CN117363537A - Antagonistic strain Wt-1 of pseudomonas syringae kiwi fruit pathogenic variety and application - Google Patents
Antagonistic strain Wt-1 of pseudomonas syringae kiwi fruit pathogenic variety and application Download PDFInfo
- Publication number
- CN117363537A CN117363537A CN202311371404.7A CN202311371404A CN117363537A CN 117363537 A CN117363537 A CN 117363537A CN 202311371404 A CN202311371404 A CN 202311371404A CN 117363537 A CN117363537 A CN 117363537A
- Authority
- CN
- China
- Prior art keywords
- kiwi fruit
- strain
- kiwi
- canker
- liquid
- 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.)
- Pending
Links
- 244000298697 Actinidia deliciosa Species 0.000 title claims abstract description 91
- 235000009436 Actinidia deliciosa Nutrition 0.000 title claims abstract description 87
- 241000589615 Pseudomonas syringae Species 0.000 title claims abstract description 19
- 230000003042 antagnostic effect Effects 0.000 title claims abstract description 17
- 230000001717 pathogenic effect Effects 0.000 title claims abstract description 17
- 239000007788 liquid Substances 0.000 claims abstract description 25
- 235000009434 Actinidia chinensis Nutrition 0.000 claims abstract description 17
- 238000000855 fermentation Methods 0.000 claims abstract description 16
- 230000004151 fermentation Effects 0.000 claims abstract description 16
- 238000010790 dilution Methods 0.000 claims abstract description 12
- 239000012895 dilution Substances 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 10
- 238000004321 preservation Methods 0.000 claims abstract description 10
- 238000009629 microbiological culture Methods 0.000 claims abstract description 4
- 244000005700 microbiome Species 0.000 claims abstract description 4
- 230000003405 preventing effect Effects 0.000 claims description 5
- 239000012880 LB liquid culture medium Substances 0.000 claims description 3
- 241000219068 Actinidia Species 0.000 claims description 2
- 241000589516 Pseudomonas Species 0.000 claims 1
- 244000297179 Syringa vulgaris Species 0.000 claims 1
- 235000004338 Syringa vulgaris Nutrition 0.000 claims 1
- 230000001580 bacterial effect Effects 0.000 abstract description 17
- 238000011282 treatment Methods 0.000 abstract description 16
- 238000012258 culturing Methods 0.000 abstract description 12
- 241000894006 Bacteria Species 0.000 abstract description 11
- 239000001963 growth medium Substances 0.000 abstract description 11
- 244000052616 bacterial pathogen Species 0.000 abstract description 10
- 241000196324 Embryophyta Species 0.000 abstract description 8
- 201000010099 disease Diseases 0.000 abstract description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 7
- 208000025865 Ulcer Diseases 0.000 abstract description 6
- 230000002265 prevention Effects 0.000 abstract description 5
- 238000005507 spraying Methods 0.000 abstract description 4
- 231100000397 ulcer Toxicity 0.000 abstract description 4
- 230000000813 microbial effect Effects 0.000 abstract description 2
- 230000008485 antagonism Effects 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 9
- 230000000844 anti-bacterial effect Effects 0.000 description 7
- 230000000443 biocontrol Effects 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 239000002609 medium Substances 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 6
- 230000005764 inhibitory process Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 235000013399 edible fruits Nutrition 0.000 description 4
- 230000007613 environmental effect Effects 0.000 description 4
- 238000000227 grinding Methods 0.000 description 4
- 239000002689 soil Substances 0.000 description 4
- IHDKBHLTKNUCCW-UHFFFAOYSA-N 1,3-thiazole 1-oxide Chemical compound O=S1C=CN=C1 IHDKBHLTKNUCCW-UHFFFAOYSA-N 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000008223 sterile water Substances 0.000 description 3
- 229960005322 streptomycin Drugs 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 244000298715 Actinidia chinensis Species 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 241001576300 endophytic bacterium Species 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000011392 neighbor-joining method Methods 0.000 description 2
- 239000002420 orchard Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000010008 shearing Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 241000219066 Actinidiaceae Species 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 244000144730 Amygdalus persica Species 0.000 description 1
- 239000010751 BS 2869 Class A2 Substances 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000723920 Bacillus subtilis B2 Species 0.000 description 1
- 241000063871 Burkholderia stagnalis Species 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 240000006509 Gynostemma pentaphyllum Species 0.000 description 1
- 235000002956 Gynostemma pentaphyllum Nutrition 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241001631271 Prunus fasciculata Species 0.000 description 1
- 235000006040 Prunus persica var persica Nutrition 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000035613 defoliation Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 235000013325 dietary fiber Nutrition 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000015203 fruit juice Nutrition 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 239000002366 mineral element Substances 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
-
- 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
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Environmental Sciences (AREA)
- Biotechnology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Virology (AREA)
- Ecology (AREA)
- Forests & Forestry (AREA)
- Pest Control & Pesticides (AREA)
- Organic Chemistry (AREA)
- Plant Pathology (AREA)
- Biochemistry (AREA)
- Agronomy & Crop Science (AREA)
- Dentistry (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Toxicology (AREA)
- Biomedical Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Botany (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an antagonistic strain Wt-1 of a pseudomonas syringae kiwi fruit pathogenic variant and application thereof, belonging to Steiner Li Bake Hold's bacteria，Preservation number: CGMCC No.27262, preservation date: 2023, 5, 4, deposit unit: china general microbiological culture Collection center, preservation address: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing. Control of kiwi ulcers using strain Wt-1The method for treating the disease comprises the following steps: inoculating the strain Wt-1 into LB culture medium for culturing, obtaining fermentation seed liquid, then performing expansion culture, and finally preparing dilution liquid for spraying plants. The strain is endophytic bacteria obtained by separating and purifying the collected healthy kiwi fruit plants, has strong antagonism on the pathogenic bacteria of the kiwi fruit bacterial canker, namely pseudomonas syringae kiwi fruit pathogeny, can obviously inhibit the growth of the pathogenic bacteria, can effectively prevent and treat the kiwi fruit canker, and provides a new microbial germplasm resource for the biological prevention and treatment of the kiwi fruit canker.
Description
Technical Field
The invention belongs to the technical field of biological control of plant protection, and particularly relates to an antagonistic strain Wt-1 of a pathogenic variation of pseudomonas syringae kiwi fruit and a method for controlling kiwi fruit canker by using the strain.
Background
Kiwi fruitActinidiaLindl) is a perennial vine belonging to the genus actinidia of the family actinidiaceae, known separately as wild peach, sheep peach, kiwifruit (Kiwifruit). The fruits contain rich dietary fibers, vitamins, mineral elements, amino acids and bioactive substances, especially the Vc content is high (90-420 mg in 100 g fresh pulp), and the fruit is a reputation of 'king' of fruits; the kiwi fruit can be eaten fresh, can be processed into jam, fruit juice and the like, has important medical care value, and has wide market prospect.
In recent years, the kiwi fruit industry has been rapidly developed in more than 30 countries worldwide, the total cultivation area is about 20 ten thousand hectares, the yield is up to 240 ten thousand tons, wherein the cultivation area and yield of Chinese kiwi fruit are the largest, respectively account for 53% and 38% of the world share, in Italy, and in New Zealand, the third. Along with the annual increase of the planting area and the gradual expansion of the production scale of the kiwi fruits, the problem of kiwi fruit diseases is also increasingly highlighted. Of these, the most typical is bacterial canker of kiwifruit, which is a disease produced by a pathogenic variant of Pseudomonas syringae kiwifruitPseudomonas syringae pv. actinidiae) The main trunk, branches, tendrils, leaves and the like of the kiwi fruit are mainly damaged, and the kiwi fruit has the characteristics of rapid transmission, strong pathogenicity, high control difficulty and the like, and is extremely easy to cause large-area death of the tree body in a short period. The disease was first discovered in japan in 1984, and has occurred in countries such as the united states, japan, italy, new zealand, korea, and chile, and areas such as the shanxi, chongqing, sichuan, and Hunan of china, causing great economic loss to the world kiwi fruit industry. For example, the export loss of the kiwi fruit canker in New Zealand 2010-2014 is reduced by 9300 hundred million New Zealand, and the kiwi fruit canker loss in China is accumulated to 3.23 hundred million yuan in 2013 only. China, new Zealand, the United states and European Plant Protection Organizations (EPPO) have respectively establishedThe kiwifruit canker is listed as a plant quarantine object and a class A2 quarantine pest to control the rapid spread of the disease.
At present, the measures for controlling the kiwi canker mainly comprise excellent resistant variety breeding, chemical control and biological control besides scientific and standard agricultural management, wherein the biological control has the characteristics of environmental friendliness, human and animal safety, sustainability, strong specificity for pathogenic bacteria and the like, meets the requirements of people on green foods and is favored by a plurality of scholars. Shao Baolin et al (2015) obtain bacillus subtilis B2 from kiwi fruit rhizosphere soil, and both pot culture and field experiments show a certain effect of preventing and treating canker; cui Ligong et al (2017) obtain bacteria (JDG 6, JDG16, JDG 23) from gynostemma pentaphylla rhizosphere soil, which can obviously reduce the occurrence of kiwi fruit canker; kim et al (2019) are separated into 3 actinomycetes from rhizosphere and leaf circumference of kiwi fruits, and have remarkable control effect on canker of kiwi fruits; wicaksono et al (2018) isolated from the medicinal plant M ā nuka to 3 endophytes inhibiting the canker Psa, can reduce the occurrence of canker in the Chinese goosebeery and the like to a certain extent. Although some achievements are achieved in the study on the biological control of kiwi fruit canker, serious disadvantages still exist: at present, most biocontrol strains are derived from soil, kiwi fruit rhizosphere and leaf circumference, and are extremely easily influenced by environmental factors (climate, temperature and humidity, soil property and the like) in the application process, so that the biocontrol effect is unstable and not ideal. Since the endophyte has specificity to the choice of host plant, and is not easy to be interfered by external environment factors. Therefore, the research and development of the kiwi fruit indigenous endophytic bacteria as the canker biocontrol strain thereof has wide application prospect.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a kiwi fruit indigenous endophytic bacterium which is an antagonistic strain of a pseudomonas syringae kiwi fruit pathogenic variety which is a kiwi fruit canker pathogenic bacterium, and the kiwi fruit canker can be effectively prevented and treated by using the strain; meanwhile, the invention also provides a method for biologically preventing and treating kiwi fruit canker by using the strain.
The inventor adopts the kiwi fruit plantation in Hunan Xiangxi area to collectSampling, collecting healthy branches and leaves of Milian No. 1 and red sun kiwi fruits, putting into an ice box, carrying the branches and leaves back to a laboratory, separating and screening the sample to obtain 1 endophytic bacterial strain inhibiting the growth of kiwi fruit canker pathogenic bacteria, namely antagonistic strain of pseudomonas syringae kiwi fruit pathogenic varieties, and identifying the antagonistic strain and then naming the antagonistic strain as Steiner Li Bake Hold's bacteria Wt-1Burkholderia stagnalis Wt-1), and is deposited with a deposit number: CGMCC No.27262, preservation date: 2023, 5, 4, deposit unit: china general microbiological culture Collection center, preservation address: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.
The isolation and screening steps of the strain Wt-1 comprise:
(1) The plantation frequently suffering from kiwi fruit canker in the western Hunan province is taken as a sampling point, the branches and leaves of healthy 'Miliang No. 1' and 'hongyang' kiwi fruit plants are collected and put into an ice box to be brought back to a laboratory for standby. Surface sterilizing the branch and leaf sample, shearing to proper size, adding into sterilized grinding bowl, adding 5-10 mL sterile water, and grinding. Standing for 5-10 min, sucking upper liquid, and sequentially performing 10 -1 Multiple of 10 -2 Multiple of 10 -3 Dilution with multiple gradients; dilutions of 0.1. 0.1 mL were applied to LB medium solid plates, each dilution was repeated 3 times, and the culture was inverted at 28 ℃.
(2) Single colony grows on the plate to be coated, colonies with different forms are picked, and the colonies are respectively inoculated on LB solid culture medium for culture by adopting a four-area streak method; repeating the purification for 3 generations, transferring the culture to an LB solid culture medium inclined surface test tube for culture, and then preserving at 4 ℃.
(3) Inoculating the pathogenic bacteria of the kiwi canker of the pseudomonas syringae and the kiwi fruits to an LB liquid culture medium for culturing 24 h, and sucking the bacterial liquid and coating the bacterial liquid on an LB solid culture medium plate for later use. And (3) selecting single colony points of the separated endophytic bacteria on the plates, inoculating 3-4 sites on each plate, culturing at the constant temperature of 28 ℃, observing the antibacterial condition, and generating a antibacterial zone, namely the antagonistic strain of the pseudomonas syringae kiwi fruit pathogenic variety.
(4) Fermenting and culturing the primary screened antagonistic strain, centrifuging, absorbing 200 mL fermentation liquor, transferring into oxford cup coated with pseudomonas syringae kiwi fruit pathogenic variant plate, observing the size of bacteriostasis zone around oxford cup, and screening strain with strong antagonistic effect. The species were identified based on morphology and 16S rRNA gene sequence analysis.
The endophytic bacteria with excellent effect of antagonizing the pseudomonas syringae and the kiwi fruit pathogenic varieties is the biocontrol strain of the kiwi fruit canker, can be used for biological control of the kiwi fruit canker, and comprises the following steps:
(1) Inoculating antagonistic strains of the pseudomonas syringae kiwi fruit pathogenic varieties into a liquid LB culture medium for culture to obtain fermentation seed liquid;
(2) Inoculating the fermentation seed liquid into a liquid LB culture medium for expansion culture to obtain a fermentation culture liquid.
(3) Preparing a fermentation culture solution into a dilution solution with a certain concentration (comprising thalli, and the bacterial concentration is more than or equal to 1.5X10) 3 cfu/mL, namely the total number of bacterial colonies per milliliter is more than or equal to 1500), aiming at the spraying of kiwi fruit plants, the effective control of kiwi fruit canker can be realized.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a novel kiwi canker biocontrol strain Wt-1, which is an indigenous endophyte strain of kiwi, namely an antagonistic strain of a pathogenic variety of kiwi, namely pseudomonas syringae and kiwi, which is identified and named as Steiner Li Bake Hold's bacteria Wt-1%Burkholderia stagnalis Wt-1）。
The strain is endophytic bacteria obtained by separating and purifying branches of the acquired kiwi fruit plants, has a strong inhibition effect on the pathogenic bacteria of the kiwi fruit bacterial canker, namely pseudomonas syringae and kiwi fruit, can obviously inhibit the growth and propagation of the pathogenic bacteria of the kiwi fruit bacterial canker, can effectively prevent and treat the kiwi fruit canker, and provides a new microbial germplasm resource for the biological prevention and treatment of the kiwi fruit canker.
The strain is a kiwi endophytic bacterium, can mutually and symbiotically with plant tissues, has small interference by external environmental factors, stable performance, convenient spraying and remarkable kiwi ulcer prevention and treatment effect, provides an environment-friendly, simple and effective method for kiwi ulcer prevention and treatment, and is beneficial to environmental protection and organic kiwi production.
Detailed Description
The method for preventing and treating the kiwi fruit canker by using the biocontrol strain Wt-1 and the application strain Wt-1 is further specifically described by combining the specific embodiments.
And 2021, taking a kiwi fruit plantation in the Hunan Xiangxi area as a sampling point, collecting samples of branches and leaves of unaffected 'Miliang No. 1' and 'hongyang' kiwi fruits, taking the samples of the kiwi fruit plantation as sampling points, taking the samples of the kiwi fruit plantation with the longitude and latitude of N26 degrees of 36'31E114 degrees of 07'33, and taking the samples of the branches and leaves of the unaffected 'Miliang No. 1' and 'hongyang' kiwi fruits back to a laboratory for standby in an ice box. Sequentially soaking with 75% ethanol for 1 min, 1% sodium hypochlorite for 5 min, and 75% ethanol for 1 min, respectively sterilizing the surfaces of the branches and leaves, shearing to proper size, placing into sterilized grinding bowl, adding 5-10 mL sterile water, and grinding. Standing for 5-10 min, sucking upper liquid, and sequentially performing 10 -1 Multiple of 10 -2 Multiple of 10 -3 And (5) performing multiple gradient dilution. Respectively coating 0.1-mL gradient dilutions on LB culture medium plates, repeating each dilution for 3 times, inversely culturing in a constant temperature incubator at 28 ℃ for 1-2 d, and growing single colony on the plates to be coated; selecting colonies with different forms, and respectively inoculating the colonies on an LB solid medium for culture by adopting a four-area streak method; repeatedly purifying for 3 generations, transferring LB solid culture medium, performing slant culture, and storing in a refrigerator at 4deg.C.
Culturing pathogenic bacteria of kiwi canker, pseudomonas syringae and kiwi fruit pathogenic variety overnight, taking 50 mu L of bacterial liquid, and coating on LB culture medium for standby. And (3) selecting single colony points of the separated endophytic bacteria on the plates, inoculating 3-4 sites on each plate, culturing at the constant temperature of 28 ℃, observing the antibacterial condition after 2-3 d, and generating an antibacterial zone, namely the antagonistic strain of the pseudomonas syringae kiwi fruit pathogenic variety.
Inoculating the strain with strong antibacterial activity into 5 mL LB liquid medium, shake culturing at 28deg.C and 180 r/min for 24 h to obtain seed liquid, then taking 100 μL, inoculating into 50 mL LB liquid medium, culturing at 28deg.C and 180 r/min for 48 h, transferring the fermentation liquid into sterile centrifuge tube, centrifuging at 4deg.C and 10000 r/min for 10 min, centrifuging, and filtering with 0.22 μm microporous membrane to obtain supernatant. Meanwhile, the pathogenic bacteria pseudomonas syringae and kiwi fruit pathogenic varieties are cultured overnight, and 50 mu L of bacterial liquid is coated on an LB culture medium for standby.
Placing oxford cup on a plate coated with pathogenic bacteria, transferring 200 μl of supernatant into oxford cup, repeating each strain for 3 times, culturing at 28deg.C at constant temperature, and observing antibacterial condition after 2-3 d to obtain a good strain with antibacterial circle diameter up to 1.5 times of colony diameter.
The strain is cultured in LB culture medium for 24 h, and has smaller colony (1-2 mm), milky white, round, smooth and complete edge, raised center and opaque colony. Extracting and separating total DNA of the bacterial strain by using a bacterial genome DNA extraction kit, and carrying out PCR amplification on 16S rRNA gene segments of the separated bacterial strain by using a bacterial universal primer PA/PB (PA: 5'-AGAGTTTGATCCTGGCTC A G-3'; PB: 5'-TTAAGGTGATCCA GCCGCA-3'), wherein the reaction system is as follows: taq PCR Mix (2×) 25 μl; 1 mu L of each forward primer and reverse primer; 2 mu L of template; sterile water was filled to 50 μl. The PCR procedure was: pre-denaturation at 95℃for 2.5 min; denaturation at 95℃for 15 s, annealing at 55℃for 30 s, elongation at 72℃for 1 min,30 cycles; extending for 10 min at 72 ℃. The PCR amplified products were subjected to agarose gel electrophoresis and then sent to the biological engineering (Shanghai) Co., ltd for sequencing. Submitting the detected sequence to GenBank (http:// www.ncbi.nlm.nih.gov) database, searching homologous sequences by Blast tool software, comparing, constructing a phylogenetic tree by using the neighbor joining method (NJ) of Mega7.0 software, and identifying and naming the strain as Steiner Li Bake Hold's bacteria Wt-1 according to phylogenetic relation analysisBurkholderia stagnalis Wt-1), and is deposited with a deposit number: CGMCC No.27262, preservation date: 2023, 5, 4, deposit unit: china general microbiological culture Collection center, preservation address: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.
The biocontrol strain for kiwi fruit canker is used for biological control of kiwi fruit canker, and comprises the following specific implementation steps:
inoculating the strain Wq-1 into 100 mL LB liquid medium, and carrying out shaking culture at 28 ℃ and 180 r/min for 24 h to obtain fermentation seed liquid; inoculating the fermentation seed liquid into 1L LB liquid medium at 1% inoculum size, shake culturing at 28deg.C and 180 r/min, and culturing whenOD 600 And harvesting the fermentation culture solution when the fermentation culture solution reaches 1.0.
The fermentation broth was prepared as a treatment group of 1,1.6% thiazolone 800-fold dilution, as a treatment group of 2, 72% agricultural streptomycin 800-fold dilution, as a treatment group of 3, and sterile clear water as a control group (treatment group of 4) at a concentration of dilution (including thalli, bacterial concentration of about 1570 cfu/mL, i.e., about 1570 total bacterial colonies per milliliter). And (3) field efficacy test: 4 red sun kiwi fruit gardens with 3-5 a tree ages are selected, 4 cells are randomly divided in each orchard for 4 treatments, 50 kiwi fruits are treated each time, 4 orchards are repeated, and 200 kiwi fruits are investigated in each group. And in the key period from fruit picking in autumn to defoliation, spraying the whole kiwi fruit plant with each corresponding medicament for 3 times at intervals of 10 d. And counting the percentage of the trunk, yellow brown or rust red ulcer disease plants in each treatment group in the high-incidence period (2-3 months) of the kiwi ulcers in the spring, taking the percentage as the disease rate of the ulcer disease rate, and calculating the field disease prevention effects of different treatments according to the following formula.
Disease preventing effect= (control morbidity-treatment morbidity)/control morbidity x 100%
Through statistics, the average inhibition rate of the field efficacy test of the strain Wt-1 group (treatment group 1) reaches 89.5%, the average inhibition rate of the field efficacy test of the 1.6% thiazolone 800-time diluent group (treatment group 2) is 75.6%, and the average inhibition rate of the field efficacy test of the 72% agricultural streptomycin 800-time diluent group (treatment group 3) is 69.7%.
Further field control experiments prove that: when the concentration of the strain Wt-1 fermentation culture solution reaches 2.5X10 3 When cfu/mL is carried out, the prevention and treatment effect of the kiwi canker is greatly enhanced, and the average inhibition rate can reach 93.4 percent, which is far higher than 1.6 percent of 800-time diluted thiazolone and 72 percent of 800-time diluted agricultural streptomycin. Therefore, the Steiner Li Bake Hold's bacillus Wt-1 has very ideal control effect on kiwi fruit canker, and is an excellent rhesusBiocontrol strain of kiwi canker.
Claims (2)
1. Antagonistic strain Wt-1 of pathogenic variant of actinidia lilac pseudomonas belongs to Steiner Li Bake Hold's bacteriaBurkholderia stagnalis) Is a biological control strain for kiwi canker, and the preservation number is as follows: CGMCC No.27262, preservation date: 2023, 5, 4, deposit unit: china general microbiological culture Collection center, preservation address: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.
2. A method for preventing and treating kiwi fruit canker by using antagonistic strain Wt-1 of pseudomonas syringae kiwi fruit pathogenic variety is characterized by comprising the following steps:
(1) Inoculating the strain Wt-1 into an LB liquid culture medium for culture to obtain fermentation seed liquid;
(2) Inoculating the fermentation seed liquid into an LB liquid culture medium for expansion culture to obtain a fermentation culture liquid;
(3) Preparing a dilution liquid with a certain concentration (the concentration of the thallus is more than or equal to 1.5X10) 3 cfu/mL), aiming at kiwi fruit plants.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311371404.7A CN117363537A (en) | 2023-10-23 | 2023-10-23 | Antagonistic strain Wt-1 of pseudomonas syringae kiwi fruit pathogenic variety and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311371404.7A CN117363537A (en) | 2023-10-23 | 2023-10-23 | Antagonistic strain Wt-1 of pseudomonas syringae kiwi fruit pathogenic variety and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117363537A true CN117363537A (en) | 2024-01-09 |
Family
ID=89388746
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311371404.7A Pending CN117363537A (en) | 2023-10-23 | 2023-10-23 | Antagonistic strain Wt-1 of pseudomonas syringae kiwi fruit pathogenic variety and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117363537A (en) |
-
2023
- 2023-10-23 CN CN202311371404.7A patent/CN117363537A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112175888B (en) | Bacillus belgii Hsg1949 and application thereof | |
| CN108148794B (en) | Bacillus subtilis DYr3.3 with broad-spectrum antibacterial activity, and preparation method and application thereof | |
| CN110628687B (en) | Streptomyces 5017 and application thereof in antagonism of phytopathogens | |
| CN108148793B (en) | Paenibacillus polymyxa DYr4.4 with broad-spectrum antibacterial activity and preparation method and application thereof | |
| CN106479938B (en) | A kind of Brevibacillus brevis bacterial strain and its application | |
| CN117165494A (en) | Kiwi fruit canker biocontrol strain Wq-1 and application thereof | |
| CN109749953B (en) | Bacillus cereus, microbial inoculum and preparation method and application thereof | |
| CN113151044B (en) | Pseudomonas for preventing and treating diseases of radix pseudostellariae and separation, screening and identification method and application | |
| CN113755389A (en) | Bacillus belgii and application thereof | |
| CN115960777B (en) | Bacillus pseudomycoides and application thereof in prevention and treatment of vegetable epidemic disease | |
| CN108441443B (en) | Strain for preventing and treating plant nematodes and application thereof | |
| CN108102992B (en) | Microbacterium aurantiacus and application thereof in prevention and treatment of tomato root-knot nematodes | |
| CN114032182B (en) | Fungus with functions of antagonizing pathogenic bacteria of garlic root rot and promoting growth | |
| CN114085788B (en) | Pseudomonas amazonensis strain and application thereof | |
| CN116240126A (en) | Multifunctional bacillus belgium SB10 and application thereof | |
| CN112322561B (en) | Klebsiella and application thereof in prevention and treatment of pear fire blight of fruit trees | |
| CN114467975A (en) | Application of staphylococcus equi in prevention and treatment of fruit and vegetable diseases | |
| CN111304135B (en) | Bacillus and application thereof in plant disease control | |
| CN109207398B (en) | Pseudomonas stutzeri and application thereof | |
| CN110607249B (en) | Streptomyces biocontrol strain and application thereof | |
| CN113416679A (en) | Bacillus methylotrophicus, microbial inoculum comprising bacillus methylotrophicus and application of bacillus methylotrophicus | |
| CN117363537A (en) | Antagonistic strain Wt-1 of pseudomonas syringae kiwi fruit pathogenic variety and application | |
| CN117106677A (en) | Bacillus saxifrage Ws-1 for antagonizing kiwi canker pathogenic bacteria and application thereof | |
| CN117363534A (en) | Streptomyces lilacinus strain Wk-1 for preventing and treating kiwi canker and application thereof | |
| KR20090105726A (en) | Bacillus megaterium isolate 22-5 controlling bacterial spot and anthracnose of red-pepper |
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 |