CN115975886B - Novel pseudomonas and application thereof in crop bacterial disease control - Google Patents
Novel pseudomonas and application thereof in crop bacterial disease control Download PDFInfo
- Publication number
- 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
- Authority
- CN
- China
- Prior art keywords
- pseudomonas
- canker
- novel
- strain
- pathogenic bacteria
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000589516 Pseudomonas Species 0.000 title claims abstract description 74
- 208000035143 Bacterial infection Diseases 0.000 title abstract description 15
- 208000022362 bacterial infectious disease Diseases 0.000 title abstract description 6
- 244000052616 bacterial pathogen Species 0.000 claims abstract description 34
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims abstract description 18
- 229910052742 iron Inorganic materials 0.000 claims abstract description 9
- 238000010521 absorption reaction Methods 0.000 claims abstract description 7
- 241000589774 Pseudomonas sp. Species 0.000 claims abstract description 6
- 230000001580 bacterial effect Effects 0.000 claims description 26
- 238000000855 fermentation Methods 0.000 claims description 22
- 230000004151 fermentation Effects 0.000 claims description 22
- 230000000813 microbial effect Effects 0.000 claims description 21
- 241000207199 Citrus Species 0.000 claims description 20
- 235000020971 citrus fruits Nutrition 0.000 claims description 20
- 235000013399 edible fruits Nutrition 0.000 claims description 20
- 235000007688 Lycopersicon esculentum Nutrition 0.000 claims description 18
- 238000002360 preparation method Methods 0.000 claims description 18
- 230000002401 inhibitory effect Effects 0.000 claims description 11
- 241000894006 Bacteria Species 0.000 claims description 8
- 241000589615 Pseudomonas syringae Species 0.000 claims description 7
- 241000208292 Solanaceae Species 0.000 claims description 7
- 239000004480 active ingredient Substances 0.000 claims description 7
- 230000001737 promoting effect Effects 0.000 claims description 7
- 241000186216 Corynebacterium Species 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- 239000013543 active substance Substances 0.000 claims description 3
- 239000003337 fertilizer Substances 0.000 claims description 3
- 239000000654 additive Substances 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims description 2
- 241000227653 Lycopersicon Species 0.000 claims 3
- 230000000996 additive effect Effects 0.000 claims 1
- 239000002054 inoculum Substances 0.000 claims 1
- 230000012010 growth Effects 0.000 abstract description 15
- 241000894007 species Species 0.000 abstract description 9
- 239000000589 Siderophore Substances 0.000 abstract description 7
- 244000005700 microbiome Species 0.000 abstract description 4
- 230000008021 deposition Effects 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 description 24
- 240000003768 Solanum lycopersicum Species 0.000 description 15
- 230000005764 inhibitory process Effects 0.000 description 14
- 229920001817 Agar Polymers 0.000 description 12
- 239000008272 agar Substances 0.000 description 12
- 239000002609 medium Substances 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 9
- 108020004465 16S ribosomal RNA Proteins 0.000 description 8
- 239000000843 powder Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 239000007788 liquid Substances 0.000 description 6
- 238000009630 liquid culture Methods 0.000 description 6
- 239000000575 pesticide Substances 0.000 description 6
- 238000004321 preservation Methods 0.000 description 6
- 230000004913 activation Effects 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 4
- 241000218899 Pseudomonas fulva Species 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000000443 biocontrol Effects 0.000 description 4
- 238000009792 diffusion process Methods 0.000 description 4
- 238000007711 solidification Methods 0.000 description 4
- 230000008023 solidification Effects 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 241000097097 Laurella Species 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000003480 eluent Substances 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- CQBDMXYZNLJUFT-UHFFFAOYSA-N 1-benzyl-2-butylimidazo[4,5-c]quinolin-4-amine Chemical compound CCCCC1=NC2=C(N)N=C3C=CC=CC3=C2N1CC1=CC=CC=C1 CQBDMXYZNLJUFT-UHFFFAOYSA-N 0.000 description 2
- 238000007900 DNA-DNA hybridization Methods 0.000 description 2
- 238000003794 Gram staining Methods 0.000 description 2
- 241000589540 Pseudomonas fluorescens Species 0.000 description 2
- 241001345077 Pseudomonas fulva NBRC 16637 = DSM 17717 Species 0.000 description 2
- 241000039933 Pseudomonas parafulva Species 0.000 description 2
- 241001345067 Pseudomonas parafulva NBRC 16636 = DSM 17004 Species 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 238000005162 X-ray Laue diffraction Methods 0.000 description 2
- 230000006978 adaptation Effects 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 244000000005 bacterial plant pathogen Species 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- 230000001788 irregular Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000002906 microbiologic effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- JGCSKOVQDXEQHI-UHFFFAOYSA-N phenazine-1-carboxylic acid Chemical compound C1=CC=C2N=C3C(C(=O)O)=CC=CC3=NC2=C1 JGCSKOVQDXEQHI-UHFFFAOYSA-N 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- INHYCNGBUDCDQQ-UHFFFAOYSA-N 1-nitro-2,3-dihydropyrrole Chemical compound [O-][N+](=O)N1CCC=C1 INHYCNGBUDCDQQ-UHFFFAOYSA-N 0.000 description 1
- 108020000946 Bacterial DNA Proteins 0.000 description 1
- 235000002566 Capsicum Nutrition 0.000 description 1
- 241001464977 Clavibacter michiganensis subsp. michiganensis Species 0.000 description 1
- 240000008067 Cucumis sativus Species 0.000 description 1
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 206010052804 Drug tolerance Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 241000598860 Garcinia hanburyi Species 0.000 description 1
- 208000012766 Growth delay Diseases 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 241001018563 Nekemias grossedentata Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 239000006002 Pepper Substances 0.000 description 1
- 235000016761 Piper aduncum Nutrition 0.000 description 1
- 235000017804 Piper guineense Nutrition 0.000 description 1
- 244000203593 Piper nigrum Species 0.000 description 1
- 235000008184 Piper nigrum Nutrition 0.000 description 1
- 241000960904 Pseudomonas sp. F Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 239000003899 bactericide agent Substances 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000009141 biological interaction Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000005562 fading Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229940117709 gamboge Drugs 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 125000001475 halogen functional group Chemical group 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000002420 orchard Substances 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000012372 quality testing Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
Landscapes
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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
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.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211689593.8A CN115975886B (en) | 2022-12-27 | 2022-12-27 | Novel pseudomonas and application thereof in crop bacterial disease control |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211689593.8A CN115975886B (en) | 2022-12-27 | 2022-12-27 | Novel pseudomonas and application thereof in crop bacterial disease control |
Publications (2)
Publication Number | Publication Date |
---|---|
CN115975886A CN115975886A (en) | 2023-04-18 |
CN115975886B true CN115975886B (en) | 2024-05-28 |
Family
ID=85961607
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202211689593.8A Active CN115975886B (en) | 2022-12-27 | 2022-12-27 | Novel pseudomonas and application thereof in crop bacterial disease control |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN115975886B (en) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106978373A (en) * | 2017-05-09 | 2017-07-25 | 福建农林大学 | One plant is used for pseudomonad and the application that bacteriosis is prevented and treated |
CN112899205A (en) * | 2021-03-31 | 2021-06-04 | 慕恩(广州)生物科技有限公司 | Pseudomonas chlororaphis MN225969 and application thereof |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
BR112015022445A2 (en) * | 2013-03-11 | 2017-07-18 | Univ Florida | Application of compounds that inhibit biofilm formation increases control of citrus cancer |
US12089595B2 (en) * | 2020-10-05 | 2024-09-17 | T3 Bioscience, Inc. | Pseudomonas strains and their metabolites to control plant diseases |
-
2022
- 2022-12-27 CN CN202211689593.8A patent/CN115975886B/en active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106978373A (en) * | 2017-05-09 | 2017-07-25 | 福建农林大学 | One plant is used for pseudomonad and the application that bacteriosis is prevented and treated |
CN112899205A (en) * | 2021-03-31 | 2021-06-04 | 慕恩(广州)生物科技有限公司 | Pseudomonas chlororaphis MN225969 and application thereof |
Non-Patent Citations (2)
Title |
---|
Pseudomonas fluorescens, a potential bacterial antagonist to control plant diseases;Girija Ganeshan,A. Manoj Kumar;Journal of Plant Interactions;20070220;第1卷(第3期);第123-134页 * |
多肽抗生素apidaecin对一些植物致病细菌的抗菌活性及对烟草原生质体毒性的研究;孙超,王晖,彭学贤;植物病理学报;20010831;第31卷(第3期);第274-279页 * |
Also Published As
Publication number | Publication date |
---|---|
CN115975886A (en) | 2023-04-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110066756B (en) | Paenibacillus kribbensis and preparation and application thereof | |
WO2021073142A1 (en) | Strain of fluorescent pseudomonas 22g5 and application thereof in preventative treatment of verticillium wilt in crops | |
CN113652382B (en) | Bacillus bailii for preventing and treating peanut southern blight, microbial agent, microbial fertilizer and application of microbial fertilizer | |
CN113755389B (en) | Bacillus bailii and application thereof | |
CN115873747B (en) | Bacillus belicus with broad-spectrum antibacterial activity and application thereof | |
CN113755397B (en) | Bacillus amyloliquefaciens with broad-spectrum antagonistic property, microbial agent thereof and application thereof | |
CN116426445B (en) | Pseudomonas bacteria NJAU-T102 and application thereof | |
CN108841748A (en) | Sinorhizobium nitrogen-fixing bacteria strain H6 and its application | |
CN115975886B (en) | Novel pseudomonas and application thereof in crop bacterial disease control | |
CN113832071B (en) | Brevibacillus halotolerans strain and application thereof in preparation of biocontrol microbial inoculum | |
CN112280714B (en) | Pseudomonas aeruginosa 8-7 and application thereof | |
CN115197853A (en) | Endophyte Epicoccum thailandicumLF-28 strain and application thereof | |
WO2021073143A1 (en) | Pseudomonas protegens xy2f4 and application thereof in prevention and treatment of crop verticillium wilt | |
CN113652374B (en) | Application of 7-hydroxy tropolone in preventing and treating crop verticillium wilt | |
CN115948299B (en) | Pseudomonas flavescens and application thereof in prevention and treatment of citrus canker | |
CN117946932A (en) | Novel flavobacterium and application thereof in prevention and treatment of citrus canker | |
CN117887589B (en) | Trichoderma FJ059 capable of parasitizing sclerotium of southern blight and application thereof | |
CN116555099B (en) | Sphingomonas bacterium NJAU-T56 with antibiotic resistance gene reduction and growth promoting functions and application thereof | |
CN116606780B (en) | Pseudomonas bacteria NJAU-T129 and application thereof | |
CN108085284B (en) | Bacillus methylotrophicus, and preparation method and application of microbial inoculum containing same | |
CN118146992A (en) | Streptomyces nojirimensis and application thereof | |
CN118909827A (en) | PQQ-GDH-mediated high-efficiency biocontrol bacterium enterobacter cholerae HTW22 and application thereof in preventing and treating plant bacterial wilt | |
CN118291331A (en) | Paenibacillus polymyxa capable of tolerating sodium selenite and application thereof | |
CN117987314A (en) | Pseudomonas paler Long Nishi and application thereof in plant disease control and ferrophosphorus absorption promotion | |
CN118370160A (en) | Method for improving tobacco black shank resistance and promoting tobacco growth |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |