CN110250207B - Citrus brown spot pathogenic bacteria bacteriostatic agent and preparation method thereof - Google Patents
Citrus brown spot pathogenic bacteria bacteriostatic agent and preparation method thereof Download PDFInfo
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- CN110250207B CN110250207B CN201910531127.9A CN201910531127A CN110250207B CN 110250207 B CN110250207 B CN 110250207B CN 201910531127 A CN201910531127 A CN 201910531127A CN 110250207 B CN110250207 B CN 110250207B
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- dihydrogen phosphate
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- 239000000022 bacteriostatic agent Substances 0.000 title claims abstract description 35
- 244000052616 bacterial pathogen Species 0.000 title claims abstract description 33
- 235000020971 citrus fruits Nutrition 0.000 title claims abstract description 29
- 241000207199 Citrus Species 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims description 9
- 241000241413 Propolis Species 0.000 claims abstract description 34
- 229940069949 propolis Drugs 0.000 claims abstract description 34
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 27
- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 27
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims abstract description 26
- 101710130006 Beta-glucanase Proteins 0.000 claims abstract description 14
- 239000000243 solution Substances 0.000 claims description 34
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 24
- 238000002156 mixing Methods 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 4
- 230000002538 fungal effect Effects 0.000 abstract description 6
- -1 flavonoid compounds Chemical class 0.000 abstract description 4
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 2
- 230000009089 cytolysis Effects 0.000 abstract description 2
- 230000034994 death Effects 0.000 abstract description 2
- 229930003935 flavonoid Natural products 0.000 abstract description 2
- 235000017173 flavonoids Nutrition 0.000 abstract description 2
- 235000007586 terpenes Nutrition 0.000 abstract description 2
- 230000005764 inhibitory process Effects 0.000 description 12
- 235000013399 edible fruits Nutrition 0.000 description 10
- 230000000694 effects Effects 0.000 description 6
- 230000035699 permeability Effects 0.000 description 5
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 244000053095 fungal pathogen Species 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 241001157813 Cercospora Species 0.000 description 2
- 241000219112 Cucumis Species 0.000 description 2
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 239000013043 chemical agent Substances 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 239000000447 pesticide residue Substances 0.000 description 2
- 230000028070 sporulation Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 241000223600 Alternaria Species 0.000 description 1
- 241000223602 Alternaria alternata Species 0.000 description 1
- 239000005739 Bordeaux mixture Substances 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 241000233639 Pythium Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 229930182692 Strobilurin Natural products 0.000 description 1
- RDXARWSSOJYNLI-UHFFFAOYSA-N [P].[K] Chemical compound [P].[K] RDXARWSSOJYNLI-UHFFFAOYSA-N 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- JHRWWRDRBPCWTF-OLQVQODUSA-N captafol Chemical compound C1C=CC[C@H]2C(=O)N(SC(Cl)(Cl)C(Cl)Cl)C(=O)[C@H]21 JHRWWRDRBPCWTF-OLQVQODUSA-N 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000001614 effect on membrane Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 208000024386 fungal infectious disease Diseases 0.000 description 1
- 244000000004 fungal plant pathogen Species 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000000575 pesticide 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
- 239000000843 powder Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 239000011782 vitamin Substances 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
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Classifications
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- 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
- A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
- A01N59/26—Phosphorus; Compounds thereof
-
- 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
- A01N61/00—Biocides, pest repellants or attractants, or plant growth regulators containing substances of unknown or undetermined composition, e.g. substances characterised only by the mode of action
-
- 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
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- Agronomy & Crop Science (AREA)
- Wood Science & Technology (AREA)
- Environmental Sciences (AREA)
- Inorganic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a bacteriostatic agent for citrus brown spot pathogenic bacteria, which is prepared from the following components in parts by weight: 0.1-0.5 part of beta-glucanase, 5-8 parts of propolis solution and 10-15 parts of potassium dihydrogen phosphate solution, aiming at the problem of preventing and treating the citrus brown spot, the invention provides a bacteriostatic agent for pathogenic bacteria of the citrus brown spot, which comprises the components of the beta-glucanase, the propolis and the potassium dihydrogen phosphate. The beta-glucanase acts on the mycelium exposed at the top of the fungal hyphae, leading to lysis and death of the fungal cells. The flavonoid compounds and pinene or terpene compounds in the propolis have strong antibacterial effect, and the propolis has high viscosity and good film forming property, and can be uniformly coated on the surface of a tree body infected with germs to form a compact medicinal film, so that the germs contacting the propolis can be effectively killed.
Description
Technical Field
The invention relates to the technical field of biological control, and particularly relates to a bacteriostatic agent for citrus brown spot pathogenic bacteria and a preparation method thereof.
Background
The citrus is rich in moisture, saccharides, organic acids, amino acids, proteins, vitamins and the like, is a fruit variety with rich nutrition and wide planting area, and has remarkable economic benefit. Cercospora citrina is a fungal infectious disease of plants caused by alternaria alternata. Alternaria pathogenic bacteria are one of citrus pathogenic fungi which are distributed most widely all over the world, and are the first plant pathogenic fungi in economy. The thallus presents brown or white velvet or powder, hyphae are gray to black and are similar to a chain grid, the strain grows rapidly, the infection is rapid, the vitality is tenacious, and the strain becomes a dominant strain to propagate and grow rapidly after the citrus is infected with diseases. Brown spot is manifested in various parts of citrus, such as susceptible parts of leaves, fruits, branches, and also may infect young shoots. The brown spot symptom can be shown in about 24 hours after the brown spot pathogenic bacteria infect the fruits, red orange brown spot pathogenic bacteria toxin is generated, and dark gray color can be generated if the leaf spot with high humidity is generated. The rapid spread of the cercospora brown spot pathogenic fungi on the leaf and fruit surfaces can cause the leaf and fruit to turn yellow and black and even fall off, resulting in reduced yield. The pathogenic bacteria can cause great economic loss caused by other kinds of fruit diseases, such as pythium disease of plant fruits.
The method for preventing and treating the citrus brown spot by using a chemical agent is an effective method at present, most fruit growers utilize captafol or a copper preparation (Bordeaux mixture) to prevent and treat the brown spot, but the pesticide has long residence time on the surfaces of melons and fruits, is not easy to degrade, and can generate spotted pesticide residue marks on the surfaces of the melons and fruits at high temperature. In recent years, strobilurin and small ornithsin are also used for controlling citrus brown spot pathogenic fungi, and can effectively and rapidly inhibit the growth of pathogenic bacteria. However, the chemical agents cannot completely inhibit the occurrence of diseases, and are likely to produce strong resistance to drugs, and the problems of pesticide residues and the like which are caused therewith are not easily seen. At present, no related patent report of the citrus brown spot bacteriostatic agent derived from natural biological extracts exists.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects of the prior art: provides a bacteriostatic agent for citrus brown spot pathogenic bacteria and a preparation method thereof.
The technical solution of the invention is as follows: a bacteriostatic agent for pathogenic bacteria of citrus brown spot is prepared from the following components in parts by weight: 0.1-0.5 part of beta-glucanase, 5-8 parts of propolis solution and 10-15 parts of potassium dihydrogen phosphate solution.
Preferably, the propolis solution is a propolis ethanol solution prepared by mixing propolis and ethanol in a volume ratio of 1: 2.
Preferably, the potassium dihydrogen phosphate solution is a potassium dihydrogen phosphate aqueous solution prepared by mixing potassium dihydrogen phosphate and water in a weight ratio of 1: 20.
The preparation method of the bacteriostatic agent for the citrus brown spot pathogenic bacteria comprises the following steps:
1) preparing a propolis solution: dissolving propolis in ethanol to obtain propolis ethanol solution, wherein the volume ratio of propolis to ethanol is 1: 2;
2) preparation of potassium dihydrogen phosphate solution: mixing potassium dihydrogen phosphate and water according to the weight ratio of 1:20 to prepare a potassium dihydrogen phosphate aqueous solution;
3) mixing beta-glucanase, propolis solution and potassium dihydrogen phosphate solution according to the following weight parts to prepare the citrus brown spot pathogenic bacteria bacteriostatic agent: 0.1-0.5 part of beta-glucanase, 5-8 parts of propolis solution and 10-15 parts of potassium dihydrogen phosphate solution.
The invention has the beneficial effects that: aiming at the problem of prevention and treatment of citrus brown spot, the invention provides a bacteriostatic agent for pathogenic bacteria of citrus brown spot, which comprises beta-glucanase, propolis and potassium dihydrogen phosphate. The beta-glucanase acts on the mycelium exposed at the top of the fungal hyphae, leading to lysis and death of the fungal cells. The flavonoid compounds and pinene or terpene compounds in the propolis have strong antibacterial effect, and the propolis has high viscosity and good film forming property, and can be uniformly coated on the surface of a tree body infected with germs to form a compact medicinal film, so that the germs contacting the propolis can be effectively killed. The monopotassium phosphate can be used as a phosphorus-potassium fertilizer for citrus fruit trees, so that the stems are hard, the loss of nutrition and water caused by the harm of pathogenic microorganisms is compensated, and the invasion of pathogenic bacteria is effectively resisted. Application tests prove that the bacteriostatic agent has an inhibition rate of 34% on spores of pathogenic bacteria of citrus brown spot, and causes thickening, twisting and breaking of hyphae of the pathogenic bacteria, and the hypha membrane has high permeability and an obvious inhibition effect.
Drawings
FIG. 1 shows the inhibition of the formation of spores of pathogenic bacteria of citrus brown spot by the bacteriostatic agent of the invention. Left: spore microscopic morphology under bacteriostatic treatment by the biological bacteriostatic agent; and (3) right: control group spore microscopic morphology
FIG. 2 is a determination of the inhibition rate of the bacteriostatic agent of the invention on the formation of spores of pathogenic bacteria of citrus brown spot.
FIG. 3 shows the effect of the bacteriostatic agent of the present invention on the hypha morphology of citrus brown spot pathogen. Left: the microscopic shape of the fungal hyphae is treated by the biological bacteriostatic agent; and (3) right: control group hyphal microscopic morphology.
FIG. 4 shows the effect of the bacteriostatic agent of the invention on the cell membrane permeability of orange brown spot pathogen.
Detailed Description
The present invention will be described in further detail with reference to the following examples, but the present invention is not limited to the following examples.
Examples
Preparing a propolis solution:
dissolving pure propolis in 95% ethanol at a volume ratio of 1:2 to obtain propolis ethanol solution.
Preparation of potassium dihydrogen phosphate solution:
and mixing the potassium dihydrogen phosphate with water according to the weight ratio of 1:20 to prepare the potassium dihydrogen phosphate aqueous solution.
Preparing a bacteriostatic agent:
mixing beta-glucanase, a propolis solution and a potassium dihydrogen phosphate solution according to the following weight part ratio to prepare the bacteriostatic agent: 0.1-0.5 part of beta-glucanase, 5-8 parts of propolis solution and 10-15 parts of potassium dihydrogen phosphate solution.
The bacteriostatic agent for the citrus brown spot pathogenic bacteria has the inhibiting effect on the citrus brown spot pathogenic bacteria:
inhibition of pathogenic spores:
cutting a round paper sheet with the diameter of 1cm, soaking the round paper sheet in the biological bacteriostatic agent for 5min, taking out the round paper sheet, symmetrically placing the round paper sheet at a position 4cm away from a pathogenic bacteria colony growing for 1d, and setting a control group. After culturing for 1-6 days, using a puncher with the diameter of 5.0mm to punch a bacterium block at a position 3-4 mm away from the edge of a bacterial colony, washing spores on the bacterium block by using sterile water, directly counting the number of spores by using a blood counting plate under an optical microscope, repeating the counting process for 3 times for each bacterium block, and calculating the relative inhibition rate by using a formula, wherein the inhibition rate of the spore formation (%) = (the number of control spores-the number of treated spores)/the number of control spores multiplied by 100%.
Influence on hyphal morphology
The hyphal morphology on the slides of the inhibition and control groups was observed under an optical microscope.
Effect on Membrane Permeability
Transferring the strain into liquid culture medium, performing shake culture at 120rpm for 5d, suction filtering the culture solution, and washing the mycelium with normal saline. 2g of hyphae were weighed into a 50 mL beaker and washed 3 times with sterile deionized water. 20 mL of deionized water and bacteriostatic agent were added and the conductivity was measured at 0, 30, 60, 90, 120 min.
Results and analysis of the experiments
And (3) determining the inhibition rate of the bacteriostatic agent on sporulation:
after 3d of culture, the filter paper sheet soaked with the bacteriostatic agent obviously inhibits the growth of pathogenic bacteria colonies, the diameter of the colonies is smaller than that of a control group, and the fungal spores are observed to shrink and deform under a microscope (figure 1). After 3 days of culture, the inhibition rate of the biological bacteriostatic agent on sporulation is reduced to 52 percent, and the inhibition rate after 6 days is 34 percent (figure 2). The biological bacteriostatic agent has the function of inhibiting the germination and growth of pathogenic bacteria spores.
Effect of bacteriostatic agents on hyphal morphology:
under the inhibition effect of the bacteriostatic agent, hyphae of pathogenic bacteria become thick and are distorted and broken, dissolving holes are formed on the surface of the hyphae, and a diaphragm shrinks (figure 3), possibly caused by degradation of hyphal cell walls by beta-glucanase in the bacteriostatic agent.
Effect of bacteriostatic agents on membrane permeability:
FIG. 4 shows that the conductivity of the bacterial suspension generally increased. The conductivity of the bacterial liquid is increased from 2.85 mu s/cm to 3.19 mu s/cm within 2h, which shows that the hyphal cell membrane permeability is increased by the treatment of the bacteriostatic agent.
The above are merely characteristic embodiments of the present invention, and do not limit the scope of the present invention in any way. All technical solutions formed by equivalent exchanges or equivalent substitutions fall within the protection scope of the present invention.
Claims (2)
1. The bacteriostatic agent for the pathogenic bacteria of the citrus brown spot is characterized by being prepared from the following components in parts by weight: 0.1-0.5 part of beta-glucanase, 5-8 parts of propolis solution and 10-15 parts of potassium dihydrogen phosphate solution;
the propolis solution is a propolis ethanol solution prepared by mixing propolis and ethanol according to a volume ratio of 1: 2;
the potassium dihydrogen phosphate solution is a potassium dihydrogen phosphate aqueous solution prepared by mixing potassium dihydrogen phosphate and water according to the weight ratio of 1: 20.
2. A method for preparing a bacteriostatic agent for citrus brown spot pathogenic bacteria according to claim 1, which comprises the following specific steps:
1) preparing a propolis solution: dissolving propolis in ethanol to obtain propolis ethanol solution, wherein the volume ratio of propolis to ethanol is 1: 2;
2) preparation of potassium dihydrogen phosphate solution: mixing potassium dihydrogen phosphate and water according to the weight ratio of 1:20 to prepare a potassium dihydrogen phosphate aqueous solution;
3) mixing beta-glucanase, propolis solution and potassium dihydrogen phosphate solution according to the following weight parts to prepare the citrus brown spot pathogenic bacteria bacteriostatic agent: 0.1-0.5 part of beta-glucanase, 5-8 parts of propolis solution and 10-15 parts of potassium dihydrogen phosphate solution.
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| CN201910531127.9A CN110250207B (en) | 2019-06-19 | 2019-06-19 | Citrus brown spot pathogenic bacteria bacteriostatic agent and preparation method thereof |
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| CN201910531127.9A CN110250207B (en) | 2019-06-19 | 2019-06-19 | Citrus brown spot pathogenic bacteria bacteriostatic agent and preparation method thereof |
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| CN110250207B true CN110250207B (en) | 2021-07-30 |
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| CN104561060A (en) * | 2014-05-30 | 2015-04-29 | 河北省科学院生物研究所 | Beta-1, 3-1, 4-dextranase gene and application thereof |
| DE102014009813A1 (en) * | 2014-07-03 | 2016-01-07 | Biopract Gmbh | Process for the prophylaxis of fungal infections in useful and ornamental plants, preferably in viticulture, and in woody plants |
| CN106376597A (en) * | 2016-08-30 | 2017-02-08 | 北京达成生物科技有限公司 | Multienzyme composite antibacterial agent |
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