CN105695368B - One plant of Te Jila bacillus and its application - Google Patents
One plant of Te Jila bacillus and its application Download PDFInfo
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- CN105695368B CN105695368B CN201610220110.8A CN201610220110A CN105695368B CN 105695368 B CN105695368 B CN 105695368B CN 201610220110 A CN201610220110 A CN 201610220110A CN 105695368 B CN105695368 B CN 105695368B
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- watermelon
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- jila bacillus
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- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 32
- 241000219109 Citrullus Species 0.000 claims abstract description 69
- 235000012828 Citrullus lanatus var citroides Nutrition 0.000 claims abstract description 63
- 241000894006 Bacteria Species 0.000 claims abstract description 38
- 230000001580 bacterial effect Effects 0.000 claims abstract description 35
- 241000196324 Embryophyta Species 0.000 claims abstract description 21
- 230000002265 prevention Effects 0.000 claims abstract description 5
- 201000010099 disease Diseases 0.000 claims description 23
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 23
- 239000001963 growth medium Substances 0.000 claims description 21
- 239000007788 liquid Substances 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 238000011218 seed culture Methods 0.000 claims description 9
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 6
- 239000002054 inoculum Substances 0.000 claims description 6
- 230000004913 activation Effects 0.000 claims description 4
- 201000004384 Alopecia Diseases 0.000 claims description 3
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- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 6
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- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
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- 108010010803 Gelatin Proteins 0.000 description 2
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- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 229910002651 NO3 Inorganic materials 0.000 description 2
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- 241000607479 Yersinia pestis Species 0.000 description 2
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 description 2
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- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 2
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- 239000012137 tryptone Substances 0.000 description 2
- 102000016938 Catalase Human genes 0.000 description 1
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- 239000005784 Fluoxastrobin Substances 0.000 description 1
- 241000508192 Fusarium oxysporum f. sp. niveum Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000005794 Hymexazol Substances 0.000 description 1
- 239000005867 Iprodione Substances 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 244000302544 Luffa aegyptiaca Species 0.000 description 1
- 235000009814 Luffa aegyptiaca Nutrition 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- 229910004616 Na2MoO4.2H2 O Inorganic materials 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
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- 239000005820 Prochloraz Substances 0.000 description 1
- 239000005822 Propiconazole Substances 0.000 description 1
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- 229920002472 Starch Polymers 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 241001123668 Verticillium dahliae Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
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- 238000001514 detection method Methods 0.000 description 1
- LVYZJEPLMYTTGH-UHFFFAOYSA-H dialuminum chloride pentahydroxide dihydrate Chemical compound [Cl-].[Al+3].[OH-].[OH-].[Al+3].[OH-].[OH-].[OH-].O.O LVYZJEPLMYTTGH-UHFFFAOYSA-H 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
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- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- UFEODZBUAFNAEU-NLRVBDNBSA-N fluoxastrobin Chemical compound C=1C=CC=C(OC=2C(=C(OC=3C(=CC=CC=3)Cl)N=CN=2)F)C=1C(=N/OC)\C1=NOCCO1 UFEODZBUAFNAEU-NLRVBDNBSA-N 0.000 description 1
- 230000001408 fungistatic effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- KGVPNLBXJKTABS-UHFFFAOYSA-N hymexazol Chemical compound CC1=CC(O)=NO1 KGVPNLBXJKTABS-UHFFFAOYSA-N 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 150000002475 indoles Chemical class 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- ONUFESLQCSAYKA-UHFFFAOYSA-N iprodione Chemical compound O=C1N(C(=O)NC(C)C)CC(=O)N1C1=CC(Cl)=CC(Cl)=C1 ONUFESLQCSAYKA-UHFFFAOYSA-N 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 229940099607 manganese chloride Drugs 0.000 description 1
- OXOKTPUZOHPXSA-UHFFFAOYSA-L manganese(2+);n-propyl-n-[2-(2,4,6-trichlorophenoxy)ethyl]imidazole-1-carboxamide;dichloride Chemical compound Cl[Mn]Cl.C1=CN=CN1C(=O)N(CCC)CCOC1=C(Cl)C=C(Cl)C=C1Cl.C1=CN=CN1C(=O)N(CCC)CCOC1=C(Cl)C=C(Cl)C=C1Cl.C1=CN=CN1C(=O)N(CCC)CCOC1=C(Cl)C=C(Cl)C=C1Cl.C1=CN=CN1C(=O)N(CCC)CCOC1=C(Cl)C=C(Cl)C=C1Cl OXOKTPUZOHPXSA-UHFFFAOYSA-L 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
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- 210000004080 milk Anatomy 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
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- 230000001590 oxidative effect Effects 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 238000013081 phylogenetic analysis Methods 0.000 description 1
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- -1 polyenoid class Chemical class 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
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- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- TVLSRXXIMLFWEO-UHFFFAOYSA-N prochloraz Chemical compound C1=CN=CN1C(=O)N(CCC)CCOC1=C(Cl)C=C(Cl)C=C1Cl TVLSRXXIMLFWEO-UHFFFAOYSA-N 0.000 description 1
- HJSRRUNWOFLQRG-UHFFFAOYSA-N propanedioic acid Chemical compound OC(=O)CC(O)=O.OC(=O)CC(O)=O HJSRRUNWOFLQRG-UHFFFAOYSA-N 0.000 description 1
- STJLVHWMYQXCPB-UHFFFAOYSA-N propiconazole Chemical compound O1C(CCC)COC1(C=1C(=CC(Cl)=CC=1)Cl)CN1N=CN=C1 STJLVHWMYQXCPB-UHFFFAOYSA-N 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
- 229960001225 rifampicin Drugs 0.000 description 1
- 239000002893 slag Substances 0.000 description 1
- FDEIWTXVNPKYDL-UHFFFAOYSA-N sodium molybdate dihydrate Chemical compound O.O.[Na+].[Na+].[O-][Mo]([O-])(=O)=O FDEIWTXVNPKYDL-UHFFFAOYSA-N 0.000 description 1
- 244000000000 soil microbiome Species 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
Abstract
The present invention provides the Te Jila bacillus WM031 that one plant of deposit number is CCTCC NO:M2016021, and what which can be stable is colonized in watermelon plant, and effectively prevents the generation of watermelon blight, in use, it is 10 that the bacterium can be prepared into bacterial content10The bacterium solution of cfu/mL carries out pouring root to the watermelon seedling of big Tanaka, arrived the purpose of prevention watermelon blight, and the microbial inoculum preventive effect is high, using simplicity, is suitable for large-scale promotion application.
Description
Technical field
The present invention relates to plant Te Jila bacillus WM031(of field of biotechnology, especially oneBacillus tequilensis WM031) and its application in watermelon blight is being prevented and treated.
Background technique
Wilt disease (Blight) is a kind of destructive disease of watermelon, widely distributed in each watermelon producing region in the world.With
The adjustment of China's agricultural structure, growth of watermelon area is increasing, and continuous cropping obstacle, which has become, restricts the important of watermelon production
Factor.Watermelon blight is one of Major Diseases of continuous cropping obstacle of watermelon, and pathogen is Fusarium oxysporum f. sp. niveum
(Fusarium oxyporum f. sp. niveum).As continuous cropping and continuous cropping, watermelon blight occur increasingly tight watermelon year by year
Weight, causes a large amount of underproduction of watermelon, and the general disease field underproduction 30%~40%, 80% or more the grave illness Tian Zeke underproduction is even had no harvest,
In the world, serious economic loss is caused in the major producing region of watermelon, seriously limits watermelon production.Although can be from screening resistance product
Kind reinforces the measures such as cultivation progress integrated control watermelon blight, but Agro-chemicals control is to control watermelon blight at present
Important and effective ways.
The country has filtered out some effective medicaments at present, such as 25% Prochloraz EC, 70% hymexazol WP, more than 50% mould prestige
The medicaments such as WP, 50% iprodione WP, 50% prochloraz-manganese chloride complex WP, 32.5% benzene first Fluoxastrobin SC and 30% benzene first propiconazole EC are to west
Cucurbit wilt has preferable control efficiency.But be used for a long time chemical bactericide not only cause the drug resistance of withered germ of water-melon by
Year increase, while the problems such as can also generate pesticide residue, with the development of modern society, people are to environmental protection and own health
Concern, Biocontrol microorganism with its low toxicity, to human health safety etc. advantages, be increasingly used in agricultural plant disease
Prevention and treatment.
Biological control is the important means of plant pest comprehensive treatment, is had to the sustainable development of realization agricultural great
Meaning, and separating and screening efficient Biocontrol microorganism then is the premise for realizing biological control.Bacillus (Bacillussp.) be
The mankind have found earliest one of bacterium, widely distributed, and the range of inhibition pathogen is very wide, resistance, has no toxic side effect, dynamic
In the biological control of plant, using than wide.Bacillus can produce peptides, lipopeptid class, phospholipid, polyenoid class, amino acid
The antibacterial materials such as class, nucleic acid, different types of antibacterial material have different biological activities, therefore secreted by bacillus
Extracellular antiseptic substance can inhibit a variety of virulence factors such as fungi, bacterium, virus.Bacillus is for plant soil-borne diseases at present
There are many example, such as obtain the life of the bacillus subtilis of Environmental Protection Agency's commercialization or the application license of limited merchandized handling in the U.S.
In anti-bacterial strain, GBO3, FZB24 may be used to the prevention and treatment of wilt disease.Wang Yaping is separated to the bacillus subtilis of sponge gourd soil
Bacterium (B.subtilus) TG26 leaching root processing watermelon and tobacco seedling, to the field efficacy point of watermelon blight and tobacco bacterial wilt
Other 73.1% and 79.6%, and have apparent yield increasing effect.Te Jila bacillus ((Bacillus tequilensis) it is withered
The close relative of careless bacillus can generate antagonism, the at present biological and ecological methods to prevent plant disease, pests, and erosion of Te Jila bacillus to plurality of plant diseases bacterial strain
Effect is concentrated on to by the fungistatic effects such as Rhizoctonia solani Kuhn, streptococcus, verticillium dahliae, about to watermelon blight
Biocontrol effect have not been reported.
Summary of the invention
In view of the above-mentioned problems, the present invention provide one plant from tomato root portion from obtained Te Jila bacillus WMO30 and
Its application in prevention and treatment watermelon blight:
One plant of Te Jila bacillus WM031(Bacillus tequilensisWM031), deposit number CCTCC
NO:M2016021；The Te Jila bacillus is gram positive bacterial strain, and thallus is in the shape of a rod, atrichia, both ends blunt circle, is produced
Raw ellipse gemma；Its bacterium colony is creamy white, and edge is irregular.
Further, the Te Jila bacillus WM031 that deposit number as described herein is CCTCC NO:M2016021 is anti-
Control the application in watermelon blight.
Further, deposit number of the present invention is the application of the Te Jila bacillus WM031 of CCTCC NO:M2016021
Specifically refer to: after watermelon transplantation of seedlings to crop field, with Te Jila bacillus WM031 bacterium solution pouring root, 400 mL/ plants, with to prevent
Control watermelon blight.
Further, Te Jila bacillus WM031 bacterium solution of the present invention is obtained by:
(1) the Te Jila bacillus WM031 that picking deposit number is CCTCC NO:M2016021 is seeded to culture presevation training
Base is supported, after 30 DEG C are continuously crossed, choose single colonie culture twice, picking single colonie is in strain activation and culture base, in 30 DEG C, 150-
200rpm shaking table shaken cultivation 12-24h；
(2) in the fermentor of Xiang Hanyou seed culture medium, according to the inoculum concentration inoculating strain of volume ratio 1:9, in 25-38
DEG C, blowing air culture 16-24h obtains liquid seeds；
(3) 10% inoculum concentration is inoculated with liquid seeds into seed culture medium by volume, and in 30-35 DEG C, 150 rpm are shaken
It is protected from light 24 h of culture under bed oscillation, obtains thalline culture living；
(4) it takes thalline culture living to be centrifuged 15 min under the conditions of 4 DEG C, 5000rpm, takes the sterile life precipitated with 0.85%
It manages salt water to rinse 3 times, being adjusted to bacterial content is 1010Cfu/mL, i.e. acquisition Te Jila bacillus WM031 bacterium solution.
Deposit number provided by the invention is the Te Jila bacillus WM031(of CCTCC NO:M2016021Bacillus tequilensisWM031) there is strong antagonism to withered germ of water-melon, PDA plate face-off experiment shows the bacterial strain
To the antibacterial band diameter 3.4cm of withered germ of water-melon, bacteriostasis rate 79.0% is 10 by concentration prepared by the bacterium7-108cfu/mL
The microbial inoculum of left and right, can stablize field planting in soil and watermelon plant.Indoor control test result shows that the microbial inoculum is inoculated with watermelon
The generation of watermelon blight can be effectively prevent after plant, disease incidence decline 50%(is shown in Fig. 4 compared with the control).Field experiment knot
Fruit shows that biocontrol microorganisms WM031 is higher than the preventive effect (73.3%) of chemical agent carbendazim to the preventive effect (76.7%) of watermelon blight.
Detailed description of the invention
Fig. 1 is bacterial strain WM031 electron microscopic picture.
Fig. 2 is the adjacent system development tree schematic diagram of the 16SrDNA sequence of bacterial strain WM031.
Fig. 3 is for bacterial strain WM05 to the inhibiting effect schematic diagram of withered germ of water-melon in PDA culture medium.
Fig. 4 is indoor control test effect schematic diagram of the bacterial strain WM05 to watermelon blight.
Specific embodiment
It is microbial withered that technical solution of the present invention produced can efficiently prevent and treat watermelon blight to endophytic bacterial agent
Disease.Below by way of the specific embodiment implementation that the present invention will be described in detail, it is therefore intended that help reader to more fully understand of the invention
Spirit Essence, but not as the restriction to the scope of the present invention.
The culture medium that embodiment is related to:
LB solid medium (culture presevation culture medium): tryptone 10g, yeast powder 5g, sodium chloride 10g, agar 20g,
Add water to 1L, pH 7.0-7.5；
LB culture solution (strain activation and culture base): tryptone 10g, yeast powder 5g, sodium chloride 10g add water to 1L, pH
7.0-7.5；
Seed culture medium (liquid, 1L): K2HPO44.8g, KH2PO43.5g, (NH4)2SO42g, MgCl2 0.16g,
CaCl20.02g, Na2MoO4.2H2O 0.0024g, FeCl30.0018g, MnCl2.2H2O 0.0015g, pH=7.0.
The above culture medium is in 121 DEG C of sterilizing 15-30 min.
LB plate: preparing the above-mentioned LB solid medium of 100ml, and after high pressure sterilization, the LB solid medium of thawing is placed in
In 55 DEG C of water-bath, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices when culture medium temperature drops to 55 DEG C, 10mlLB solid medium is poured into a sterilizes culture dish,
Lid is opened, in the UV lamp according to 10-15 minute, is sealed and is inverted with sealed membrane and be put in 4 DEG C of refrigerators after cooling down, it is spare.
Sweet mung bean soup culture medium: 300g mung bean Yu Shuizhong boils 10 minutes, after being removed slag with 4 layers of filtered through gauze, is settled to 1000
ML, 121 DEG C of sterilizing 20min of high pressure.
PDA plate: peeling potatoes 200g, which is cut into small pieces, to be put into pot, and 1000ml water is added, and lasting 20- is boiled in heating
30min, filter and remove residue, addition 20g agar powder melt to agar, 20g glucose are added, water is added to supply 4 layers of gauze while hot
1000ml, after 121 DEG C of sterilizing 15-30 min, taking-up is cooled to 55 DEG C, and every 10mlLB culture medium pours into a sterilizes culture dish
In, open lid, in the UV lamp according to 10-15 minute, cool down after sealed with sealed membrane and be inverted be put in it is spare in 4 DEG C of refrigerators.
Withered germ of water-melon (Fusarium oxyporum f. sp. niveum) by the Jiangsu academy of agricultural sciences place of production Shi Jiansuo
Environmental Studies room, which saves, to be provided；
Watermelon seed is Soviet Union's honey five, is purchased from Jiang Shu seedling Science and Technology Ltd., Jiangsu Province
The acquisition of 1 bacterial strain of embodiment and bacterium solution preparation
1, the acquisition of bacterial strain
(1) sample acquires: the tomato plant in acquisition Nanjing Jiangsu Province Agriculture Science Institute Vegetable Base greenhouse
Its surface is attached to dust soil with tap water and rinsed well by root proper amount of fresh plant sample, natural air drying, then by plant
Root tissue carries out surface sterilization: and then successively impregnate 1 min and 8%NaClO immersion, 4 min with 75% alcohol and carry out surface sterilization
Processing, sterile washing 4 times；
(2) separation screening: root, stem and leaf is cut off with sterile scissors, and root and stem are cut into the segment of 1 cm or so, is placed in LB
On plate；Root adds water to be ground to paste, is coated with LB plate after standing 10 min, is placed in 30 DEG C of 48 h of culture；
(3) it purifies: after thering is culture to grow, being purified using plate streaking partition method, picking is grown under maximum concentration
Bacterium colony cross on enriched medium, until separation obtain pure culture.
One plant of pure culture is obtained through the above steps, and applicant is by it from bacterial strain WM031 is named as, and electromicroscopic photograph is such as
Shown in Fig. 1；The bacterial strain is gram positive bacterial strain, and thallus is in the shape of a rod, atrichia, and both ends blunt circle can generate oval gemma.
The bacterial strain its bacterium colony on LB culture medium is creamy white, and edge is irregular.Can 25 DEG C ~ 40 DEG C at a temperature of grow, it is most suitable
Growth temperature is 32 DEG C；Growing pH range is 4.0 ~ 9.0；Optimal pH is 6.5, and specific physicochemical property is as shown in table 1:
1 bacterial strain WM031 physicochemical property of table
| Test item Test item | As a result Results | Test item Test item | As a result Results |
| Strain morphology Morphology | Rod-shaped rod | Gluconate Gluconate | + |
| Gram-reaction Gram reaction | + | Malonate Malonate | - |
| Catalase Catalase | + | Oxidizing ferment Oxidase | + |
| V-P test | + | M.R test | + |
| Nitrate reduction Nitrate reduction | + | Citrate Simmon ' s citrate | + |
| Litmus milk | + | Gelatin liquefaction gelatin liquefaction | + |
| Indoles Indole | + | Starch Hydrolysis | + |
*+represent to react and be positive ,-represent and react be negative (+positive reaction ,-negative
Reaction)
The 16S rDNA Phylogenetic Analysis figure of bacterial strain WM031 is as shown in Fig. 2, in Genbank(network address http: //
Www.ncbi.nlm.nih.gov/ it) is carried out with blast program with the 16S rDNA gene order for having logged in bacterium bacterial strain on website
Compare, the results showed that with Bacillus tequilensis. similitude highest, can achieve 99% or more.
The bacterial strain is preserved in China typical culture collection center (CCTCC), address on January 7th, 2016 by applicant
For Wuhan University, Wuhan, China city, postcode 430072, which is Te Jila bacillus WM031,Bacillus tequilensisWM031, deposit number are CCTCC NO:M 2016021.
2, WM031 bacterium solution is prepared
(1) actication of culture: picking bacterial strain WM031 to culture presevation culture medium, 30 DEG C are continuously crossed, choose single colonie culture two
After secondary, picking single colonie is in strain activation and culture base, in 30 DEG C, 150-200 r/min shaking table shaken cultivation 12-24h；
(2) prepared by liquid seeds: into the fermentor of the seed culture medium equipped with high-temperature sterilization, according to 10% (V/V's)
Inoculum concentration is inoculated with WM031, and in 25-38 DEG C, blowing air culture 16-24h obtains liquid seeds.
(3) it liquid state fermentation: into the seed culture medium equipped with high-temperature sterilization, is inoculated with according to the inoculum concentration of volume fraction 10%
Liquid seeds are protected from light 24 h(logarithmic growth phases of culture under 30-35 DEG C, 150 r/min), obtain thalline culture living.
(4) prepared by microbial inoculum: taking thalline culture living to be centrifuged 15 min under 4 DEG C, 5000 r/min, takes precipitating 0.85%
Sterile saline is rinsed 3 times, and appropriate seed culture medium is added and adjusts bacterial content to 1010Cfu/mL or so, as WM031 bacterium
Liquid；It is added glycerol (volume fraction 50%), filling preservation.500-1000 times is diluted when use.
Inhibiting effect of the 2 bacterial strain WM031 of embodiment to withered germ of water-melon
(1) withered germ of water-melon is inoculated on PDA plate, after plate is covered in 28 DEG C of cultures, with the punching of 5 mm of diameter
Bacteria cake is made in device, and by pure culture biscuits involvng inoculation in the center of PDA plate, while point connects on four angle points away from 30 mm of center or so
WM031 bacterium solution (concentration 108Cfu/mL, 100 μ L) the WM031 bacterium solution that obtains of embodiment 1 is as experimental group, while only to connect disease
The plate of fungal pathogens compares, and each processing 3 repeats；
(2) PDA plate is placed in 28 DEG C of environment and cultivates 7 d, cover with plate, measurement disease to control group disease fungus bacterium colony
Fungal pathogens colony diameter, and bacteriostasis rate is calculated as follows:
Bacteriostasis rate (%)=[(A-B)/(A-5)] * 100%；
(note: A is control group disease fungus colony diameter, and B is processing group disease fungus colony diameter).
(3) antibacterial result: for cultivation results as shown in figure 3, wherein Fig. 3 A is experimental group cultivation results schematic diagram, Fig. 3 B is pair
According to a group culture result schematic diagram, it is seen then that bacterial strain WM031 is to the antibacterial band diameter 3.4cm of withered germ of water-melon, bacteriostasis rate
79.0%。
3 bacterial strain WM031 colonizing in soil and watermelon plant of embodiment
1 μ g/mL rifampin is added into LB plate, then accesses the WM031 bacterial strain that embodiment 1 obtains, is stepped up benefit
For the flat concentration of good fortune to 300 μ g/mL, growth can be stablized and physiological property and the consistent mutant strain of original strain by filtering out；?
It transferred on LB plate for 10 generations, observes colonial morphology, be then forwarded in the LB culture medium that 300 μ g/mL rifampins are added and mark.
1, WM031 is colonized in the soil
(1) it will be inoculated in LB culture solution with the labeled bacterial strain WM031 of 300 μ g/mL rifampins, in 32 DEG C, 180 r/
Min, shaking table vibrate for 24 hours, and it is about 10 that bacteria containing amount, which is made,8The labeled strain bacteria suspension of cfu/mL；
(2) naturally native (being derived from Nanjing, Jiangsu Province Agriculture Science Institute experimental field) and sterilized soil are loaded in flowerpot respectively, often
Basin 1kg soil, 100 mL labeled strain bacteria suspensions are injected into soil and are mixed thoroughly；
(3) it places at room temperature, the bacterium in 7 days separation soil, soil first after gradient dilution, takes 10-3、10-4、
10-5Soil dilution liquid spread plate, calculate bacteria containing amount；
(4) testing result: the colonization amount of soil and WM031 in sterilized soil can reach 10 naturally after 28 days4Cfu/g soil with
On, showing WM031 bacterial strain in the soil has stronger colonization ability.
2, bacterial strain WM031 is colonized test in watermelon plant
(1) full consistent watermelon seed is chosen, successively 3 min and 8% NaClO is impregnated with 75 % alcohol and impregnates 5
Min carries out surface sterilization processing to seed, examines disinfection result with the sterile water coated plate that last time is cleaned；
Seed is placed in the culture dish for being covered with filter paper by (2) 50 DEG C of sterile waters after impregnating 2 h, and sharp good fortune is added with 10 mL/ wares
WM031 bacterial strain after flat label impregnates 24 h of seed, while being control with physiological saline.
(3) growth, regular sample detection bacterial strain in nutritive cube be will move into after sprouting and colonize situation in watermelon root and stem；
(4) the result shows that, colonizing for WM031 can be detected in the root and stem of watermelon plant after 28 days, colonization amount in root
It is higher, 10 can be reached4 Cfu/g, the colonization amount in stem can reach 103 Cfu/g or more, this illustrates that WM031 bacterial strain has in watermelon
There is stronger colonization ability.
4 WM031 of embodiment prevents and treats watermelon blight pot experiment
The preparation of withered germ of water-melon spore suspension: withered germ of water-melon cultivates 6-7d on PDA plate, is with aperture
The punch of 20mm punches on culture medium, takes 10 band bacterium culture mediums to be added in 200mL sterilizing sweet mung bean soup culture medium, in 28
DEG C, 180 r/min, shaking table shaken cultivation 7d, with sterile gauze (4 layers) filter and remove residue, filtrate under 4 DEG C, 5000 r/min from
10 min of the heart goes supernatant taking precipitate (withered germ of water-melon conidium), after being resuspended with liquid potato culture, with going out
The dilution of bacterium deionized water makes spore concentration 1 × 106A/mL, 28 DEG C of incubation 60min, it is spare.
Pot experiment carries out in the heliogreenhouse of Jiangsu Province Agriculture Science Institute, and experimental cultivar is that Soviet Union No. five, of honey are set at 3
Reason: A.WM031 bacterium solution, B. withered germ of water-melon, C. WM031 bacterium solution+withered germ of water-melon, every processing 3 repeat, every repetition 12
Strain seedling.
Tests concrete steps are as follows:
(1) after the surface of the seed being sterilized, sterile water soaked overnight, 32 DEG C of 2 d vernalization of constant temperature.Hole tray is seeded in after showing money or valuables one carries unintentionally,
When seedling it is long to 2 true leaves when, transplanting to 10 cm of internal diameter, high 15 cm, in the plastic cup of built-in 300 g sterile soil.
(2) after a week, root fills 30 ml 10 in processing group A and processing group C for transplanting8Cfu/mL biocontrol microorganisms bacterium solution.
(3) after a week, (the every plastic cup inoculation 50 of withered germ of water-melon spore suspension is inoculated in processing group B, processing group C
ML).After being inoculated with disease fungi, plant incidence is investigated, disease symptom (leaf occurs in the seedling to only connect disease fungus group 60%
It is both morbidity that piece or vines wilting area, which are more than the 1/4 of whole strain) after, terminate test.
Test result is as shown in figure 4, wherein Fig. 4 A is only to spray WM031 bacterium solution treatment effect；Fig. 4 B is only to be inoculated with to wither
Germ processing result；Fig. 4 C is to spray WM031 bacterium solution and wilt processing result；As it can be seen that being inoculated with WM031 bacterium solution in C processing
The generation of watermelon blight can be effectively prevent after watermelon plant afterwards, see Table 2 for details for disease incidence decline 50% compared with control (B).
2 WM031 of table prevents and treats watermelon blight results from pot experiment test
| Processing | Morbidity strain number | Disease incidence (%) |
| Handle A | 0 | 0 |
| Handle B | 36 | 100 |
| Handle C | 18 | 50 |
5 WM031 of embodiment prevents and treats watermelon blight field experiment
By plantation after the watermelon seed vernalization of disinfection in sterilizing hole tray, it is colonized and arrives when watermelon seedling grows 3-4 piece true leaf
Crop field, field plot trial are carried out in Jiangsu Province Agriculture Science Institute trial zone greenhouse.After watermelon seedling is colonized 7 d, every plant
Western melon root pours 400mL withered germ of water-melon spore suspension (spore concentration 1 × 106A/mL).Test sets 4 processing,
Carbendazim (50% 800 times of carbendazol wettable powder liquid), WM031 bacterium solution (1 × 108Cfu/mL) and control blank culture solution
(seed culture medium after sterilizing), 30 plants of watermelon seedlings of every processing, 3 repetitions.Pouring withered germ of water-melon spore suspension
The 2nd d it is each processing respectively western melon root pour 400 mL.Incidence (blade or vines wilting face are investigated after 20 d
It is both morbidity that product, which is more than the 1/4 of whole strain), disease incidence and preventive effect are calculated, as a result see Table 3 for details.
3 WM031 of table prevents and treats watermelon blight field experiment result
| Processing | Morbidity strain number | Disease incidence (%) | Preventive effect (%) |
| Control | 85 | 94.4 | - |
| Carbendazim | 24 | 26.7 | 73.3 |
| WM031 | 21 | 23.3 | 76.7 |
Field experiment the result shows that, it is more that biocontrol microorganisms WM031 is slightly above chemical agent to the preventive effect (76.7%) of watermelon blight
The preventive effect (73.3%) of bacterium spirit, show biocontrol microorganisms WM031 can be used as prevention and treatment watermelon blight disinfectant use in agriculture developed benefit
With.
Claims (3)
1. one plant of Te Jila bacillus WM031(Bacillus tequilensisWM031), deposit number CCTCC
NO:M2016021；The Te Jila bacillus is gram positive bacterial strain, and thallus is in the shape of a rod, atrichia, both ends blunt circle, is produced
Raw ellipse gemma；Its bacterium colony is creamy white, and edge is irregular.
2. application of the Te Jila bacillus WM031 as described in claim 1 in prevention and treatment watermelon blight, specific steps
Are as follows: after watermelon transplantation of seedlings to crop field, with Te Jila bacillus WM031 bacterium solution pouring root, 400 mL/ plants, to prevent and treat watermelon
Wilt disease.
3. applying according to claim 2, which is characterized in that the Te Jila bacillus WM031 bacterium solution is obtained by
:
(1) picking deposit number is that the Te Jila bacillus WM031 of CCTCC NO:M2016021 is seeded to culture presevation culture
Base, after 30 DEG C are continuously crossed, choose single colonie culture twice, picking single colonie is in strain activation and culture base, in 30 DEG C, 150-
200rpm shaking table shaken cultivation 12-24h；
(2) in the fermentor of Xiang Hanyou seed culture medium, according to the inoculum concentration inoculating strain of volume ratio 1:9, in 25-38 DEG C, lead to
Air jet flow 16-24h, obtains liquid seeds；
(3) 10% inoculum concentration is inoculated with liquid seeds into seed culture medium by volume, and in 30-35 DEG C, 150 rpm shaking tables shake
It is protected from light 24 h of culture under swinging, obtains thalline culture living；
(4) it takes thalline culture living to be centrifuged 15 min under the conditions of 4 DEG C, 5000rpm, takes the sterile physiological salt precipitated with 0.85%
Water rinses 3 times, and being adjusted to bacterial content is 1010Cfu/mL, i.e. acquisition Te Jila bacillus WM031 bacterium solution.
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| CN107568248A (en) * | 2017-10-10 | 2018-01-12 | 石河子大学 | A kind of controlling plant diseases simultaneously strengthen microbial bacterial agent of plant stress-resistance and its preparation method and application |
| CN109536425B (en) * | 2019-01-24 | 2020-06-30 | 河南科技大学 | Bacillus tequilensis and application thereof |
| CN110402929A (en) * | 2019-08-21 | 2019-11-05 | 浙江省桐庐汇丰生物科技有限公司 | A kind of bactericidal composition and its application containing jinggangmeisu |
| CR20220686A (en) * | 2020-06-17 | 2023-06-01 | Bioconsortia Inc | BENEFICIAL MICROBES FOR AGRICULTURE, MICROBIAL COMPOSITIONS AND CONSORTIA |
| CN111718872A (en) * | 2020-06-19 | 2020-09-29 | 河南农贝得农业科技有限公司 | Microbial agent for crop blight and preparation method thereof |
| CN111944716B (en) * | 2020-08-03 | 2022-03-08 | 北京恩格兰环境技术有限责任公司 | Special compound microbial agent for tobacco seedling culture and preparation method and application thereof |
| CN112646755B (en) * | 2021-01-22 | 2022-06-28 | 西北农林科技大学 | Bacillus tequilensis and application thereof |
| CN115975847B (en) * | 2022-08-08 | 2023-09-26 | 兰州交通大学 | Bacillus tertiaryanae for preventing and treating root rot of angelica sinensis and application thereof |
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