CN115161239A - 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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CN115161239A
CN115161239A CN202210875568.2A CN202210875568A CN115161239A CN 115161239 A CN115161239 A CN 115161239A CN 202210875568 A CN202210875568 A CN 202210875568A CN 115161239 A CN115161239 A CN 115161239A
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bacillus velezensis
gibberellin
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CN115161239B (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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    • 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
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    • 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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    • A—HUMAN NECESSITIES
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Abstract

The invention discloses a special fermentation medium for bacillus velezensis, which comprises the following components in part by weight: 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 sodium chloride and the balance of water. The fermentation liquor prepared by the special fermentation medium disclosed by the invention 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 invention relates to the field of agriculture, in particular to a special fermentation medium for bacillus velezensis bacteria, a fermentation method for the bacillus velezensis bacteria, fermentation liquor and an acaricide.
Background
Among the current agricultural pest control, phytophagous mites are one of the most difficult pest groups recognized in the world, the main group of which is spider mites, commonly known as red spiders, belonging to the phylum Arthropoda (arthopoda), arachnida (Arachnida), acari subclass (Acari), euacarida (Acariformes), spider mite superfamily (Tetranychoidea). The spider mites are world pest mites, are small in size, short in generation, short in development period, fast in propagation, strong in adaptability, high in mutation rate, extremely easy to generate drug resistance, and extremely difficult to control, so that the situation that the mites of crops are more and more seriously damaged and the 'small insects are in a great disaster' is caused, the agricultural production is greatly lost, the agricultural production increase and the farmer income increase are influenced, and the spider mites are an important big problem to be solved urgently in the current agricultural production.
Chemical control is still the main strategy for controlling mite damage in the production of horticultural crops in China, but the abuse and misuse of acaricide can easily cause the generation of drug resistance of spider mites, thus greatly reducing the field control effect of chemical pesticides. Statistics in 2008 show that spider mites generate drug resistance to active ingredients in 92 of the current medicament, and the drug resistance comprises neurotoxin insecticides such as organic 24976. The drug resistance of the tetranychus urticae is much more serious than that of other field pests such as diamondback moth, aphid and the like, so that the tetranychus urticae is regarded as the most drug resistant species by scientific personnel. The drug resistance of Tetranychus Urticae Koch is higher than that of other mites on greenhouse vegetables and open fruit trees. For example, the resistance times of the tetranychus urticae to abamectin, dicofol, propargite and the like are more than 10 times of that of other mites. The whole genome DNA research of two-spotted spider mite shows that the detoxication gene in vivo is 3 times as much as that of other animals, 39 genes of one drug resistance gene family are contained, and only 14 insects and vertebrates exist. Therefore, development of control strategies based on biological control is an important approach to sustainable control.
Among various control means of pest mites, biological control has been a focus and hot spot of research, playing an important role in pest control strategies. At present, the biological control research on the aspects of pest mite natural enemies, botanical pesticides, tetranychus urticae and the like has made some progress and results. The natural enemies of the tetranychus urticae are various, phytoseiidae and predatory mite are two types which are more researched and applied, and particularly, the phytoseiidae is industrially produced on a large scale in many countries. The netherlands have 60% of greenhouses that use phytoseiid persimmons to control tetranychus urticae on cucumbers, whereas british, finland, sweden and danish have exceeded 70%. The plant protection institute of agricultural academy of sciences of Fujian province initially establishes the first mass breeding base of predatory mites in China, and the popularization and application area of the plant protection base in China reaches 13.3 ten thousand hectares. However, the use of natural enemies is limited in application and development because of the disadvantages that dominant populations are not easy to establish, the cost for maintaining the natural enemy populations is high, the chemical control cannot be carried out together with the chemical control, and the like. The active ingredients of the botanical pesticide are safe and low in toxicity, most of the botanical pesticide is a plant secondary metabolite with acaricidal activity, more than 200 plants which are measured for the acaricidal activity are reported, but the active ingredients with low activity are not clear, so that the botanical pesticide has little development and utilization value.
Various kinds of cytoplasmic-inherited intracellular symbiotic bacteria are widely distributed in arthropods and are classified into primary symbiotic bacteria (primary symbiot) and secondary symbiotic bacteria (secondary symbiot). The nascent symbiotic bacteria are necessary for the survival of the host, are co-evolved with the host, are widely distributed in the histiocyte of the host, are mainly vertically transmitted through a mother system, and can be horizontally transmitted at the same time. The secondary symbionts are not essential for host survival, may be beneficial to the host and may also be harmful, and the harmful may be referred to as spider mite pathogens. The tetranychus urticae is subjected to a long evolutionary process, and interdependence and restriction relations are formed between the tetranychus urticae and pathogenic bacteria. How to utilize the pathogenic bacteria and bioactive substances thereof has become a main attack direction of many laboratories in developed countries in recent years.
Spider mites have relatively few pathogenic bacteria in nature, and are recorded mainly by Wolbachia, and originally found in the ovary of culex pipiens (culex pipiens), and can infect various arthropods such as insects and mites. The bacteria can cause abnormal host reproductive behaviors, such as cytoplasm incompatibility, parthenogenesis, emasculation, feminization and the like. The strain with similar activity also can be Candidatus cardium, which is called cardium for short, is discovered from parasitoid wasps of Encarsia, and can cause leaf mite feminization, induce parthenogenesis, induce cytoplasm incompatibility, influence host fitness and change the spawning behavior of hosts. Verticillium lecanii Zimm can be used for preventing and treating aphid, whitefly and mite. The Acremonium hansfordii strain is obtained by separating larvae of a natural disease-susceptible peach aphid (Myzuspicate Sulzer), has an infection effect on cabbage caterpillar, peach aphid and two-spotted spider mite, and after the host infects the Acremonium, the in-vivo protein of the Acremonium hansfordii strain is consumed, and the tissues and organs of the synthesized protein are damaged, so that the in-vivo detoxification enzyme and the protective enzyme are out of balance. The research cases and the utilization of the pathogenic bacteria of the tetranychus urticae are relatively few, but the pathogenic bacteria of the tetranychus urticae are utilized to 'control mites with bacteria', so that the population density can be safely and effectively adjusted, the tetranychus urticae extract plays an important role in biological control and is an important component of biological control work.
On the premise that the existing pests generally generate drug resistance and pay attention to the continuity and harmlessness of comprehensive control of the pests, the microbial resources are developed, strains with high pathogenicity are screened, and the consumption of chemical pesticides is reduced, which is the development direction of the current advanced agriculture.
Disclosure of Invention
Based on the problems, the invention provides the special fermentation medium for the bacillus velezensis bacteria, and the special fermentation medium is diluted with fermentation liquor prepared by matching the bacillus velezensis bacteria, and then used as an active ingredient for killing mites, so that the mite-killing effect is good.
A special fermentation medium for bacillus velezensis bacteria 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% 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 sodium chloride and the balance of water;
the bacillus velezensis strain is preserved in common microorganisms of China Committee for culture Collection of microorganisms with the preservation number of 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 membrane residue is 0.5wt%, the monopotassium phosphate is 0.1wt%, the calcium carbonate is 0.15wt%, the magnesium sulfate heptahydrate is 0.15wt%, the sodium chloride is 0.02wt%, the sodium chloride is 0.1wt%, and the balance is water.
In one or more embodiments herein, the yeast powder is 2wt%, the soy protein flour is 1.5wt%, the molasses is 2wt%, the gibberellin membrane raffinate is 0.5wt%, 0.2wt% of monopotassium phosphate, 0.3wt% of calcium carbonate, 0.15wt% of magnesium sulfate heptahydrate, 0.03wt% of sodium chloride, 0.07wt% of natural enemy and the balance of 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 membrane residue is 0.5wt%, the monopotassium phosphate is 0.15wt%, the calcium carbonate is 0.2wt%, the magnesium sulfate heptahydrate is 0.1wt%, the sodium chloride is 0.02wt%, the sodium chloride is 0.06wt%, and the balance is water.
In one or more embodiments of the present application, the gibberellin membrane raffinate is prepared by:
(1) preparing gibberellin fermentation liquor;
(2) filtering the gibberellin fermentation liquor obtained in the step (1) through a membrane to obtain clear liquid and gibberellin membrane residual liquid.
Based on the fermentation medium, the invention also provides a fermentation method of the bacillus velezensis bacteria.
A fermentation method of Bacillus velezensis bacteria comprises the following steps:
s1, taking a strain with the preservation number of CGMCC No.24996 and the name of bacillus velezensis;
s2, marking the strain on a solid plate of a culture medium for activation, and picking a single colony after culture;
s3, inoculating the picked single colony into a shake flask culture medium for culture and then transferring into a small-scale fermentation tank;
s4, transferring the cultured small-scale fermentation tank into a fermentation tank;
s5, putting the culture medium into a fermentation tank after the culture;
and S6, obtaining fermentation liquor.
The bacillus velezensis strain is named as LMFSSMJ-1, and the morphological characteristics of the LMFSSMJ-1 are as follows: gram-positive bacteria, wherein the bacteria are rod-shaped, the two ends of the bacteria are blunt and round, the bacteria appear singly, in pairs or in strings, the bacteria have spores, the spores are oval or round, and the bacteria can move and generate flagellum peritomatically from the middle end to the secondary end; the strain is in a round shape on a nutrient agar culture medium, has irregular edges, obvious folds, a diameter of 2-3 mm, slight bulges, white color and non-transparency.
Based on the fermentation method, the application also provides fermentation.
The fermentation liquor is prepared by the fermentation method of the high-efficiency acaricidal strain.
Based on the fermentation liquor, the application also provides an acaricide.
An acaricide comprising a fermented liquid as an active ingredient, the fermented liquid being the above-mentioned fermented liquid.
The invention has the following principle and beneficial effects:
the fermentation broth prepared by matching the special fermentation medium and bacillus velezensis bacteria is used as an active ingredient of the acaricide, and the acaricide rate reaches 92% in field experiments.
Biological preservation Instructions
The collection bacteria is classified, named Latin, and named Bacillus velezensis, and is preserved in China general microbiological culture Collection center (address: no. 3 of West Lu No. 1 of Beijing, chaoyang, and Microbiol research institute of Chinese academy of sciences, postal code: 1000101) at 30 months 05 and 2022, and the preservation number is CGMCC No.24996.
Detailed Description
The present invention will be further explained below.
Example 1
An original strain is separated from citrus leaves in Qingshen county of Meishan city (the preservation number of the original strain is CGMCC No.24996, and the name of the original strain is bacillus velezensis).
The preservation number is CGMCC No.24996, the bacterial strain named Bacillus velezensis (Bacillus belgii) is named LMFSSMJ-1, and the morphological characteristics of the bacterial strain of LMFSSMJ-1 are as follows: gram-positive bacteria, which are rod-shaped, have two blunt ends, appear singly, in pairs or in strings, have spores, are oval or circular, grow from the middle end to the secondary end, can move, and grow around flagellum. The strain is in a round shape on a nutrient agar culture medium, has irregular edges, obvious folds, a diameter of 2-3 mm, slight bulges, white color and non-transparency.
Example 2
A special fermentation medium with the preservation number of CGMCC No.24996 and the name of Bacillus velezensis (Bacillus belief) in example 1 has a formula shown in Table 1.
Example 3
A special fermentation medium with the preservation number of CGMCC No.24996 and the name of Bacillus velezensis (Bacillus belief) in example 1 has a formula shown in Table 1.
Example 4
A special fermentation medium with the preservation number of CGMCC No.24996 and the name of Bacillus velezensis (Bacillus belgii) in example 1 has the formula shown in Table 1.
Comparative example 1
A fermentation medium having the formula shown in Table 1.
Comparative example 2
A fermentation medium having the formula shown in Table 1.
Comparative example 3
A fermentation medium having the formula shown in Table 1.
TABLE 1 (wt%)
Figure BDA0003762305050000071
In Table 1, a gibberellin membrane raffinate was prepared by the following steps:
(1) the fermentation method of CN113957115A (a fermentation method for high yield of gibberellic acid) in example 3 is used to prepare gibberellin fermentation broth.
(2) And (3) filtering the gibberellin fermentation liquor in the step (1) through a tubular membrane to obtain clear liquid and membrane residual liquid, wherein the clear liquid is used for producing GA3 through subsequent extraction, and the membrane residual liquid is the gibberellin membrane residual liquid in the table 1. The gibberellin membrane residual liquid is yellow viscous liquid, the main components of the gibberellin membrane residual liquid are water, microbial mycelium, culture medium which is not metabolized and utilized completely, 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 protein, various amino acids, nucleic acid, lipid, major elements, trace elements and the like.
Example 5
The ingredients of the fermentation media of examples 1 to 3 and comparative examples 1 to 3 were mixed uniformly to prepare fermentation solutions 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 strain in the example 1 stored in an ultra-low temperature refrigerator at minus 80 ℃.
And S2, marking on an agar culture medium solid plate for activation, and picking out a single colony after culturing for 2 days.
S3, inoculating the picked single colony in a shake flask culture medium, culturing for 16h in a shaking table with the temperature of 30 ℃ and the rpm of 200, and transferring into a small-scale fermentation tank.
S4, inoculating 1% of bacterial liquid into a small-scale fermentation tank with the liquid loading capacity of 30L, adjusting the fermentation parameters, controlling the rotating speed to be 300rpm, the temperature to be 30 ℃, the pH to be 7 and the ventilation flow to be 1vvm, culturing for 16h, and transferring into a 5T fermentation tank.
S5: fermenting and culturing inPerforming fermentation culture under the condition of 0.04Mpa, culturing for 72h, and discharging into the tank. The fermentation conditions are as follows: the temperature is 30-35 ℃, the pH value is 6-8, and the ventilation volume is 100-120 m 3 The rotation speed is 200-300rpm. The fermentation medium was one of the fermentation media of examples 1-3 and comparative examples 1-3.
S6: obtaining the fermentation liquor.
In the present embodiment, the first and second electrodes are,
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: peptone 2%, glucose 2.5%, potassium dihydrogen phosphate 0.1%, calcium carbonate 0.02%, magnesium sulfate heptahydrate 0.005%, manganese sulfate 0.002%, sodium chloride 0.002%, and water in balance.
The components of the small test culture medium are 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 sodium silicate and the balance of water.
Example 6
The fermentation solutions of example 5 were diluted 100 times and 150 times, respectively, and applied as spraying solutions, while clear water and 20% abamectin-spirodiclofen were diluted 3000 times as a control for spraying.
Selecting a citrus orchard with basically consistent growth vigor, more basic tetranychid population and serious illness, arranging three cells for each plant, selecting 6 plants in each cell, selecting 6 leaves in each plant according to the directions of south, east and north, numbering the leaves and marking the leaves.
The whole plant of the selected citrus plant is sprayed, the front and back surfaces of all the leaves need to be completely sprayed, and the back surfaces of the leaves and the grooves of the branches are specially noticed, and the liquid medicine is preferably dripped into the leaves.
The number of the live leaf mites of the marked leaves was investigated 1d after application, and the control effect was calculated, with the results shown in table 2 below.
The calculation method is as follows: tetranychus decline rate = (number of live mites base-number of live mites after drug)/number of live mites base = 100%. The control effect = (the tetranychus decline rate of the treatment group +/-the tetranychus decline rate of the control group)/(100 +/-the tetranychus decline rate of the control group) × 100%.
TABLE 2
Figure BDA0003762305050000101
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (8)

1. A special fermentation culture medium for bacillus velezensis is characterized by comprising the following components in parts by weight:
2-3 wt% of yeast powder, 1-2 wt% of soybean protein powder, 2-4 wt% of molasses, 0.5-1 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 sodium chloride and the balance of water;
the bacillus velezensis strain is preserved in common microorganisms of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.24996.
2. The special fermentation medium for bacillus velezensis bacteria as claimed in claim 1, wherein the yeast powder is 3wt%, the soybean protein powder is 1wt%, the molasses is 3wt%, the gibberellin membrane raffinate is 0.5wt%, the monopotassium phosphate is 0.1wt%, the calcium carbonate is 0.15wt%, the magnesium sulfate heptahydrate is 0.15wt%, the sodium chloride is 0.02wt%, the sodium chloride is 0.1wt%, and the balance is water.
3. The special fermentation medium for bacillus velezensis bacteria as claimed in claim 1, wherein the yeast powder is 2wt%, the soybean protein powder is 1.5wt%, the molasses is 2wt%, the gibberellin membrane raffinate is 0.5wt%, the monopotassium phosphate is 0.2wt%, the calcium carbonate is 0.3wt%, the magnesium sulfate heptahydrate is 0.15wt%, the sodium chloride is 0.03wt%, the natural enemy is 0.07wt%, and the balance is water.
4. The special fermentation medium for bacillus velezensis bacteria as claimed in claim 1, wherein the yeast powder is 2wt%, the soybean protein powder is 2wt%, the molasses is 4wt%, the gibberellin membrane raffinate is 0.5wt%, the monopotassium phosphate is 0.15wt%, the calcium carbonate is 0.2wt%, the magnesium sulfate heptahydrate is 0.1wt%, the sodium chloride is 0.02wt%, the natural enemy is 0.06wt%, and the balance is water.
5. The special fermentation medium for Bacillus velezensis bacteria according to any one of claims 1 to 4, wherein the gibberellin membrane raffinate is prepared by:
(1) preparing gibberellin fermentation liquor;
(2) filtering the gibberellin fermentation liquor obtained in the step (1) through a membrane to obtain clear liquid and gibberellin membrane residual liquid.
6. A fermentation method of Bacillus velezensis bacteria comprises the following steps:
s1, taking a strain, wherein the strain is a strain with the preservation number of CGMCC No.24996 and the name of bacillus velezensis;
s2, marking the strain on a solid plate of a culture medium for activation, and picking a single colony after culture;
s3, inoculating the selected single colony in a shake flask culture medium for culture and then transferring the single colony into a small-scale fermentation tank;
s4, transferring the cultured product into a fermentation tank after culturing for 16h in the small fermentation tank;
s5, putting the strain into a tank after the strain is cultured in a fermentation tank, wherein a fermentation culture medium in the fermentation tank culture is the special fermentation culture medium for the bacillus velezensis bacteria in any one of claims 1 to 4;
and S6, obtaining fermentation liquor.
7. A fermentation broth produced by the fermentation method of Bacillus velezensis according to claim 6.
8. An acaricide comprising a fermentation broth as an active ingredient, characterized in that the fermentation broth is the fermentation broth according to claim 7.
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CN115161240A (en) * 2022-07-25 2022-10-11 四川龙蟒福生科技有限责任公司 Bacillus velezensis strain and application thereof

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