CN115161239B - Special fermentation medium for bacillus velezensis bacteria and application thereof - Google Patents

Special fermentation medium for bacillus velezensis bacteria and application thereof Download PDF

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CN115161239B
CN115161239B CN202210875568.2A CN202210875568A CN115161239B CN 115161239 B CN115161239 B CN 115161239B CN 202210875568 A CN202210875568 A CN 202210875568A CN 115161239 B CN115161239 B CN 115161239B
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CN115161239A (en
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李玉婷
王敏
熊仁科
景飞江
左建英
申文熹
杨怀亮
李宏达
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Lomon Bio Technology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
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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
    • 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
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    • A01P7/02—Acaricides
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    • C12R2001/07—Bacillus

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Abstract

The application discloses a special fermentation medium for bacillus velezensis bacteria, which comprises the following components: 2 to 3 weight percent of yeast powder, 1 to 2 weight percent of soybean protein powder, 2 to 4 weight percent of molasses, 0.5 to 1 weight percent of gibberellin membrane residual liquid, 0.1 to 0.4 weight percent of monopotassium phosphate, 0.1 to 0.4 weight percent of calcium carbonate, 0.1 to 0.2 weight percent of magnesium sulfate heptahydrate, 0.02 to 0.05 weight percent of sodium chloride, 0.06 to 0.10 weight percent of dichlord and the balance of water. The fermentation liquor prepared by the special fermentation medium is diluted and used as an active ingredient for killing mites, and the mite killing effect is good.

Description

Special fermentation medium for bacillus velezensis bacteria and application thereof
Technical Field
The application relates to the field of agriculture, in particular to a special fermentation medium for bacillus velezensis bacteria, a bacillus velezensis bacteria fermentation method, a fermentation liquid and an acaricide.
Background
Currently, among agricultural pest control, phytophagous mites are one of the most difficult pest groups recognized worldwide, the main category of which is spider mites, commonly known as red spiders, belonging to the phylum Arthropoda (Arthropoda), arachnidae (Arachnida), acarina (Acari), acarina (Acariforms), she Manzong (Tetranychoidea). The spider mites are world pest mites, and are very easy to generate drug resistance due to small individuals, short generation, short development period, rapid propagation, strong adaptability and high mutation rate, are extremely difficult to control, cause the situation that the mites of crops grow increasingly hard and become a big disaster, cause great loss in agricultural production, influence the agricultural yield increase and the peasant yield increase, and become an important and urgent problem to be solved in the current agricultural production.
Chemical control is still a main countermeasure for controlling mites in the production of horticultural crops in China, but misuse and misuse of acaricides are extremely easy to cause the generation of drug resistance of spider mites, and the field control effect of chemical pesticides is greatly reduced. The statistics in 2008 show that spider mites have developed resistance to 92 active ingredients in current medicaments, including neurotoxin insecticides such as organic acid esters and pyrethroids, selective acaricides such as mitochondrial electron transfer inhibitors and organotin and the like. The spider mite resistance is much worse than other field pests such as plutella xylostella, aphid, etc., so that scientists consider spider mites as the most resistant species. Wherein, the resistance of the tetranychus urticae (Tetranychus Urticae Koch) is obviously higher than that of other mites, both on greenhouse vegetables and on open-air fruit trees. For example, the resistance multiple of two-spotted spider mites to avermectin, trichlorfon, cyromazine and the like is more than 10 times that of other mites. The whole genome DNA study of Tetranychus urticae found that the detoxification genes in the body are 3 times as many as those of other animals, and that the detoxification genes contain 39 genes of a drug resistance gene family, and that insects and vertebrates are only 14. Therefore, development of control strategies based on biological control is an important way to achieve sustainable control.
Among various means of controlling mites, biological control has been an important point and hot spot of research, playing an important role in pest control strategies. At present, some progress and achievements are made on the aspects of biocontrol researches on natural enemies of mites, plant-derived pesticides, spider mite pathogenic bacteria and the like. The natural enemies of the spider mites are various, the phytoseiid mites and the mite eating ladybug are two kinds with more researches and applications, and especially the phytoseiid mites are industrially produced in large scale in a plurality of countries. 60% of the greenhouse phytoseiid mites control the cucumber spider mites in the netherlands, while the uk, finland, sweden and denmark have exceeded 70%. The plant protection institute of the agricultural academy of sciences of Fujian province initially establishes a mass propagation base for first predatory mites in China, and the popularization and application area of the mass propagation base in China reaches 13.3 ten thousand hectares. However, natural enemy utilization is limited in application and development due to the defects of difficult establishment of dominant population, high cost for maintaining natural enemy population, inability of comprehensive control together with chemical control and the like. The plant pesticide has safe and low toxicity effective components, is a plant secondary metabolic substance with acaricidal activity, and has been reported to be almost 200 plants with acaricidal activity measurement, but the plant pesticide has a few development and utilization values due to the fact that the active low active components are undefined.
Various cytoplasmic inherited intracellular symbiotic bacteria are widely distributed in arthropods, and are classified into primary symbiotic bacteria (primary symbiotic) and secondary symbiotic bacteria (secondary symbiont). The symbiotic bacteria are necessary for the existence of the host, co-evolve with the host, are widely distributed in tissue cells of the host, are mainly vertically transmitted through a mother line, and can also be horizontally transmitted. The secondary symbiotic bacteria are not necessary for the survival of the host, may be beneficial or harmful to the host, and the harmful may be referred to as spider mite pathogens. The pathogenic bacteria of the spider mites and the spider mites have a interdependence and restriction relationship through a long evolution process. How to utilize the pathogenic bacteria and the bioactive substances thereof has become the main attack direction of many laboratories in developed countries in recent years.
The pathogenic bacteria of spider mites in nature are relatively few, the most important record is Wolbachia (Wolbachia), and the spider mites are originally found in the ovaries of culex spinosa (Culexpipiens) and can infect various arthropods such as insects, mites and the like. Such bacteria can trigger abnormal host reproduction behavior, such as cytokinesis, parthenogenesis, emasculation, and feminization. Strains with similar activities, also Candidatus Cardinium, abbreviated as Cardinium, are found from the parasitic wasp of the genus Aphis (Encarsia) and are capable of inducing the feminization of spider mites, inducing parthenogenesis, inducing cytoplasmic incompatibility, affecting host fitness and altering the oviposition behavior of the host. Verticillium lecanii (Verticillium lecanii Zimm) can be used for controlling aphids, whiteflies and mites. Acremonium strain (Acremonium hansfordii) is separated from naturally infected larvae of green peach aphid (Myzuspersicae Sulzer) and has an infection effect on cabbage caterpillar, green peach aphid and spider mite, and after a host infects the Acremonium strain, the in-vivo protein is consumed, and tissues and organs of the synthesized protein are destroyed to unbalance in-vivo detoxification enzyme and protective enzyme. The research cases and the utilization of the spider mite pathogenic bacteria are relatively less, but the spider mite pathogenic bacteria are utilized to treat the spider mites with bacteria, so that the spider mite density can be safely and effectively regulated, and the spider mite pathogenic bacteria plays an important role in biocontrol and is an important component of biocontrol work.
Under the premise that the current pests generally generate drug resistance and the sustainability and nuisance free performance of comprehensive pest control are focused, the microbial resources are developed, strains with high pathogenicity are screened, and the use amount of chemical pesticides is reduced in the development direction of the current advanced agriculture.
Disclosure of Invention
Based on the problems, the application provides a special fermentation medium for bacillus velezensis bacteria, which is diluted with a fermentation liquid prepared by combining bacillus velezensis bacteria and has good mite killing effect as an active ingredient.
A special fermentation medium for bacillus velezensis bacteria, the special fermentation medium for bacillus velezensis bacteria comprising:
2 to 3 weight percent of yeast powder, 1 to 2 weight percent of soybean protein powder, 2 to 4 weight percent of molasses, 0.5 to 1 weight percent of gibberellin membrane residual liquid, 0.1 to 0.4 weight percent of monopotassium phosphate, 0.1 to 0.4 weight percent of calcium carbonate, 0.1 to 0.2 weight percent of magnesium sulfate heptahydrate, 0.02 to 0.05 weight percent of sodium chloride, 0.06 to 0.10 weight percent of dichlord and the balance of water;
the bacillus velezensis strain is preserved in common microorganism of China Committee for culture Collection of microorganisms, and the preservation number is CGMCC No.24996.
In one or more embodiments of the present application, the yeast powder is 3wt%, the soy protein powder is 1wt%, the molasses is 3wt%, the gibberellin film raffinate is 0.5wt%, the potassium dihydrogen phosphate is 0.1wt%, the calcium carbonate is 0.15wt%, the magnesium sulfate heptahydrate is 0.15wt%, the sodium chloride is 0.02wt%, the dichlord is 0.1wt%, and the balance is water.
In one or more embodiments of the present application, the yeast powder is 2wt%, the soy protein powder is 1.5wt%, the molasses is 2wt%, the gibberellin film raffinate is 0.5wt%, the potassium dihydrogen phosphate is 0.2wt%, the calcium carbonate is 0.3wt%, the magnesium sulfate heptahydrate is 0.15wt%, the sodium chloride is 0.03wt%, the dichlord is 0.07wt%, and the balance is water.
In one or more embodiments of the present application, the yeast powder is 2wt%, the soy protein powder is 2wt%, the molasses is 4wt%, the gibberellin film raffinate is 0.5wt%, the potassium dihydrogen phosphate is 0.15wt%, the calcium carbonate is 0.2wt%, the magnesium sulfate heptahydrate is 0.1wt%, the sodium chloride is 0.02wt%, the dichlord is 0.06wt%, and the balance is water.
In one or more embodiments of the application, the gibberellin film residue is prepared by the following method:
(1) preparing gibberellin fermentation liquor;
(2) filtering the gibberellin fermentation broth of (1) by a membrane to obtain clear liquid and gibberellin membrane residual liquid.
Based on the fermentation medium, the application also provides a fermentation method of bacillus velezensis bacteria.
A fermentation process of bacillus velezensis bacteria comprising the steps of:
s1, taking a strain with a preservation number of CGMCC No.24996 and a name of bacillus velezensis;
s2, marking strains on a solid plate of a culture medium for activation, and picking single bacterial colonies after culturing;
s3, inoculating the picked single colony into a shake flask culture medium for culture, and transferring the single colony into a small-scale fermentation tank;
s4, transferring the cultured small-scale fermentation tank into a fermentation tank;
s5, discharging the fermented product from the fermentation tank after culturing;
s6, obtaining fermentation liquor.
The bacillus velezensis strain is named as LMFSSMJ-1, and the thallus shape of the LMFSSMJ-1 is characterized in that: gram positive bacteria, which are rod-shaped, have rounded ends, are single, paired or in strings, and have spores which are oval or round, can move from the middle end to the secondary end and grow all around; the single colony of the strain is round on a nutrient agar culture medium, the edge is irregular, obvious folds are formed, the diameter is 2-3 mm, the strain is micro-raised, and the strain is white and opaque in color.
Based on the fermentation method, the application also provides fermentation.
The fermentation liquor is prepared by the fermentation method of the efficient mite-killing strain.
Based on the fermentation broth, the application also provides an acaricide.
An acaricide comprising a fermentation broth as an active ingredient, which is the above fermentation broth.
The application has the following principle and beneficial effects:
the fermentation liquor prepared by the cooperation of the special fermentation medium and bacillus velezensis bacteria is used as an active ingredient of the acaricide, and the acaricide rate reaches 92% in a field experiment.
Description of biological preservation
The Latin chemical name is bacillus velezensis, and is preserved in China general microbiological culture Collection center (address: north Chen West Lu No. 1, 3 of the area of Charpy, beijing, china center of science, postal code: 1000101) for 30 months in 2022, with a preservation number of CGMCC No.24996.
Detailed Description
The present application will be further described below.
Example 1
The original strain (the preservation number of the original strain is CGMCC No.24996 and the name is bacillus velezensis (Bacillus bailii)) is separated from citrus leaves in Qingshen county of the eyebrow mountain city.
The strain with the preservation number of CGMCC No.24996 and the name of bacillus velezensis (Bacillus bailii) is named as LMFSSMJ-1, and the thallus shape of LMFSSMJ-1 is characterized in that: gram positive bacteria, which are rod-shaped, have rounded ends, are single, paired or in strings, and have spores which are oval or round, can move from the middle end to the secondary end and grow circumferentially. The single colony of the strain is round on a nutrient agar culture medium, the edge is irregular, obvious folds are formed, the diameter is 2-3 mm, the strain is micro-raised, and the strain is white and opaque in color.
Example 2
A special fermentation medium with the preservation number of CGMCC No.24996 and the name of bacillus velezensis (Bacillus bailii) of example 1 is shown in the formula of Table 1.
Example 3
A special fermentation medium with the preservation number of CGMCC No.24996 and the name of bacillus velezensis (Bacillus bailii) of example 1 is shown in the formula of Table 1.
Example 4
A special fermentation medium with the preservation number of CGMCC No.24996 and the name of bacillus velezensis (Bacillus bailii) of example 1 is shown in the formula of Table 1.
Comparative example 1
A fermentation medium having a formulation as set forth in table 1.
Comparative example 2
A fermentation medium having a formulation as set forth in table 1.
Comparative example 3
A fermentation medium having a formulation as set forth in table 1.
TABLE 1 (wt%)
In Table 1, gibberellin membrane raffinates are prepared by the following steps:
(1) gibberellin fermentation broth was prepared according to CN113957115a (a fermentation method for high gibberellic acid production) fermentation method of example 3.
(2) Filtering the gibberellin fermentation broth of (1) through a tube membrane to obtain a clear solution and a membrane raffinate, wherein the clear solution is used for subsequent extraction to produce GA3, and the membrane raffinate is the gibberellin membrane raffinate in Table 1. The gibberellin membrane residual liquid is yellow viscous liquid, and the main components of the gibberellin membrane residual liquid are water, microbial mycelium, a culture medium which is not used by metabolism, inorganic salt and a small amount of gibberellin residues, the dry matter content is about 10%, and the gibberellin membrane residual liquid is rich in crude proteins, various amino acids, nucleic acids, lipids, macroelements, microelements and the like.
Example 5
The components of the fermentation media of examples 1 to 3 and comparative examples 1 to 3 were mixed uniformly, and fermentation broths were prepared according to the following steps S1 to S6 of the fermentation method.
S1, taking out the strain with the preservation number of CGMCC No.24996 and the name of bacillus velezensis strains of the embodiment 1 preserved in an ultralow temperature refrigerator at the temperature of minus 80 ℃.
S2, streaking on an agar culture medium solid plate for activation, and after 2 days of culture, picking single colonies.
S3, inoculating the picked single colony into a shake flask culture medium, culturing for 16 hours in a shaking table at the temperature of 30 ℃ and at 200rpm, and transferring into a small-scale fermentation tank.
S4, inoculating 1% of bacterial liquid into a small test fermentation tank with the liquid loading amount of 30L, adjusting the fermentation parameters, rotating at 300rpm, controlling the temperature to 30 ℃, controlling the pH to 7, controlling the aeration flow to 1vvm, culturing for 16h, and transferring into a 5T fermentation tank.
S5: fermenting and culturing inFermenting and culturing under the condition of 0.04Mpa for 72h, and discharging. The fermentation conditions are as follows: the temperature is 30-35 ℃, the pH is 6-8, and the ventilation rate is 100-120 m 3 And/h, the rotating speed is 200-300rpm. The fermentation medium was one of the fermentation media of examples 1 to 3 and comparative examples 1 to 3.
S6: obtaining fermentation liquor.
In the present embodiment of the present application,
the agar medium comprises the following components: 10g of peptone, 3g of beef extract, 5g of sodium chloride, 15-20g of agar and 1000ml of distilled water;
the shake flask culture medium comprises the following components: 2% of peptone, 2.5% of glucose, 0.1% of potassium dihydrogen phosphate, 0.02% of calcium carbonate, 0.005% of magnesium sulfate heptahydrate, 0.002% of manganese sulfate, 0.002% of sodium chloride and the balance of water.
The composition of the test medium is as follows: 2wt% of yeast powder, 1.5wt% of soybean protein powder, 2wt% of molasses, 0.5wt% of gibberellin membrane residual liquid, 0.2wt% of monopotassium phosphate, 0.3wt% of calcium carbonate, 0.15wt% of magnesium sulfate heptahydrate, 0.03wt% of sodium chloride, 0.07wt% of dichlormid and the balance of water.
Example 6
The fermentation broths of example 5 were diluted 100-fold and 150-fold respectively and applied as spray broths, while clear water and 20% abamectin and spirodiclofen were diluted 3000-fold and sprayed as controls.
Selecting a citrus garden with basically consistent growth vigor, a plurality of spider mite port cardinality and serious disease conditions, arranging three cells on each plant, selecting 6 plants in each cell, selecting 6 leaves in each plant according to the southeast and northwest directions, numbering the leaves and listing the leaves, and marking.
The whole citrus plant is sprayed, and all the front and back sides of the leaf need to be completely sprayed, and special attention is paid to the back sides of the leaf and the grooves of the branches, so that the leaf is suitable for dripping the liquid medicine.
The number of spider mites marking the leaf blades was investigated 1d after the application, and the control effect was calculated, and the results are shown in table 2 below.
The calculation method is as follows: tetranychus telangiectasis rate = (number of live mites-number of live mites after drug)/number of live mites 100%. Control effect= (treatment group spider mite reduction rate ± control group spider mite reduction rate)/(100+ -control group spider mite reduction rate) ×100%.
TABLE 2
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims (6)

1. A fermentation method of bacillus belgium, comprising the following steps:
s1, taking a strain which is CGMCC No.24996 and is named as bacillus bailiibacillus velezensis）；
S2, marking strains on a solid plate of a culture medium for activation, and picking single bacterial colonies after culturing;
s3, inoculating the picked single colony into a shake flask culture medium for culture, and transferring the single colony into a small-scale fermentation tank;
s4, culturing in a small-scale fermentation tank for 16 hours, and transferring the small-scale fermentation tank into the fermentation tank;
s5, discharging the fermentation medium after the fermentation tank is cultured, wherein the fermentation medium in the fermentation tank is a special fermentation medium for bacillus bailii;
s6, obtaining fermentation liquor;
the special fermentation medium for bacillus bailii comprises the following components:
2-3 wt% of yeast powder, 1-2 wt% of soybean protein powder, 2-4 wt% of molasses, 0.5-1 wt wt% of gibberellin membrane residual liquid, 0.1-0.4 wt% of monopotassium phosphate, 0.1-0.4 wt% of calcium carbonate, 0.1-0.2 wt% of magnesium sulfate heptahydrate, 0.02-0.05 wt% of sodium chloride, 0.06-0.10 wt% of dichlord and the balance of water;
the gibberellin membrane raffinate is prepared by the following method:
(1) preparing gibberellin fermentation liquor;
(2) filtering the gibberellin fermentation broth of (1) by a membrane to obtain clear liquid and gibberellin membrane residual liquid.
2. The fermentation method of bacillus beljalis according to claim 1, wherein the yeast powder is 3wt%, the soybean protein powder is 1wt%, the molasses is 3wt%, the gibberellin film raffinate is 0.5wt%, the potassium dihydrogen phosphate is 0.1wt%, the calcium carbonate is 0.15wt%, the magnesium sulfate heptahydrate is 0.15wt%, the sodium chloride is 0.02wt%, the dichlord is 0.1wt%, and the balance is water.
3. The method for fermenting bacillus beljalis according to claim 1, wherein the yeast powder is 2wt%, the soybean protein powder is 1.5wt%, the molasses is 2wt%, the gibberellin membrane residual liquid is 0.5wt%, the potassium dihydrogen phosphate is 0.2wt%, the calcium carbonate is 0.3wt%, the magnesium sulfate heptahydrate is 0.15wt%, the sodium chloride is 0.03wt%, the dichlord is 0.07wt%, and the balance is water.
4. The fermentation method of bacillus beljalis according to claim 1, wherein the yeast powder is 2wt%, the soybean protein powder is 2wt%, the molasses is 4wt%, the gibberellin film raffinate is 0.5wt%, the potassium dihydrogen phosphate is 0.15wt%, the calcium carbonate is 0.2wt%, the magnesium sulfate heptahydrate is 0.1wt%, the sodium chloride is 0.02wt%, the dichlord is 0.06wt%, and the balance is water.
5. A fermentation broth, characterized in that it is prepared by the fermentation method of bacillus beljalis according to any one of claims 1 to 4.
6. An acaricide comprising a fermentation broth as an active ingredient, wherein the fermentation broth is the fermentation broth of claim 5.
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