CN108410740B - Compound microbial preparation and application thereof in prevention and treatment of phytophthora - Google Patents
Compound microbial preparation and application thereof in prevention and treatment of phytophthora Download PDFInfo
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- CN108410740B CN108410740B CN201810280288.0A CN201810280288A CN108410740B CN 108410740 B CN108410740 B CN 108410740B CN 201810280288 A CN201810280288 A CN 201810280288A CN 108410740 B CN108410740 B CN 108410740B
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- 238000002360 preparation method Methods 0.000 title claims abstract description 27
- 230000000813 microbial effect Effects 0.000 title claims abstract description 19
- 150000001875 compounds Chemical class 0.000 title claims abstract description 18
- 241000233614 Phytophthora Species 0.000 title abstract description 11
- 230000002265 prevention Effects 0.000 title description 8
- 238000000855 fermentation Methods 0.000 claims abstract description 69
- 230000004151 fermentation Effects 0.000 claims abstract description 69
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 27
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 27
- 241000190950 Rhodopseudomonas palustris Species 0.000 claims abstract description 23
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 18
- 239000000843 powder Substances 0.000 claims abstract description 13
- 241000223259 Trichoderma Species 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 11
- 238000002156 mixing Methods 0.000 claims abstract description 6
- 230000001580 bacterial effect Effects 0.000 claims abstract description 5
- 230000008569 process Effects 0.000 claims abstract description 5
- 238000009777 vacuum freeze-drying Methods 0.000 claims abstract description 5
- 239000001963 growth medium Substances 0.000 claims description 52
- 241000378866 Trichoderma koningii Species 0.000 claims description 26
- 238000012258 culturing Methods 0.000 claims description 24
- 238000011081 inoculation Methods 0.000 claims description 20
- 239000002609 medium Substances 0.000 claims description 17
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 16
- 238000011218 seed culture Methods 0.000 claims description 15
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 12
- 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 11
- 229940041514 candida albicans extract Drugs 0.000 claims description 11
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- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 11
- 239000012138 yeast extract Substances 0.000 claims description 11
- 238000009630 liquid culture Methods 0.000 claims description 10
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- 239000001888 Peptone Substances 0.000 claims description 8
- 108010080698 Peptones Proteins 0.000 claims description 8
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 8
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 7
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 7
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 7
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 7
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- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 7
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 7
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- 239000000284 extract Substances 0.000 claims description 4
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 4
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 4
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 4
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- 239000004455 soybean meal Substances 0.000 claims description 4
- 230000000694 effects Effects 0.000 abstract description 7
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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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- 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
- A01N63/22—Bacillus
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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
- 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
- A01N63/27—Pseudomonas
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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
- 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
- A01N63/32—Yeast
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- 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
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
The invention belongs to the technical field of microorganisms, and discloses a compound microbial preparation which is prepared by the following processes: uniformly mixing Trichoderma koningii-myceliophthora yeast mixed fermentation liquor, Rhodopseudomonas palustris fermentation liquor and Bacillus subtilis fermentation liquor according to the volume ratio of 5-8:2-3:2-3, and then carrying out vacuum freeze drying to obtain the bacterial powder. The compound microbial preparation provided by the invention adopts four biocontrol strains, can realize synergistic symbiosis, is reasonable in compatibility and free from antagonism, greatly improves the phytophthora control effect, is harmless to human, livestock and crops, is environment-friendly, is relatively simple in preparation process, convenient and fast in use method and has a wide application prospect.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a compound microbial preparation and application thereof in prevention and treatment of phytophthora.
Background
Phytophthora (Phytophthora), Peronosporales (Peronosporales) pathogenic bacteria, lower sporangium peduncles and hyphae have no obvious difference, have high difference, sporangium is lemon-shaped or oval, have papilla at the top, cause potato late blight, taro epidemic disease and the like. Hyphae are not isolated and have multiple cores at first, many branches are acute-angled, and are often constricted at the branches, are 2-10 μm thick, and some of them can produce haustoria, sometimes form large hypha bulges. The sporangium stem is not greatly different from hypha to be obviously differentiated, and is irregularly branched, coaxially branched or emerges from the inner layer of the sporangium. The sporangium has large morphological change, generally being oval, inverted pear-shaped and elliptical, and also having irregular shape and large size change; mastoid, hemimastoid or mastoid-free on the top; typically, they are alone born on the sporocyst stalk, occasionally internationally; shedding or non-shedding after maturation, with long (> 20 μm), medium (5-20 μm) or short (< 5 μm) stalks; can directly germinate to produce germ tubes or indirectly germinate to produce zoospores. Zoospores are oval or broad bean shaped, and have a lateral double flagellum, and form a cell wall after resting, and are spherical, called resting spores. Chlamydospores are mostly spherical, thin-walled or thick-walled, colorless to dark-colored, terminal or intergrown. Spherical or nearly spherical, funnel-shaped, smooth or textured wall, colorless, yellow to brown. The male organs are different in size and shape, colorless, and are either enclosed (male-piercing) or laterally born. Oospore is spherical, thick-walled or thin-walled, colorless to light-colored, full or not full.
Pepper production is affected by a variety of pests, with phytophthora capsici being one of the major soil-borne pests. The phytophthora capsici is damaged at the green part and the root of the ground of the full growth period of the capsicum, the base part of a stem which is ill in the seedling period is in a dark green water stain shape and is soft rot or lodging, namely, the base part of the stem which is ill in the seedling period is black brown, seedlings wither and die, the root and stem tissues of wooden young stems are rotten, stems and leaves are withered rapidly, and seedlings are withered. The basal part of the main stem and the branch of the adult plant are damaged, less brown spots are generated at first, then the adult plant rapidly expands to the periphery, including the whole stem, and the diseased part is brown at the later stage and has obvious boundary line with the healthy tissue. The infected roots of the plants at the roots are reduced, the lateral roots are light brown or dark brown, and the plants are rotten at the later stage. The whole plant of the main stem or the root is dead, the lateral branches are damaged, and the branches on the lateral branches are dead. The leaves are soaked in water and spread inwards from the edge, and then are light brown. The disease of the floral organs is manifested as browning, soft rot and abscission. Fruits usually attack pedicles, and are watery spots in early stage, soft and rotten when wet, and the boundary between diseases and health is obvious. If the environment is suitable, the environment is rapidly expanded outwards, which leads to fruit rot.
Phytophthora capsici is caused by the flagellata subphylum fungus Phytophthora capsici. The pathogenic bacteria mainly take oospores and thick tapestry spores as main infection sources to live through the winter on diseased residues or soil and seeds, wherein the diseased residues in the soil have high bacteria carrying rate. Under proper conditions, the germs after overwintering are transmitted to the stem base or plants near the ground through rainwater splashing or irrigation water, so that the diseases are caused. Therefore, phytophthora capsici becomes a devastating disease with short disease period and rapid epidemic speed. High temperature, high humidity, large number of rainfall days and large rainfall, and is beneficial to the occurrence of diseases. The existing control method mainly comprises the following steps: agricultural control, chemical control, and biological control. The agricultural prevention and control mainly comprises reasonable stubble ploughing and scientific planting. The reasonable stubble ploughing is a considerable cultivation measure in agricultural production, and the reasonable cultivation system and layout are very beneficial to the growth of the pepper. Chemical control includes soil application and field application; however, chemical drugs are likely to remain and cause environmental pollution, and the more serious and poorer the chemical control effect due to the generation of antagonistic bacteria. Antagonistic microorganisms are mainly adopted for biological prevention and treatment, so that the biological prevention and treatment agent is environment-friendly and cannot cause pollution. In the prior art, single microbial agents are more common, such as streptomyces, bacillus subtilis and the like, but a single bacterial strain often has the defects of low control effect, slow drug effect and the like, and a plurality of research institutes have conducted research on compound biocontrol agents at present. The compound biocontrol preparation generally refers to a preparation comprising more than two microorganisms, and the selection of strains in the compound biocontrol preparation is more critical and difficult; if the selection is careless, the strains can play antagonistic roles instead.
Disclosure of Invention
In order to overcome the defects of poor control effect, slow pesticide effect and the like of a single biocontrol microbial inoculum in the prior art, the invention provides a compound microbial preparation and application thereof in controlling phytophthora.
The invention is realized by the following technical scheme:
a compound microbial preparation is prepared by the following processes: uniformly mixing Trichoderma koningii-myceliophthora yeast mixed fermentation liquor, Rhodopseudomonas palustris fermentation liquor and Bacillus subtilis fermentation liquor according to the volume ratio of 5-8:2-3:2-3, and then carrying out vacuum freeze drying to obtain the bacterial powder.
Further, the compound microbial preparation is used for preventing and treating phytophthora.
Further, the phytophthora is phytophthora capsici.
Preferably, the first and second electrodes are formed of a metal,
the trichoderma koningii-trichosporon mixed fermentation liquor is prepared according to the following steps:
1) inoculating the hyphomycete on a YPDA culture medium for culturing to obtain a single colony; selecting single colony, inoculating to YPD liquid culture medium, culturing at 30 deg.C for 24 hr to obtain Trichosporon sinensis seed liquid;
2) inoculating Trichoderma koningii to a PDA culture medium for culture by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a PDA liquid culture medium, and performing shake cultivation at 30 ℃ and 200rpm for 36h to obtain Trichoderma koningii seed liquid for later use;
3) inoculating the trichoderma koningii seed liquid into a fermentation culture medium according to the inoculation amount of 10%, culturing for 6 hours at 30 ℃, then inoculating the filamentous spore yeast seed liquid into the fermentation culture medium according to the inoculation amount of 5%, and continuously culturing for 18 hours at 30 ℃ to obtain the trichoderma koningii-filamentous spore yeast mixed fermentation liquid.
Preferably, the first and second electrodes are formed of a metal,
the rhodopseudomonas palustris fermentation liquor is prepared by the following steps:
performing streak culture on rhodopseudomonas palustris on an LB (Langerhans) plate to obtain a single colony; selecting a single colony to be inoculated into a seed culture medium to be cultured to a logarithmic growth phase, wherein the culture conditions are as follows: at 30-32 ℃, illuminating 3000-; then inoculating the strain into a fermentation culture medium according to the inoculation amount of 10 percent, and culturing for 24 hours at the temperature of 30-32 ℃ to obtain the rhodopseudomonas palustris fermentation liquor.
Preferably, the first and second electrodes are formed of a metal,
the bacillus subtilis fermentation liquor is prepared according to the following steps:
carrying out streak culture on bacillus subtilis on a beef extract peptone agar culture medium to obtain a single colony; selecting single colony, inoculating to seed culture medium, shake culturing at 30 deg.C and 200rpm to logarithmic phase, and making into seed solution; then inoculating the bacillus subtilis into a fermentation medium according to the inoculation amount of 8%, and culturing for 36h at 30 ℃ to obtain the bacillus subtilis fermentation liquor.
Preferably, the first and second electrodes are formed of a metal,
the fermentation medium comprises the following components: 16g/L of corn straw powder, 10g/L of soybean meal, 5g/L of ammonium chloride, 2g/L of dipotassium phosphate, 1g/L of monopotassium phosphate, 0.5g/L of sodium chloride, 0.1g/L of magnesium sulfate and 0.01g/L of ferrous sulfate.
Preferably, the first and second electrodes are formed of a metal,
the rhodopseudomonas palustris seed culture medium and the fermentation culture medium comprise the following components: 8g/L glucose, 5g/L yeast extract, 1g/L ammonium sulfate, 0.2g/L potassium dihydrogen phosphate, 0.2g/L dipotassium hydrogen phosphate, 0.1g/L sodium bicarbonate and 0.01g/L magnesium sulfate.
Preferably, the first and second electrodes are formed of a metal,
the bacillus subtilis seed culture medium and the fermentation culture medium both comprise the following components: 10g/L glucose, 5g/L yeast extract, 3g/L ammonium sulfate, 1g/L uremia, 0.1g/L monopotassium phosphate, 0.1g/L dipotassium phosphate, 0.02g/L calcium chloride and 0.01g/L ferrous sulfate.
Compared with the prior art, the invention has the advantages that the following aspects are mainly included but not limited:
the trichoderma koningii and the filamentous spore yeast have no antagonistic action and can symbiotic with each other, the trichoderma koningii can secrete cellulase, and the straw cellulose is decomposed into a carbon source which can be utilized by the filamentous spore yeast, so that the filamentous spore yeast grows by utilizing the carbon source, and the fermentation cost is reduced; inoculating Trichoderma koningii first for 6 hr and inoculating saccharomycete; the saccharomycetes can promote the growth of trichoderma koningii and greatly improve the enzyme yield of the trichoderma, so that the ability of resisting botrytis cinerea is improved. The rhodopseudomonas palustris and the bacillus subtilis can secrete various antibacterial substances and can compete with pathogenic bacteria for nutrition and space; the compound microbial preparation adopts four biocontrol strains, can realize synergistic symbiosis, has reasonable compatibility and no mutual antagonism, and greatly improves the effect of preventing and controlling phytophthora; the preparation of the invention is harmless to human and livestock, harmless to crops, environment-friendly, relatively simple in preparation process, convenient and fast in use method and wide in application prospect.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the technical solutions in the present application will be clearly and completely described below with reference to specific embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A compound microbial preparation comprises Trichosporon, Trichoderma koningii, Bacillus subtilis, and Rhodopseudomonas palustris.
Specifically, the compound microbial preparation is prepared according to the following process:
inoculating the hyphomycete on a YPDA culture medium for culturing to obtain a single colony; selecting a single colony, inoculating the single colony on an YPD liquid culture medium for culture, and culturing at 30 ℃ for 24 hours to obtain a seed solution for later use;
the YPD medium formula comprises: 20g of glucose, 20g of peptone and 10g of yeast extract powder, and supplementing distilled water to 1000 mL;
the YPDA culture medium formula comprises: 20g of glucose, 20g of peptone, 10g of yeast extract powder and 20g of agar, and supplementing distilled water to 1000 mL;
inoculating Trichoderma koningii to a PDA culture medium for culture by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a PDA liquid culture medium for culturing, and performing shake culture at 30 ℃ and 200rpm for 36 hours to obtain a seed solution for later use;
firstly, inoculating Trichoderma koningii seed liquid according to the inoculation amount of 10% (the inoculation density is 1 multiplied by 10)7CFU/ml) was inoculated into a fermentation medium, cultured at 30 ℃ for 6 hours, and then a filamentous yeast seed solution was inoculated in an amount of 5% (inoculation density of 1X 10)7CFU/ml) is inoculated into a fermentation medium, and the mixture is continuously cultured for 18 hours at the temperature of 30 ℃ to obtain Trichoderma koningii-myceliophthora mixed fermentation liquor; the fermentation medium comprises the following components: 16g/L of corn straw powder, 10g/L of soybean meal, 5g/L of ammonium chloride, 2g/L of dipotassium phosphate, 1g/L of monopotassium phosphate, 0.5g/L of sodium chloride, 0.1g/L of magnesium sulfate and 0.01g/L of ferrous sulfate;
performing streak culture on rhodopseudomonas palustris on an LB (Langerhans) plate to obtain a single colony; selecting a single colony to be inoculated into a seed culture medium to be cultured to a logarithmic growth phase, wherein the culture conditions are as follows: at 30-32 ℃, illuminating 3000-; then according to the inoculation amount of 10% (the inoculation density is 3 multiplied by 10)7CFU/ml) is inoculated into a fermentation medium, and the fermentation medium is cultured for 24 hours at the temperature of 30-32 ℃ to obtain rhodopseudomonas palustris fermentation liquor; the seed culture medium and the fermentation culture medium comprise the following components: 8g/L glucose, 5g/L yeast extract, 1g/L ammonium sulfate, 0.2g/L potassium dihydrogen phosphate, 0.2g/L dipotassium hydrogen phosphate, 0.1g/L sodium bicarbonate and 0.01g/L magnesium sulfate;
carrying out streak culture on bacillus subtilis on a beef extract peptone agar culture medium to obtain a single colony; selecting single colony, inoculating to seed culture medium, shake culturing at 30 deg.C and 200rpm to logarithmic phase, and making into seed solution; then according to the inoculation amount of 8% (the inoculation density is 5X 10)6CFU/ml) is inoculated into a fermentation medium, and the mixture is cultured for 36 hours at the temperature of 30 ℃ to obtain bacillus subtilis fermentation liquor; the seed culture medium and the fermentation culture medium comprise the following components: 10g/L glucose, 5g/L yeast extract, 3g/L ammonium sulfate, 1g/L uremia, 0.1g/L monopotassium phosphate, 0.1g/L dipotassium phosphate, 0.02g/L calcium chloride and 0.01g/L ferrous sulfate;
uniformly mixing Trichoderma koningii-myceliophthora yeast mixed fermentation liquor, Rhodopseudomonas palustris fermentation liquor and Bacillus subtilis fermentation liquor according to the volume ratio of 8:3:3, and then carrying out vacuum freeze drying to obtain the bacterial powder.
The Trichoderma koningii is ATCC 66766; the filamentous spore yeast is ATCC 201110; the rhodopseudomonas palustris is ATCC 17001; the Bacillus subtilis is ATCC 6633.
Example 2
A compound microbial preparation comprises Trichosporon, Trichoderma koningii, Bacillus subtilis, and Rhodopseudomonas palustris.
Specifically, the compound microbial preparation is prepared according to the following process:
inoculating the hyphomycete on a YPDA culture medium for culturing to obtain a single colony; selecting a single colony, inoculating the single colony on an YPD liquid culture medium for culture, and culturing at 30 ℃ for 24 hours to obtain a seed solution for later use;
the YPD medium formula comprises: 20g of glucose, 20g of peptone and 10g of yeast extract powder, and supplementing distilled water to 1000 mL;
the YPDA culture medium formula comprises: 20g of glucose, 20g of peptone, 10g of yeast extract powder and 20g of agar, and supplementing distilled water to 1000 mL;
inoculating Trichoderma koningii to a PDA culture medium for culture by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a PDA liquid culture medium for culturing, and performing shake culture at 30 ℃ and 200rpm for 36 hours to obtain a seed solution for later use;
firstly, inoculating Trichoderma koningii seed liquid according to the inoculation amount of 10% (the inoculation density is 1 multiplied by 10)7CFU/ml) was inoculated into a fermentation medium, cultured at 30 ℃ for 6 hours, and then a filamentous yeast seed solution was inoculated in an amount of 5% (inoculation density of 1X 10)7CFU/ml) is inoculated into a fermentation medium, and the mixture is continuously cultured for 18 hours at the temperature of 30 ℃ to obtain Trichoderma koningii-myceliophthora mixed fermentation liquor; the fermentation medium comprises the following components: 16g/L of corn straw powder, 10g/L of soybean meal, 5g/L of ammonium chloride, 2g/L of dipotassium phosphate, 1g/L of monopotassium phosphate, 0.5g/L of sodium chloride, 0.1g/L of magnesium sulfate and 0.01g/L of ferrous sulfate;
performing streak culture on rhodopseudomonas palustris on an LB (Langerhans) plate to obtain a single colony; selecting a single colony to be inoculated into a seed culture medium to be cultured to a logarithmic growth phase, wherein the culture conditions are as follows: at 30-32 ℃, illuminating 3000-; then according to 10% inoculum size (inoculum density 3X 10)7CFU/ml) is inoculated into a fermentation medium, and the fermentation medium is cultured for 24 hours at the temperature of 30-32 ℃ to obtain rhodopseudomonas palustris fermentation liquor; the seed culture medium and the fermentation culture medium comprise the following components: 8g/L glucose, 5g/L yeast extract, 1g/L ammonium sulfate, 0.2g/L potassium dihydrogen phosphate, 0.2g/L dipotassium hydrogen phosphate, 0.1g/L sodium bicarbonate and 0.01g/L magnesium sulfate;
carrying out streak culture on bacillus subtilis on a beef extract peptone agar culture medium to obtain a single colony; selecting single colony, inoculating to seed culture medium, shake culturing at 30 deg.C and 200rpm to logarithmic phase, and making into seed solution; then according to the inoculation amount of 8% (the inoculation density is 5X 10)6CFU/ml) is inoculated into a fermentation medium, and the mixture is cultured for 36 hours at the temperature of 30 ℃ to obtain bacillus subtilis fermentation liquor; the seed culture medium and the fermentation culture medium comprise the following components: 10g/L glucose, 5g/L yeast extract, 3g/L ammonium sulfate, 1g/L uremia, 0.1g/L monopotassium phosphate, 0.1g/L dipotassium phosphate, 0.02g/L calcium chloride and 0.01g/L ferrous sulfate;
uniformly mixing Trichoderma koningii-myceliophthora yeast mixed fermentation liquor, Rhodopseudomonas palustris fermentation liquor and Bacillus subtilis fermentation liquor according to the volume ratio of 5:2:2, and then carrying out vacuum freeze drying to obtain the strain powder.
The Trichoderma koningii is ATCC 66766; the filamentous spore yeast is ATCC 201110; the rhodopseudomonas palustris is ATCC 17001; the Bacillus subtilis is ATCC 6633.
Example 3
The compound microbial preparation has the effect of preventing and treating phytophthora capsici
Grouping: experimental groups: example 1; control group 1: the same procedure as in example 1 was repeated except that only the mixed fermentation broth of Trichoderma koningii and Trichosporon mentagroides was used; control group 2: the same procedure as in example 1 was repeated except that Rhodopseudomonas palustris fermentation broth and Bacillus subtilis fermentation broth were used; control group 3: separately fermenting trichoderma koningii and myceliophthora, and mixing the four kinds of fermentation liquor, the rest is the same as the example 1; blank control: equal amount of clear water.
Preparation pretreatment: the preparation is added into water with the weight of 100 times, and is stirred uniformly for standby.
Soil treatment: solid culturing Phytophthora capsici for 4 days, inoculating into V8 liquid culture medium, culturing at 25 deg.C for 4 days, decanting the liquid culture medium, weighing mycelium, and inoculating into soil of each cell according to the amount of 10 g/kg soil.
The treatment method comprises the following steps: the hot pepper test field is divided into five cells, and each cell corresponds to each group; soaking Capsici fructus seedling (cornu bovis Seu Bubali Capsici) in the diluent of each preparation for 60min, taking out, and transplanting to each cell;
detection standard: the degree of phytophthora blight is classified as grade 6. 0 level is that the root system is not attacked; grade 1 is that the incidence of root system is less than or equal to 30 percent and the leaves are normal; grade 2 is 30%, the incidence rate of roots is less than or equal to 60%, and the leaves are normal; grade 3 is 60% < the root disease incidence is less than or equal to 80%; the leaves turn yellow, the incidence rate of roots is more than 80% in 4 grades, the leaves wither, the whole plants die in 5 grades, and the leaves are dry. The disease index and the prevention and treatment effect are calculated according to the following formula, wherein the disease index is [ (disease-grade plant number multiplied by representative value)/(total plant number multiplied by highest disease-grade representative value) ] multiplied by 100. The control rate is [ (control disease index-treatment disease index)/control disease index ] × 100%. Sampling after 21 days, counting the disease degree, and calculating the disease index; see table 1. The total fresh weight of the plants and the fresh weight of the roots were also examined, as shown in Table 2.
TABLE 1
| Group of | Index of disease condition | The prevention and treatment rate% |
| Experimental group | 9.3 | 89.2 |
| Control group 1 | 27.1 | 68.6 |
| Control group 2 | 32.6 | 62.3 |
| Control group 3 | 14.9 | 72.8 |
| Blank control | 86.5 | --- |
TABLE 2
| Group of | Total fresh weight of plant g | Fresh weight of root g |
| Experimental group | 129.6 | 33.5 |
| Control group 1 | 104.6 | 25.1 |
| Control group 2 | 96.1 | 23.7 |
| Control group 3 | 113.2 | 28.9 |
| Blank control | 81.4 | 19.8 |
And (4) conclusion: as shown in table 1, the experimental group has the highest control rate of phytophthora capsici, which is 89.2%, and is significantly higher than the control group 1-2, and also higher than the control group 3 in a separate fermentation mode, which indicates that the myceliophthora and trichoderma koningii can be fermented together, trichoderma koningii is inoculated first, and the cellulose is decomposed into a carbon source which can be used by the myceliophthora, so that the subsequent inoculated myceliophthora is grown by using the carbon source, the fermentation cost is reduced, the yeast in turn can promote the growth of trichoderma koningii, and the enzyme yield of trichoderma koningii is greatly improved, thereby improving the ability of antagonizing botrytis cinerea. The plants absorb and utilize mineral nutrients from soil by root systems to maintain the growth and the propagation of the plants, and the more developed the root systems, the better the growth of the plants; the plant represents the biomass of the plant and can be used as an important index for evaluating the yield and quality of the fruit; as shown in Table 2, the total fresh weight and root fresh weight of the plants of the experimental group and the control groups 1-3 are obviously higher than those of the blank control, wherein the weight gain of the experimental group is the most obvious, and the fruit yield and quality of the experimental group are also better than those of other groups.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (4)
1. The compound microbial preparation is characterized by being prepared according to the following process: uniformly mixing Trichoderma koningii-myceliophthora yeast mixed fermentation liquor, Rhodopseudomonas palustris fermentation liquor and Bacillus subtilis fermentation liquor according to the volume ratio of 5-8:2-3:2-3, and then carrying out vacuum freeze drying to obtain bacterial powder; said Trichoderma koningii (f) (Trichoderma koningii) Is ATCC 66766; the filamentous spore yeast (A), (B), (C)Trichosproom sp.) ATCC 201110; the Rhodopseudomonas palustris (A), (B)Rhodopseudanonas palustris) ATCC 17001; the Bacillus subtilis (A), (B) and (C)Bacillus subtilis) Is ATCC 6633.
2. The microbial preparation according to claim 1, wherein the trichoderma koningii-myceliophthora mixed fermentation broth is prepared by the following steps:
1) inoculating the hyphomycete on a YPDA culture medium for culturing to obtain a single colony; selecting single colony, inoculating to YPD liquid culture medium, culturing at 30 deg.C for 24 hr to obtain Trichosporon sinensis seed liquid;
2) inoculating Trichoderma koningii to a PDA culture medium for culture by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a PDA liquid culture medium, and performing shake cultivation at 30 ℃ and 200rpm for 36h to obtain Trichoderma koningii seed liquid for later use;
3) firstly, inoculating trichoderma koningii seed liquid into a fermentation culture medium according to the inoculation amount of 10%, culturing for 6 hours at 30 ℃, then inoculating the filamentous spore yeast seed liquid into the fermentation culture medium according to the inoculation amount of 5%, and continuously culturing for 18 hours at 30 ℃ to obtain trichoderma koningii-filamentous spore yeast mixed fermentation liquid; the fermentation medium comprises the following components: 16g/L of corn straw powder, 10g/L of soybean meal, 5g/L of ammonium chloride, 2g/L of dipotassium phosphate, 1g/L of monopotassium phosphate, 0.5g/L of sodium chloride, 0.1g/L of magnesium sulfate and 0.01g/L of ferrous sulfate.
3. The microbial preparation of claim 1, wherein the Rhodopseudomonas palustris fermentation broth is prepared by the following steps:
performing streak culture on rhodopseudomonas palustris on an LB (Langerhans) plate to obtain a single colony; selecting a single colony to be inoculated into a seed culture medium to be cultured to a logarithmic growth phase, wherein the culture conditions are as follows: at 30-32 ℃, illuminating 3000-; then inoculating the strain into a fermentation culture medium according to the inoculation amount of 10 percent, and culturing for 24 hours at the temperature of 30-32 ℃ to obtain rhodopseudomonas palustris fermentation liquor; the seed culture medium and the fermentation culture medium comprise the following components: 8g/L glucose, 5g/L yeast extract, 1g/L ammonium sulfate, 0.2g/L potassium dihydrogen phosphate, 0.2g/L dipotassium hydrogen phosphate, 0.1g/L sodium bicarbonate and 0.01g/L magnesium sulfate.
4. The microbial preparation of claim 1, wherein the bacillus subtilis fermentation broth is prepared by the following steps:
carrying out streak culture on bacillus subtilis on a beef extract peptone agar culture medium to obtain a single colony; selecting single colony, inoculating to seed culture medium, shake culturing at 30 deg.C and 200rpm to logarithmic phase, and making into seed solution; then inoculating the bacillus subtilis into a fermentation medium according to the inoculation amount of 8%, and culturing for 36h at 30 ℃ to obtain the bacillus subtilis fermentation liquor.
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