CN116590152A - Fusarium brick and application thereof - Google Patents
Fusarium brick and application thereof Download PDFInfo
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- CN116590152A CN116590152A CN202310544085.9A CN202310544085A CN116590152A CN 116590152 A CN116590152 A CN 116590152A CN 202310544085 A CN202310544085 A CN 202310544085A CN 116590152 A CN116590152 A CN 116590152A
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- fusarium
- banana
- dpi
- wilt
- ynf2101
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- 241000223218 Fusarium Species 0.000 title claims abstract description 40
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 title claims abstract description 39
- 239000011449 brick Substances 0.000 title description 7
- 241000234295 Musa Species 0.000 claims abstract description 89
- 235000018290 Musa x paradisiaca Nutrition 0.000 claims abstract description 86
- 241000223221 Fusarium oxysporum Species 0.000 claims abstract description 39
- 240000000905 Nymphoides indica Species 0.000 claims abstract description 38
- 235000017590 Nymphoides indica Nutrition 0.000 claims abstract description 38
- 241000221779 Fusarium sambucinum Species 0.000 claims abstract description 20
- 230000001737 promoting effect Effects 0.000 claims abstract description 17
- MYSWGUAQZAJSOK-UHFFFAOYSA-N ciprofloxacin Chemical compound C12=CC(N3CCNCC3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 MYSWGUAQZAJSOK-UHFFFAOYSA-N 0.000 claims abstract description 15
- 241000223197 Fusarium lateritium Species 0.000 claims abstract description 8
- 230000002792 vascular Effects 0.000 claims description 42
- 238000000034 method Methods 0.000 claims description 26
- 239000001963 growth medium Substances 0.000 claims description 22
- 239000007788 liquid Substances 0.000 claims description 19
- 239000002068 microbial inoculum Substances 0.000 claims description 18
- 239000002609 medium Substances 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 9
- 238000009630 liquid culture Methods 0.000 claims description 9
- 241000567178 Fusarium venenatum Species 0.000 claims description 7
- 230000008635 plant growth Effects 0.000 claims description 7
- 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 claims description 5
- 239000008103 glucose Substances 0.000 claims description 5
- 244000061456 Solanum tuberosum Species 0.000 claims description 4
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 4
- 230000002401 inhibitory effect Effects 0.000 claims description 4
- 230000000813 microbial effect Effects 0.000 claims description 2
- 239000002054 inoculum Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 35
- 230000012010 growth Effects 0.000 abstract description 21
- 238000004321 preservation Methods 0.000 abstract description 6
- 244000005700 microbiome Species 0.000 abstract description 3
- 238000011282 treatment Methods 0.000 description 43
- 241000196324 Embryophyta Species 0.000 description 33
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 23
- 244000052769 pathogen Species 0.000 description 23
- 244000052616 bacterial pathogen Species 0.000 description 22
- 238000000855 fermentation Methods 0.000 description 15
- 230000004151 fermentation Effects 0.000 description 15
- 230000001717 pathogenic effect Effects 0.000 description 11
- 238000011081 inoculation Methods 0.000 description 9
- 239000007787 solid Substances 0.000 description 9
- 230000001580 bacterial effect Effects 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 230000005764 inhibitory process Effects 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 6
- 238000003973 irrigation Methods 0.000 description 6
- 230000002262 irrigation Effects 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 230000002265 prevention Effects 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 241000233866 Fungi Species 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 230000009545 invasion Effects 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
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- 208000015181 infectious disease Diseases 0.000 description 3
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- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
- 239000008223 sterile water Substances 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000003385 bacteriostatic effect Effects 0.000 description 2
- 235000021015 bananas Nutrition 0.000 description 2
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- 241000894007 species Species 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 241001477352 Bengalia Species 0.000 description 1
- 241000016649 Copaifera officinalis Species 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 108091023242 Internal transcribed spacer Proteins 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 241000845082 Panama Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 230000009418 agronomic effect Effects 0.000 description 1
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- 238000000137 annealing Methods 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 208000027697 autoimmune lymphoproliferative syndrome due to CTLA4 haploinsuffiency Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010876 biochemical test Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000010455 vermiculite Substances 0.000 description 1
- 235000019354 vermiculite Nutrition 0.000 description 1
- 229910052902 vermiculite Inorganic materials 0.000 description 1
- 230000003442 weekly effect Effects 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- 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
- A01N63/30—Microbial fungi; 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
- A01P21/00—Plant growth regulators
-
- 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
- A01P3/00—Fungicides
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- 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
- C12N1/14—Fungi; Culture media therefor
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- 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
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/77—Fusarium
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Pest Control & Pesticides (AREA)
- Virology (AREA)
- Botany (AREA)
- Plant Pathology (AREA)
- Environmental Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
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Abstract
The invention relates to the field of microorganisms, and discloses Fusarium rubrum (Fusarium lateritium) and application thereof, wherein the Fusarium rubrum has a preservation number of CCTCC NO: M20221723. Fusarium roseum can inhibit the activity of the fusarium oxysporum gulf of banana fusarium, so that banana fusarium wilt can be prevented and treated, and in addition, the Fusarium roseum has a remarkable growth promoting effect on banana plants.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to Fusarium roseum and application thereof.
Background
Bananas (Musa spp.) are important fruits in tropical and subtropical regions of the world, 130 countries are totally occupied worldwide for planting bananas, and the planting amount on the world is inferior to rice, wheat and corn, and is an important food crop in developing countries. Banana wilt (Fusarium Wilt of banana) caused by fusarium oxysporum copaiba specialization (Fusarium oxysporum f.sp.cube, foc) is a worldwide destructive disease, also known as panama disease, yellow mosaic disease. Pathogenic bacteria survive in the soil for years, generally by over 20% and severely field is even dead. The disease causes the wilting and drooping of the plant stems and leaves to be all withered, and is one of the plant soil-borne diseases with the greatest hazard, the greatest distribution and the most serious loss in the world. Infection of banana vascular wilt external symptoms: the disease plant in the adult stage presents special yellow in the lower leaf and the outer leaf sheath, and occurs in the edge of the leaf in the early stage, and then expands gradually to the middle rib, which is obviously compared with the dark green part of the leaf. There are also whole leaves that yellow, the susceptible leaves rapidly wither, turn brown from yellow and dry, the last top leaves of which are often withdrawn late or not withdrawn, and the disease plants die. Infection of internal symptoms of banana vascular wilt: the etiology of banana wilt belongs to vascular bundle diseases, internal symptoms are obvious, vascular bundles with yellow-red lesions are formed at and around the central medulla, the lesions are darker at the base of the stems, the root wooden ducts become reddish brown and gradually become black brown and dry, and the corms become black brown and gradually decay, so that the banana wilt has special odor.
Banana vascular wilt was first found in australia in 1874 and outbreaks in several countries in south america in 1935-1939 have been spread to all banana distribution areas worldwide, except for the new banbuia, the south pacific islands and some mediterranean coastal countries. In 1960, banana wilt occurs in the places of China such as Guangxi nan Ning, \37013 Ning, bobai and the like, and the banana wilt is spread over the provinces of China such as Guangdong, guangxi, fujian, yunnan, hainan and the like for banana planting. Banana wilt seriously restricts the development of banana industry, and the incidence rate can reach more than 90% when serious, so that banana gardens are abandoned. Research and exploration of a method for efficiently and safely preventing banana vascular wilt is urgent. Banana wilt is taken as a soil-borne disease, can be transmitted through seedlings with diseases and different media (water, air, soil, tools and the like), and the wilt pathogen chlamydospores can survive continuously for thirty years in infected soil, so far, no effective prevention and control measures exist. At present, the control of banana vascular wilt mainly comprises biological control, breeding of disease-resistant varieties, chemical control, rotation, molecular biology and other methods. Although the breeding of the disease-resistant variety achieves a certain disease-resistant effect, the period is long, the cost is high, and the disease resistance and the agronomic characters are difficult to be considered; the chemical control achieves better control effect, but the problems of pesticide residue, environmental pollution and the like are difficult to solve; because of a large number of uncontrollable factors of the field environment, the methods such as rotation and molecular biology and the like do not achieve ideal control effects in production practice. The green prevention and control technology of banana wilt is an urgent need for maintaining ecological environment and ecological balance, and biological prevention and control are carried out by utilizing beneficial microorganisms, so that the technology is one of the effective measures for preventing and controlling banana wilt at present. Therefore, it is important to find a method for preventing and controlling banana vascular wilt which is efficient and meets the requirements of environmental protection and agricultural sustainable development.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provides Fusarium rubrum and application thereof.
In order to achieve the aim, the invention provides a fusarium venenatum (Fusarium lateritium) with a preservation number of CCTCC NO: M20221723.
The second aspect of the invention provides a microbial inoculum comprising the Fusarium rubrum.
The third aspect of the invention provides the application of the fusarium oxysporum or the microbial inoculum in inhibiting fusarium oxysporum cubeba specialization (Fusarium oxysporum f.sp.cube).
In a fourth aspect, the present invention provides a method for controlling banana vascular wilt and promoting banana plant growth, the method comprising: the fusarium venenatum or the microbial inoculum is cultured and then applied to banana plants.
In a fifth aspect, the invention provides the use of Fusarium rubrum or the microbial inoculum or the method in preventing and treating banana vascular wilt and promoting banana plant growth.
The Fusarium roseum disclosed by the invention can inhibit the activity of the fusarium oxysporum dedicated type of banana fusarium wilt pathogenic bacteria, so that banana fusarium wilt can be prevented and treated, and in addition, the Fusarium roseum has a remarkable growth promoting effect on banana plants.
Preservation of organisms
Fusarium roseum (Fusarium lateritium) of the invention is preserved in China center for type culture Collection (address: china, WU-Han, university of Wu Han, postal code: 430072) (abbreviated as CCTCC of preservation unit) at 4/11/2022, and the preservation number is CCTCC NO: M20221723.
Drawings
FIG. 1 shows the bacteriostatic effect of Fusarium roseum on banana vascular wilt pathogens;
FIG. 2 shows the growth of pathogenic bacteria of banana vascular wilt;
FIG. 3 is YNF2101+TR4 plants and bulb growth;
FIG. 4 shows CK+TR4 plants and bulb growth;
FIG. 5 is 45dpi YNF2101 plant and subsurface growth;
FIG. 6 shows 45dpi CK plants and subsurface growth.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
The invention provides a fusarium venenatum (Fusarium lateritium), and the fusarium venenatum has a preservation number of CCTCC NO: M20221723.
Fusarium roseum of the invention is derived from endophytic fungi separated from banana bulbs of wild resource species. On the plate culture medium, the colony is flocculent, the hypha is compact, the white is gray, and the edge of the colony is neat. Conidiophores are generated on aerial mycelium, are colorless and have two types of spores of size and shape. The large conidium is straight or sickle-shaped, the top cell is bent or bird's beak-shaped, and the basal part has buds, which are mostly 3-5 separated. Small conidia are oval or fusiform, single cell or with 1 septum.
The second aspect of the invention provides a microbial inoculum comprising the Fusarium rubrum.
The third aspect of the invention provides the application of the fusarium oxysporum or the microbial inoculum in inhibiting fusarium oxysporum cubeba specialization (Fusarium oxysporum f.sp.cube).
In the present invention, the form of the microbial inoculum may be a form of microbial inoculum conventional in the art, for example, may be a solid, liquid or semisolid form.
In the invention, the microbial inoculum contains the Fusarium rubrum.
In the invention, the number of living bacteria in the microbial inoculum can be selected in a wider range, so long as the requirements of related standards are met. Taking liquid microbial inoculum as an example, the content of living microbial cells in the microbial inoculum is 1 multiplied by 10 6 cfu/mL and above.
The preparation method of the microbial inoculum can refer to the conventional preparation method in the field, and is not described herein.
In a fourth aspect, the present invention provides a method for controlling banana vascular wilt and promoting banana plant growth, the method comprising: the fusarium venenatum or the microbial inoculum is cultured and then applied to banana plants.
Preferably, the preparation method of the fermentation broth comprises the following steps: inoculating the Fusarium roseum into a liquid culture medium for culture.
In the present invention, the liquid medium is not limited, and can be used for smooth fermentation of Fusarium roseum, preferably PDA liquid medium, more preferably, the liquid medium contains: glucose 10-20g/L, peeled potato 150-250g/L. More preferably, the pH of the liquid medium is from 6 to 8. Further preferably, the culturing conditions include: the time is 3-7 days, and the temperature is 25-34 ℃.
In the invention, in order to enable the fusarium oxysporum to ferment better, the shaking can be carried out in the culture process, and the shaking speed is 200-300r/min.
In the invention, the fusarium oxysporum can be activated before being inoculated into a liquid culture medium for culture, and the activation mode is that the fusarium oxysporum is inoculated into a solid culture medium. Inoculating the strain into liquid culture medium after growing out of the strain.
In the invention, the solid culture medium is not limited, and can enable Fusarium rubrum to be activated smoothly, preferably a PDA solid culture medium, more preferably the solid culture medium contains 10-20g/L glucose, 150-250g/L peeled potatoes and 10-20g/L agar. More preferably, the pH of the solid medium is from 6 to 8. Further preferably, the culture conditions of the solid medium include: the time is 1-5 days, and the temperature is 25-34 ℃.
In a fifth aspect, the invention provides the use of Fusarium rubrum or the microbial inoculum or the method in preventing and treating banana vascular wilt and promoting banana plant growth.
Examples
1. Preparation and identification
(1) Material source
The Fusarium brick (Fusarium lateritium) CCTCC NO: M20221723 is endophytic fungi separated from banana corms of wild source species of Yunnan, lushan, yunnan province. Numbered YNF2101.
(2) Culture medium
Bengalhong medium: 5g of peptone, 1g of monopotassium phosphate, 0.5g of magnesium sulfate, 10g of glucose, 0.1g of chloramphenicol, 0.033g of Bengalum red, 20g of agar, 1000mL of distilled water and pH 7.
PDA medium: 15g of glucose, 15g of agar, 200g of peeled potatoes, 1000mL of distilled water and pH 7. Agar-free PDA liquid culture medium was used.
(3) Methods of obtaining, identifying and culturing
Sample disinfection treatment: taking banana plant bulbs in advance, washing with sterile water, then carrying out surface disinfection for 60s by using 75 weight percent ethanol, and finally washing with sterile water and airing.
And (3) separating and culturing: after the banana bulbs are disinfected, the root system is split by a sterile knife under the sterile condition, the internal tissues of the bulbs are smashed and ground into liquid in a sterile mortar, a sterile inoculating loop is used for streak culture on a Bengalia red culture medium, the bangalia red culture medium is subjected to constant temperature culture for 3d at 28 ℃, and after single colonies are selected and purified on a PDA culture medium for 3 times, the bangalia red culture medium is put in a refrigerator at 4 ℃ for standby.
And (5) classifying and identifying: and (3) adopting a fungus culture method, and observing culture characters and morphological characteristics of the fungus obtained by separation and performing ITS amplification and sequence analysis. The observation and identification of culture traits and morphological characteristics were carried out in reference (Fangzhong Dai plant disease research method [ M ]. Beijing: china agricultural Press, 1998.).
ITS amplification and sequence analysis: genomic DNA was extracted using a fungal genomic DNA extraction kit, and the partial sequence of fungal rDNA-ITS gene was amplified by PCR using the universal primers ITS1/ITS4 (Kunming division of Beijing qing department Biotechnology Co., ltd.) (5'-TCCGTAGGTGAACCTGCGG-3' (SEQ ID NO: 1)/5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID NO: 2)), and the PCR product was sent to the Beijing qing department Biotechnology Co., ltd., sequencing. PCR amplification was performed using this as a template. The PCR reaction system was 50. Mu.L: 2 XMastermix 25. Mu.L, DNA template 1. Mu.L, upstream and downstream primers 1. Mu.L each, double distilled water 22. Mu.L. The reaction amplification conditions are as follows: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 1min for 33 cycles; extending at 72℃for 10min. After sequencing the PCR products, BLAST alignment was performed on the sequencing results at NCBI website, and phylogenetic tree was constructed using MEGA7.0 software using the adjacency method. Classification search reference: chai Zhaoxiang, li Jinhua, xin Jianfeng. Fusarium roseum biological characterization research [ J ]. Plant pathogenicity journal 2004 (5): 409-413. In the recipe, plant diseases research method [ M ] Beijing, china agricultural Press, 1998.
PCR amplification is carried out by taking the total genome DNA of the strain as a template to obtain a PCR product of about 600kb, and Blast comparison is carried out on the sequencing result in NCBI, so that the sequence similarity of the strain and the strain Fusarium lateritium is 90%. The phylogenetic tree is constructed through MEGA5.0 software, and the result shows that the bacterial strain has the closest relationship with Fusarium rubrum (Fusarium lateritium) and is clustered into one. The strain is determined to be Fusarium rubrum through culture characteristics, morphological observation, physiological and biochemical tests and ITS sequence analysis. The strain is numbered YNF2101.
2. Detection of
(1) The invention relates to a test for determining the bacteriostatic activity of Fusarium brick on banana vascular wilt pathogenic bacteria
The bacterial inhibition activity of the separated and purified endophytic fungus Fusarium rubrum is measured by using a conventional counter culture method by taking a banana fusarium wilt pathogen No. 4 race (Foc for short) as an indicator bacterium. The pathogenic bacteria of banana vascular wilt are Fusarium oxysporum Guba specialization (Fusarium oxysporum f.sp.cube) (Foc) No. 4 physiological race strain Foc-1, which is separated, identified and stored by banana research team of agricultural environmental resource institute of the agricultural sciences of Yunnan province. The banana fusarium wilt pathogenic bacteria culture medium is PDA culture medium.
The bacterial inhibition rate of the bacterial strain is measured by adopting a flat plate counter method, banana fusarium wilt pathogenic bacteria are transferred onto a PDA flat plate, culturing is carried out for 7d at 28 ℃, a bacterial cake with the diameter of 5mm is taken along the edge of a bacterial colony by a sterilized puncher, the bacterial cake is inoculated in the center of the PDA flat plate, then fusarium brick hyphae are picked up by an inoculating loop and are symmetrically connected with 4 points at the position 25mm away from the center of the PDA flat plate, the treatment is repeated for 3 times, the PDA flat plate which is only inoculated with the pathogenic bacteria to be measured is used as a reference, the bacterial inhibition effect is observed after culturing is carried out for 7d at 28 ℃, and the diameter of the banana fusarium wilt pathogenic bacteria (Foc-1) is measured by a cross method. Antibacterial ratio (%) = (control pathogen colony diameter-treated pathogen colony diameter)/control pathogen colony diameter x 100.
Results: the diameters of the 3 times of pathogenic bacteria colonies are respectively 2.60cm, 2.45cm and 2.55cm; the diameters of 3 pathogenic bacteria colonies of the control are 8.85cm, 8.90cm and 8.85cm respectively, the 3 repeated bacteriostasis rates are 70.62%, 72.47% and 71.19%, and the average bacteriostasis rate of Fusarium rubrum on banana vascular wilt pathogens is 71.43%. It has strong inhibiting effect on the growth of pathogenic bacteria of banana vascular wilt.
(2) Fusarium roseum potted plant control effect and growth promotion effect determination for banana vascular wilt
The preparation method of the banana seedlings for the test comprises the following steps: in a plastic greenhouse, cleaning the culture medium of the root of a tissue-cultured Brazilian banana seedling (the tissue-cultured seedling of banana research laboratory of agricultural environmental resource institute of the agricultural sciences of Yunnan province), transplanting the banana seedling into a seedling bag, and transplanting the banana seedling into a plastic basin with the diameter of 11cm and the height of 12cm and taking vermiculite as a matrix after 3-4 leaves (about 1 month) of the banana seedling grow out. After transplanting, water is sprayed frequently for moisturizing, and fertilizer is applied weekly (each plant is applied by 2 g/time of compound fertilizer dissolved in water, and the mass fraction of N-P in the compound fertilizer is calculated 2 O 5 -K 2 The content of O is 15-15 wt%) 1-2 times. After 5-6 leaves (about 1 month) of banana seedlings grow out, the banana seedlings are ready for use.
Preparation of a test Strain, fusarium roseum fermentation broth in the present invention: inoculating the preserved strain YNF2101 on a solid PDA culture medium plate by streaking, placing the solid PDA culture medium plate in a constant temperature incubator at 28 ℃ for activation culture for 3d, inoculating activated antagonistic fungus cakes into a liquid PDA culture medium, shake-culturing the liquid PDA culture medium at 28 ℃ with a 250r/min shaking table for 5d, and filtering the culture solution with 4 layers of sterile gauze to obtain a strain fermentation broth. Preparing sterile water to obtain 1×10 concentration 6 cfu/mL of the strain fermentation broth is ready for use.
The method for determining the potted plant control effect and growth promotion effect of banana vascular wilt comprises the following steps: the strain fermentation liquor (Fusarium roseum fermentation liquor: concentration is 1X 10) for resisting banana vascular wilt 6 cfu/mL) was irrigated onto roots of the potted banana plants of consistent growth vigor, 40mL each, and each was irrigated against40mL PDA liquid culture. After 7d, the concentration was 1X 10 6 cfu/mL of banana vascular wilt pathogenic bacteria spore liquid is irrigated onto the roots of potted banana plants, 40mL of each plant is irrigated, and PDA liquid culture liquid is used as a control.
Not inoculated with banana vascular wilt pathogen: the test equipment (1) irrigates PDA liquid culture solution (CK) and (2) irrigates fusarium oxysporum (YNF 2101) to inoculate banana fusarium wilt pathogenic bacteria: the test equipment (3) irrigates PDA liquid culture solution, banana fusarium wilt pathogenic bacteria spore solution (CK+TR 4) and (4) irrigates Fusarium oxysporum fermented solution, banana fusarium wilt pathogenic bacteria spore solution (YNF 2101+TR 4). Investigation of the biological information indexes of the plants treated in the steps (1) and (2), and calculation of the growth promoting effect; investigation of the disease conditions of treatment (3) and (4), statistics of the disease indexes of each treatment, and calculation of the prevention and treatment effects. The above 4 treatments were performed simultaneously, each with 3 replicates of 10 banana seedlings. Measuring the bioinformatics indexes such as plant height, pseudostem diameter, leaf number and the like of banana plants in each treatment (0 dpi CK, 0dpi YNF2101, 0dpi CK+TR4, 0dpi YNF2101+TR4) on the same day when the fusarium oxysporum (R) brick fermentation liquor is poured, and measuring the bioinformatics indexes such as plant height, pseudostem diameter, leaf number, fresh weight of overground parts, fresh weight of underground parts and the like of banana plants in each treatment (45 dpi CK, 45dpi YNF2101, 45dpi CK+TR4 and 45dpi YNF2101+TR4) after inoculating banana fusarium wilt pathogen for 45d, and calculating the growth promoting effect of the fusarium oxysporum on the banana plants by investigating the bioinformatics indexes treated in (1) and (2); and (3) calculating the growth promoting effect of Fusarium roseum on banana plants under the condition of banana fusarium wilt pathogen infection by investigating the bioinformatics indexes treated in the steps (3) and (4).
Measurement method
Plant height: measuring the distance from the ground to the intersection of the top two petioles;
the diameter of the false stem is measured by a vernier caliper to measure the diameter of the base of the false stem which is about 1cm away from the ground;
leaf length and leaf width: measuring the leaf length and leaf width of the first expanded leaf;
the drug effect is calculated by adopting the following two formulas:
table 1 shows the classification standard of banana vascular wilt disease
TABLE 1
Results:
leaf disease index and control effect of fusarium oxysporum fermented liquid on 3 repeated leaf disease indexes of banana vascular wilt are respectively as follows: 20.00, 17.50, on average 19.17; the leaf disease index for the 3 replicates of the control was: 60.00, 67.50, 72.50, on average 66.67; the potted plant prevention and control effects of the leaves are 66.67%, 70.37% and 75.86% respectively, and the average is 70.97%. The specific control effect is shown in Table 2.
Plants and bulbs of YNF2101+TR4 are shown in FIG. 3, and plants and bulbs of CK+TR4 are shown in FIG. 4.
Disease finger and control effect of bulb: the bulb disease indexes of the fusarium oxysporum fermented liquid for 3 times of banana vascular wilt are respectively as follows: 10.00, 12.50, 5.00, average 9.17; the bulb disease index for the 3 replicates of the control was: 70.00%, 85.00%, 77.5%, average 77.50; the potted plant prevention and control effects of the corms are 85.71%, 85.29%, 93.55% respectively, and the average is 88.18%. The specific control effect is shown in Table 2.
TABLE 2
Note that: a and b are symbols commonly used in the statistical arts to represent significance, the same column having the same symbol indicates no significance, and the absence of the same symbol indicates significant difference.
Growth promoting effect on the number of leaves: the number of blades for treatment CK (0 dpi CK) was 4.23, the number of blades for treatment YNF2101 (0 dpi YNF 2101) was 4.10, the number of blades for CK+TR4 (0 dpi CK+TR4) was 4.27, and the number of blades for treatment YNF2101+TR4 (0 dpi YNF 2101+TR4) was 4.03; the number of leaves for treatment CK (45 dpi CK) was 5.03, the number of leaves for treatment YNF2101 (45 dpi YNF 2101) was 5.47, the number of leaves for treatment CK+TR4 (45 dpi CK+TR4) was 3.3, and the number of leaves for treatment YNF2101+TR4 (45dpi YNF2101+TR4) was 4.97. Analysis of results: without inoculation with banana vascular wilt pathogens, the leaf number of the treatment (45 dpi YNF 2101) was significantly different from that of the control (45 dpi CK) and from that of all the treated leaves inoculated with banana vascular wilt pathogens. The fusarium oxysporum f.sp.brick fermentation liquid has no inhibition effect on the growth of banana plant leaves, can inhibit the invasion hazard of banana fusarium wilt pathogenic bacteria on banana plants, and has promotion effect on the number of banana plant leaves. The specific growth-promoting effect is shown in Table 3. Plants treated with YNF2101 (45 dpi YNF 2101) are shown in FIG. 5, and plants of CK (45 dpi CK) are shown in FIG. 6.
Growth promoting effect on plant height: the height of treated CK (0 dpi CK) is 14.91cm, the height of treated YNF2101 (0 dpi YNF 2101) is 14.02cm, the height of CK+TR4 (0 dpi CK+TR4) is 14.57cm, and the height of treated YNF2101+TR4 (0 dpi YNF 2101+TR4) is 14.34cm; the height of treated CK (45 dpi CK) was 21.70cm, the height of treated YNF2101 (45 dpi YNF 2101) was 25.08cm, the height of treated CK+TR4 (45 dpi CK+TR4) was 15.30cm, and the height of treated YNF2101+TR4 (45dpi YNF2101+TR4) was 21.85cm. Analysis of results: without inoculation of banana vascular wilt pathogens, the plant height of Fusarium roseum (45 dpi YNF 2101) was significantly different from the control (45 dpi CK) and from all treatments with banana vascular wilt pathogens. The fusarium oxysporum f.bricius fermentation liquid has no inhibition effect on the plant height growth of banana plants, can inhibit invasion hazard of banana fusarium wilt pathogenic bacteria to banana plants, and has obvious promotion effect on the plant height of banana plants. The specific growth-promoting effect is shown in Table 3.
Promoting growth of pseudo-stem thickness: the diameter of the dummy stem of treatment CK (0 dpi CK) is 1.49cm, the diameter of the dummy stem of treatment irrigation YNF2101 (0 dpi YNF2216+CK) is 1.52cm, the diameter of the dummy stem of CK+TR4 (0 dpi CK+TR4) is 1.49cm, and the diameter of the dummy stem of treatment YNF2101+TR4 (0 dpi YNF2101+TR4) is 1.50cm; 1.86cm for treatment CK (45 dpi CK), 2.10cm for treatment YNF2101 (45 dpi YNF 2101), 1.68cm for treatment CK+TR4 (45 dpi CK+TR4), and 2.10cm for treatment YNF2101+TR4 (45dpi YNF2101+TR4). Analysis of results: in the treatment without inoculation of banana vascular wilt pathogens, the pseudostem diameter of the treatment with Fusarium roseum (45 dpi YNF 2101) was significantly different from the control (45 dpi CK) and from the control with inoculation of banana vascular wilt pathogens. The fusarium oxysporum f.bricius fermentation liquid has no inhibition effect on the diameter of the banana plant pseudostem, can inhibit invasion hazard of banana fusarium wilt pathogenic bacteria to the banana plant, and has obvious promotion effect on the diameter of the banana plant pseudostem. The specific growth-promoting effect is shown in Table 3.
Influence on fresh weight of the aerial parts of banana plants: the fresh weight of the aerial part of the treated CK (45 dpi CK) is 43.86g, the fresh weight of the treated YNF2101 (45 dpi YNF 2101) is 44.90g, the fresh weight of the treated CK+TR4 (45 dpi CK+TR4) is 33.90g, and the fresh weight of the treated YNF2101+TR4 (45dpi YNF2101+TR4) is 44.13g. Analysis of results: in the treatment without inoculation of banana vascular wilt pathogens, the fresh weight of the overground part of the treatment (45 dpi YNF 2101) with Fusarium oxysporum (Fusarium oxysporum) was significantly different from that of the control (45 dpi CK) and from that of the control treatment with inoculation of banana vascular wilt pathogens. The fusarium oxysporum fermentation liquor has a remarkable promoting effect on the fresh weight of the overground part of banana plants. The specific growth-promoting effect is shown in Table 3.
Influence on the fresh weight of the underground part of banana plants: the fresh weight of the underground part of treated CK (45 dpi CK) was 28.54g, the fresh weight of treated irrigation YNF2101 (45 dpi YNF 2101) was 30.37g, the fresh weight of treated CK+TR4 (45 dpi CK+TR4) was 27.80g, and the fresh weight of treated YNF2101+TR4 (45dpi YNF2101+TR4) was 29.30g. Analysis of results: in the treatment without inoculation of banana vascular wilt pathogens, the fresh weight of the lower part of the treatment (45 dpi YNF 2101) of Fusarium roseum was significantly different from that of the control (45 dpi CK) and from all treatments with inoculation of banana vascular wilt pathogens and all treatments on the day of irrigation. The fusarium oxysporum fermentation liquor has a remarkable promoting effect on the fresh weight of the lower part of the banana plant. The specific growth-promoting effect is shown in Table 3. The underground part of the processed YNF2101 (45 dpi YNF 2101) is shown in FIG. 5, and the underground part of the CK (45 dpi CK) is shown in FIG. 6.
Leaf length and leaf width effects on first expanded leaf of banana plant: the leaf length and leaf width of the first expanded leaf of treatment CK (0 dpi CK) are 21.05 and 10.65cm, respectively, the leaf length and leaf width of the first expanded leaf of treatment irrigation YNF2101 (0 dpi YNF2101) are 20.42 and 10.20cm, respectively, the leaf length and leaf width of the first expanded leaf of CK+TR4 (0 dpi CK+TR4) are 19.91 and 9.56cm, respectively, and the leaf length and leaf width of the first expanded leaf of treatment YNF2101+TR4 (0 dpi YNF2101+TR4) are 19.01 and 9.82cm, respectively; the leaf length and leaf width of the first expanded leaf of treatment CK (45 dpi CK) were 22.90 and 11.12cm, respectively, the leaf length and leaf width of the first expanded leaf of treatment YNF2101 (45 dpi YNF 2101) were 13.32 and 11.85cm, respectively, the leaf length and leaf width of the first expanded leaf of treatment CK+TR4 (45 dpi CK+TR4) were 20.68 and 10.24cm, respectively, and the leaf length and leaf width of the first expanded leaf of treatment YNF2101+TR4 (45dpi YNF2101+TR4) were 23.47 and 12.02cm, respectively. Analysis of results: in the treatment without inoculation of banana vascular wilt pathogen, the leaf length and leaf width of the treatment with Fusarium roseum (45 dpi YNF 2101) was significantly different from the control (45 dpi CK+TR4) without irrigation of banana vascular wilt pathogen, and from all treatments on the day of irrigation. The fusarium oxysporum fermentation liquor has obvious promotion effect on the leaf length and the leaf width of the first expanded leaf of the banana plant. The specific growth-promoting effect is shown in Table 3. Plants treated with YNF2101 (45 dpi YNF 2101) are shown in FIG. 5, and plants of CK (45 dpi CK) are shown in FIG. 6.
TABLE 3 Table 3
Note that: a. b, c, d and e are symbols commonly used in the statistical arts to represent significance, and the same column has the same symbol indicating no significance, and the absence of the same symbol indicates a significance.
The above results show that under the condition that banana fusarium wilt pathogenic bacteria are not invaded and banana fusarium wilt pathogenic bacteria are invaded, compared with a control, banana plants treated by the fusarium oxysporum fermenting liquid have obvious differences from the control in biological information indexes such as plant height, leaf number, pseudostem diameter, fresh weight of overground part and underground part, and the like, and the results show that: the fusarium oxysporum f.bricius fermentation liquor has no inhibition effect on banana plant growth, can inhibit invasion hazard of banana fusarium wilt pathogenic bacteria on banana plants, and has obvious growth promotion effect on plant height, leaf number, pseudostem diameter, fresh weight of overground part, underground part and the like of banana plants. The Fusarium oxysporum provided by the invention can inhibit the activity of the fusarium oxysporum dedicated type fusarium oxysporum, which is a pathogen of banana vascular wilt, so that banana vascular wilt can be prevented and treated, and in addition, the Fusarium oxysporum provided by the invention has a remarkable growth promoting effect on banana plants.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (9)
1. Fusarium rubrum (Fusarium lateritium) is characterized in that the collection number of the Fusarium rubrum is CCTCC NO: M20221723.
2. A microbial inoculant comprising the fusarium venenatum of claim 1.
3. Use of fusarium oxysporum according to claim 1 or of the microbial inoculum according to claim 2 for inhibiting fusarium oxysporum gulum specialization (Fusarium oxysporum f.sp.cube).
4. A method of controlling banana vascular wilt and promoting banana plant growth, the method comprising: the fusarium venenatum of claim 1 or the microbial inoculum of claim 2 is applied to banana plants after culturing.
5. The method of claim 4, wherein the method of culturing comprises: inoculating the Fusarium roseum into a liquid culture medium.
6. The method of claim 5, wherein the liquid medium comprises: glucose 10-20g/L, peeled potato 150-250g/L.
7. The method of claim 5 or 6, wherein the pH of the liquid medium is 6-8.
8. The method of claim 5 or 6, wherein the culturing conditions comprise: the time is 3-7 days, and the temperature is 25-34 ℃.
9. Use of fusarium oxysporum of claim 1 or the microbial inoculum of claim 2 or the method of any one of claims 4-8 for controlling banana vascular wilt and promoting banana plant growth.
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| CN112899201A (en) * | 2021-03-16 | 2021-06-04 | 云南省农业科学院农业环境资源研究所 | Bacillus belgii, application thereof and method for preventing and treating banana wilt |
| CN113025522A (en) * | 2021-03-16 | 2021-06-25 | 云南省农业科学院农业环境资源研究所 | Bacillus amyloliquefaciens, application thereof and method for preventing and/or treating banana vascular wilt |
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| CN112899201A (en) * | 2021-03-16 | 2021-06-04 | 云南省农业科学院农业环境资源研究所 | Bacillus belgii, application thereof and method for preventing and treating banana wilt |
| CN113025522A (en) * | 2021-03-16 | 2021-06-25 | 云南省农业科学院农业环境资源研究所 | Bacillus amyloliquefaciens, application thereof and method for preventing and/or treating banana vascular wilt |
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