the length is 1532bp. The 16S rRNA gene sequence of strain F0096 was submitted to EzBioCloud database (www.ezbiocloud.net) for sequence homology alignment. The comparison result shows that: the highest similarity of strain F0096 to the 16SrRNA gene sequence of model strain Pseudomonas fulva NBRC 16637 T (accession number, BBIQ 01000036) was 99.66%; similarity to the 16S rRNA gene sequence of Pseudomonas parafulva NBRC 16636 T (accession number BBIU 01000051) was 99.52%; the similarity of the 16S rRNA gene with other model strains of Pseudomonas is lower than 99.00%. These analyses showed that: strains F0096 had a closest phylogenetic relationship with Pseudomonas fulva and Pseudomonas parafulva. However, the similarity of the 16S rRNA gene sequences of the strains F0096 and the model strains of two species Pseudomonas fulva and Pseudomonas parafulva is close, and the classification status of the strain F0096 cannot be accurately determined only by 16S rRNA gene sequencing analysis at present.
Example 3: novel whole gene sequence analysis of pseudomonas F0096
The genome of the novel Pseudomonas F0096 extracted in example 2 was used and sent to Shanghai Meiji Biotechnology Co.Ltd. Sequencing was performed by constructing an Illumina PE (-400 bp) library using the Illumina Novaseq platform. After quality testing of the original Reads, the obtained high quality data were subjected to genome sequence splicing using SPAdes v 3.15.3. And filtering to remove low-quality contig sequences according to the fact that the coverage of spliced contigs is more than or equal to 100 and the length is more than or equal to 500bp as parameters. After removal of low quality sequences, quality control was performed on the strain F0096 genome using software CheckM v 1.1.3. The results show that: the strain F0096 genome has an integrity of 99.30% and a pollution degree of 0.48%. Typically, the genome is considered to be of high quality when its integrity is greater than or equal to 95% and contamination is less than or equal to 5%. Therefore, the genome of the strain F0096 is a high-quality genome, and the accuracy of analysis can be ensured. The genome of strain F0096 was analyzed using software QUAST v 5.0.2. The results show that: the genome sequence of the strain F0096 contains 44 contigs, the total length of the genome is 5013616bp, the length of N50 is 235436bp, and the content of the genomic DNA G+C is 63.59%.
70% Of the digitized DNA-DNA hybridization values (DIGITAL DNA-DNA hybridization, dDDH) are the gold standard for the classification and identification of prokaryotes. The Genome sequence of strain F0096 was calculated using an online tool Genome-to-Genome Distance Calculator 3.0.0 (https:// ggdc. Dsmz. De/ggdc. Php#) to be compared to the Genome sequences of the closely related species model strain Pseudomonas fulva NBRC 16637 T (Genome accession number: BBIQ 00000000) and Pseudomonas parafulva NBRC 16636 T (Genome accession number: BBIU 000000000000) for the values of dDDH, 24.3 and 25.1 respectively, both dDDH values being below the 70% species division criteria, indicating that the two species of strain F0096 are different from those of Pseudomonas fulva and Pseudomonas fulva. The genomic phylogenetic tree of strain F0096 and the kindred reference model strain was constructed using software UBCG v 3.0.0. As a result, strain F0096 forms an independent phylogenetic branch, and the self-expanding support value is 100, which indicates that strain F0096 has independent classification status, as shown in FIG. 2. The strain F0096 is a potential new species in the genus Pseudomonas, which is thus designated as Pseudomonas sp.) F0096, combined with dDDH and the results of systematic genomic developmental analysis. The strain was deposited at the cantonese microbiological strain collection center (GDMCC), address: 30, building 5, post code of 30 # 100 college in Guangzhou city View area martyr: 510070, the preservation number is GDMCC No:63027, and the preservation date is 2022, 12 months and 2 days.
Example 4: novel pseudomonas F0096 growth performance identification
Inoculating novel pseudomonas F0096 into NB liquid culture medium for activation, culturing for 24 hours, and collecting fermentation liquor as seed liquor. 180. Mu.L of NB liquid medium was added to each 96-well cell culture plate as basal medium, then 20. Mu.L of seed solution and NB liquid medium (as control) were added to each well, 3 replicates were set, and after shaking, they were placed on a thermostatic shaker at 28℃and incubated at 180rpm for 48h. During the incubation, the absorbance of OD600 of each well was measured every two hours using a spectrophotometer. As shown in figure 3, after the novel pseudomonas F0096 is inoculated in NB liquid medium, 0-2 h is a growth delay period of the strain F0096, and the number of cells is not obviously increased because the cells need to adapt to new growth environments. The bacterial strain F0096 grows in logarithmic phase in 2-10h, and the cell number of the bacterial strain is obviously increased. After 10h, the growth of strain F0096 enters stationary phase until the end of the test. The measurement result of the growth performance of the novel pseudomonas F0096 shows that: the strain F0096 has the advantages of high growth speed, high metabolic capability, short fermentation period and simple culture conditions, has the basic characteristics of excellent production strains, and is a strain with excellent potential fermentation performance.
Example 5: identification of inhibition of novel pseudomonas F0096 on citrus canker pathogenic bacteria
The inhibition of the pathogenic bacteria of citrus canker by the novel Pseudomonas F0096 is determined by a double-layer flat-plate agar diffusion method. The pathogenic bacteria Xcc of citrus canker and the novel pseudomonas F0096 are inoculated to NB liquid culture medium for activation, and fermentation liquid is respectively collected after 24 hours of culture. The bottom NA culture medium (agar powder content 1.6%) was prepared, and the thickness of the culture medium was moderate. Oxford cups were placed on the bottom NA medium, 3 per plate. 1mL of Xcc fermentation broth is taken, 300mL of NA culture medium (agar powder content 1%) is added, the final concentration of Xcc is 10 6~107 cfu/mL, and after solidification, an oxford cup is taken out, namely the upper layer culture medium. 30 mu L of novel pseudomonas F0096 fermentation broth is taken and added into the hole, and is cultured for 36 hours at 28 ℃, and the diameter of the inhibition zone is measured by using a vernier caliper.
As shown in FIG. 4, after the addition of the novel Pseudomonas F0096, the citrus canker pathogenic bacteria Xcc formed clear transparent rings around the holes, with diameters of 22.96, 21.69 and 22.31mm, respectively, and an average value of 23.32mm. The results show that: the novel pseudomonas F0096 has remarkable inhibiting effect on the growth of pathogenic bacteria Xcc of citrus canker.
Example 6: identification of inhibition of novel pseudomonas F0096 on bacterial wilt pathogens
The inhibition of bacterial wilt pathogenic bacteria by novel pseudomonas F0096 is determined by a double-layer flat-plate agar diffusion method. Bacterial wilt pathogenic bacteria of the Solanaceae Laue fungus GMI 1000 and novel Pseudomonas F0096 are inoculated to NB liquid culture medium for activation, and fermentation liquid is respectively collected after 24 hours of culture. The bottom NA culture medium (agar powder content 1.6%) was prepared, and the thickness of the culture medium was moderate. Oxford cups were placed on the bottom NA medium, 3 per plate. 1mL of GMI 1000 fermentation broth is taken, 300mL of NA culture medium (agar powder content 1%) is added, the final concentration of GMI 1000 is 10 6~107 cfu/mL, and after solidification, an oxford cup is taken out, namely the upper layer culture medium. 30 mu L of novel pseudomonas F0096 fermentation broth is taken and added into the hole, and is cultured for 36 hours at 28 ℃, and the diameter of the inhibition zone is measured by using a vernier caliper.
As shown in FIG. 5, after the addition of novel Pseudomonas F0096, bacterial wilt pathogenic bacteria, L.solanaceae, GMI 1000, formed clear transparent rings around the wells with diameters of 24.36, 23.27 and 24.08mm, respectively, and average value of 23.90mm. The results show that: the novel pseudomonas F0096 has remarkable inhibition effect on the growth of bacterial wilt pathogenic bacteria Laurella GMI 1000 of Solanaceae.
Example 7: identification of inhibition of novel pseudomonas F0096 on tomato canker pathogenic bacteria
The inhibition of the bacterial pathogen of tomato canker by Pseudomonas F0096 was determined by double-layer plate agar diffusion. The pathogenic bacteria of tomato canker, namely corynebacterium michiganensis GDMCC 1.858 and novel pseudomonas F0096, are inoculated into NB liquid culture medium for activation, and fermentation liquor is respectively collected after 24 hours of culture. The bottom NA culture medium (agar powder content 1.6%) was prepared, and the thickness of the culture medium was moderate. Oxford cups were placed on the bottom NA medium, 3 per plate. 1mL GDMCC 1.858 fermentation liquor is taken and added with 300mL NA culture medium (agar powder content 1%), the final concentration of GDMCC 1.858 is 10 6~107 cfu/mL, and after solidification, an oxford cup is taken out, namely the upper layer culture medium. 30 mu L of novel pseudomonas F0096 fermentation broth is taken and added into the hole, and is cultured for 36 hours at 28 ℃, and the diameter of the inhibition zone is measured by using a vernier caliper.
As shown in FIG. 6, after the addition of novel Pseudomonas F0096, corynebacterium michiganensis GDMCC1.858, a pathogenic bacterial species of tomato canker, formed clear transparent circles around the wells with diameters of 20.42, 17.89 and 19.13mm, respectively, with an average value of 19.14mm. The results show that: the novel pseudomonas F0096 has remarkable inhibiting effect on the growth of corynebacterium michiganensis GDMCC1.858 which is pathogenic to tomato canker.
Example 8: identification of inhibition of novel pseudomonas F0096 on fruit tree canker pathogenic bacteria
The inhibition of the novel Pseudomonas F0096 on the pathogenic bacteria of the canker of the fruit tree is measured by adopting a double-layer flat-plate agar diffusion method. The pathogenic bacteria of the canker of the fruit tree, namely pseudomonas syringae GDMCC 1.330.330 and novel pseudomonas F0096, are inoculated into NB liquid culture medium for activation, and fermentation liquid is respectively collected after 24 hours of culture. The bottom NA culture medium (agar powder content 1.6%) was prepared, and the thickness of the culture medium was moderate. Oxford cups were placed on the bottom NA medium, 3 per plate. 1mL GDMCC 1.330.330 fermentation broth is taken, 300mL NA culture medium (agar powder content 1%), the final concentration of GDMCC 1.330.330 is 10 6~107 cfu/mL, and the oxford cup is taken out after solidification, namely the upper layer culture medium. 30 mu L of novel pseudomonas F0096 fermentation broth is taken and added into the hole, and is cultured for 36 hours at 28 ℃, and the diameter of the inhibition zone is measured by using a vernier caliper.
As shown in FIG. 7, after the novel Pseudomonas F0096 is added, the pathogenic bacteria of fruit tree canker, pseudomonas syringae GDMCC 1.330.330, form clear transparent rings around the holes, the diameters of which are 15.03, 15.55 and 15.64mm respectively, and the average value of which is 15.41mm. The results show that: the novel pseudomonas F0096 has remarkable inhibition effect on the growth of pseudomonas syringae GDMCC 1.330.330 which is the pathogenic bacteria of fruit tree canker.
Example 9: novel pseudomonas F0096 siderophore identification
The novel Pseudomonas F0096 is inoculated in NB liquid culture medium, cultured for 36h at 30 ℃ and 200rpm, 1mL of fermentation broth is sucked into a 1.5mL centrifuge tube, and centrifuged for 2min at 12000 rpm. 100. Mu.L of the supernatant was pipetted into a 96-well plate while adding 100. Mu.L of CAS detection solution and incubated in an incubator at 28℃for 1h. The absorbance at 630nm was measured by an enzyme-labeled instrument and was designated as OD A. For the blank, 100. Mu.L of NB broth was added, together with 100. Mu.L of CAS detection solution, and after incubation under the same conditions, the absorbance at 630nm was measured and recorded as OD B. The capacity of the novel pseudomonas F0096 for producing the iron carrier is judged through the ratio of OD A to OD B, and the smaller the ratio is, the stronger the capacity of the novel pseudomonas F0096 for producing the iron carrier is. The ratio of OD A to OD B was greater than 1, indicating that the strain was unable to produce siderophores; if the ratio is less than 1, it indicates that the strain is capable of producing siderophores. The ratio of OD A to OD B of novel pseudomonas F0096 was 0.91353, indicating that it was capable of producing siderophores.
The results show that the novel Pseudomonas sp.f 0096 is a potential novel species of Pseudomonas, has the capability of obviously inhibiting the growth of pathogenic bacteria Xcc of citrus canker, pathogenic bacteria solanaceae Laue bacteria GMI 1000 of bacterial wilt, pathogenic bacteria of tomato canker, corynebacterium michiganensis GDMCC 1.858, pathogenic bacteria of fruit tree canker, pseudomonas syringae GDMCC 1.330.330 and the like, has excellent fermentation performance, is a broad-spectrum and efficient biocontrol functional strain, can be applied to the control of citrus canker, bacterial wilt, tomato canker and fruit tree canker by direct spraying, or can be used for the production of microbial fertilizers, pesticides, microbial bactericides and the like, and further can be applied to the control of the crop diseases. The novel Pseudomonas sp. F0096 is capable of producing siderophores and has great application potential in promoting iron absorption of crops.
SEQ ID NO.1
TGAAGAGTTT GATCATGGCT CAGATTGAAC GCTGGCGGCA GGCCTAACAC ATGCAAGTCG 60
AGCGGATGAG AGGAGCTTGC TTCTCGATTC AGCGGCGGAC GGGTGAGTAA TGCCTAGGAA 120
TCTGCCTGGT AGTGGGGGAC AACGTTTCGA AAGGAACGCT AATACCGCAT ACGTCCTACG 180
GGAGAAAGCA GGGGACCTTC GGGCCTTGCG CTATCAGATG AGCCTAGGTC GGATTAGCTA 240
GTTGGTGAGG TAATGGCTCA CCAAGGCGAC GATCCGTAAC TGGTCTGAGA GGATGATCAG 300
TCACACTGGA ACTGAGACAC GGTCCAGACT CCTACGGGAG GCAGCAGTGG GGAATATTGG 360
ACAATGGGCG AAAGCCTGAT CCAGCCATGC CGCGTGTGTG AAGAAGGTCT TCGGATTGTA 420
AAGCACTTTA AGTTGGGAGG AAGGGTTGTA GATTAATACT CTGCAATTTT GACGTTACCG 480
ACAGAATAAG CACCGGCTAA CTCTGTGCCA GCAGCCGCGG TAATACAGAG GGTGCAAGCG 540
TTAATCGGAA TTACTGGGCG TAAAGCGCGC GTAGGTGGTT TGTTAAGTTG GATGTGAAAG 600
CCCCGGGCTC AACCTGGGAA CTGCATCCAA AACTGGCAAG CTAGAGTACG GTAGAGGGTG 660
GTGGAATTTC CTGTGTAGCG GTGAAATGCG TAGATATAGG AAGGAACACC AGTGGCGAAG 720
GCGACCACCT GGACTGATAC TGACACTGAG GTGCGAAAGC GTGGGGAGCA AACAGGATTA 780
GATACCCTGG TAGTCCACGC CGTAAACGAT GTCAACTAGC CGTTGGAATC CTTGAGATTT 840
TAGTGGCGCA GCTAACGCAT TAAGTTGACC GCCTGGGGAG TACGGCCGCA AGGTTAAAAC 900
TCAAATGAAT TGACGGGGGC CCGCACAAGC GGTGGAGCAT GTGGTTTAAT TCGAAGCAAC 960
GCGAAGAACC TTACCAGGCC TTGACATGCA GAGAACTTTC CAGAGATGGA TTGGTGCCTT 1020
CGGGAACTCT GACACAGGTG CTGCATGGCT GTCGTCAGCT CGTGTCGTGA GATGTTGGGT 1080
TAAGTCCCGT AACGAGCGCA ACCCTTGTCC TTAGTTACCA GCACGTCATG GTGGGCACTC 1140
TAAGGAGACT GCCGGTGACA AACCGGAGGA AGGTGGGGAT GACGTCAAGT CATCATGGCC 1200
CTTACGGCCT GGGCTACACA CGTGCTACAA TGGTCGGTAC AGAGGGTTGC CAAGCCGCGA 1260
GGTGGAGCTA ATCTCACAAA ACCGATCGTA GTCCGGATCG CAGTCTGCAA CTCGACTGCG 1320
TGAAGTCGGA ATCGCTAGTA ATCGCGAATC AGAATGTCGC GGTGAATACG TTCCCGGGCC 1380
TTGTACACAC CGCCCGTCAC ACCATGGGAG TGGGTTGCAC CAGAAGTAGC TAGTCTAACC 1440
TTCGGGGGGA CGGTTACCAC GGTGTGATTC ATGACTGGGG TGAAGTCGTA ACAAGGTAGC 1500
CGTAGGGGAA CCTGCGGCTG GATCACCTCC TT 1532
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.

Claims (10)

1. Pseudomonas sp.) F0096, accession number: GDMCC No: 63027.
2. Use of a fermentation broth of pseudomonas F0096 according to claim 1 for the preparation of a medicament for the control of citrus canker, bacterial wilt, tomato canker and fruit tree canker.
3. Use of pseudomonas F0096 according to claim 1 for the preparation of a microbial inoculant or fertilizer additive for promoting iron absorption in crops.
4. A microbial preparation comprising the fermentation broth of Pseudomonas F0096 according to claim 1 as an active ingredient.
5. The microbial preparation according to claim 4, wherein the microbial preparation is a microbial preparation for controlling bacterial wilt.
6. The microbial preparation according to claim 4, wherein the microbial preparation is a microbial preparation for controlling tomato canker.
7. The microbial preparation according to claim 4, wherein the microbial preparation is a microbial preparation for controlling canker of fruit trees.
8. A microbial live bacteria preparation for promoting iron absorption in crops, characterized by comprising the pseudomonas F0096 according to claim 1 as an active ingredient.
9. An active substance for inhibiting pathogenic bacteria of citrus canker, pathogenic bacteria of the family solanaceae, corynebacterium michiganensis and pathogenic bacteria of the family fruit tree canker, pseudomonas syringae, characterized in that the active substance is a fermentation broth of pseudomonas F0096.
10. A method for controlling citrus canker, bacterial wilt, tomato canker and fruit tree canker, characterized in that the fermentation broth of pseudomonas F0096 according to claim 1 is sprayed onto the area to be controlled.
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