CN117264847B - Bacillus bailii YTQ, compound agent and application thereof in improving low-temperature freeze injury resistance of plants - Google Patents
Bacillus bailii YTQ, compound agent and application thereof in improving low-temperature freeze injury resistance of plants Download PDFInfo
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- CN117264847B CN117264847B CN202311446207.7A CN202311446207A CN117264847B CN 117264847 B CN117264847 B CN 117264847B CN 202311446207 A CN202311446207 A CN 202311446207A CN 117264847 B CN117264847 B CN 117264847B
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
-
- 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
- A01P21/00—Plant growth regulators
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
Abstract
The invention relates to the technical field of biocontrol bacteria, in particular to bacillus beijerinckii YTQ, a compound agent and application thereof in improving the low-temperature freezing injury resistance of plants. The bacillus belicus YTQ is a strain separated, purified and screened from cherry orchard rhizosphere soil of the agricultural science institute of tobacco stage city in Shandong province, and the antibacterial substances and plant growth regulators contained in fermentation bacteria liquid can improve the disease resistance and low-temperature freeze injury resistance of plants. Furthermore, the compound agent formed by the bacillus belicus YTQ fermentation liquor, sodium alginate, fulvic acid, calcium sugar alcohol, magnesium sulfate and zinc sulfate according to a specific proportion can obviously improve the low-temperature freezing injury resistance of plants, has a control effect on the low-temperature freezing injury of the plants which is obviously better than that of the common Biyan powder, and provides a new technical support for cherry prevention and treatment.
Description
Technical Field
The invention relates to the technical field of biocontrol bacteria, in particular to bacillus beijerinckii YTQ, a compound agent and application thereof in improving the low-temperature freezing injury resistance of plants.
Background
Cherry is one of the earliest mature fruits in northern deciduous fruit trees, is popular with consumers and cultivars due to delicious taste, short growing period and high economic benefit, and is one of the fastest growing tree species in northern areas of China. The cherry belongs to warm and cold-resistant tree species, and is suitable for cultivation in areas with the average annual temperature of 10-15 ℃. The winter temperature variation amplitude in northern areas of China is large, and the phenomena of freezing death of small branches, frostbite of flower buds and even freezing death of the whole plant are often caused by low temperature and spring frost in winter, so that the research on the technology for effectively defending the frost in low temperature and spring in winter has become a key factor to be solved in cherry production.
At present, physical methods such as trunk binding cloth plastic, trunk white coating, irrigation, smoking, ignition and the like of an orchard before frost are mainly adopted for preventing and controlling cherry low-temperature freeze injury, but the methods are limited by objective conditions of the orchard, and have certain difficulty in implementation.
Disclosure of Invention
In order to solve the problems, the invention provides bacillus beijerinckii YTQ, a compound agent and application thereof in improving the low-temperature freezing injury resistance of plants. The bacillus belicus YTQ3 provided by the invention has the function of improving the low-temperature freezing injury resistance of plants, and can prevent and treat the low-temperature freezing injury of cherry by spraying the bacillus belicus YTQ Shi Beilai and the compound agent thereof, and has the advantages of simplicity, easiness, low cost and quick response.
In order to achieve the above object, the present invention provides the following technical solutions:
The invention provides bacillus beleiensis (Bacillus velezensis) YTQ3, wherein the preservation number of bacillus beleiensis YTQ is CCTCC No: m20211465.
The invention provides a compound agent for improving the low-temperature freezing injury resistance of plants, which comprises the following components: fermentation liquor, sodium alginate, fulvic acid, calcium sugar alkoxide, magnesium sulfate and zinc sulfate; the fermentation liquor is the fermentation liquor of bacillus bailii YTQ according to the technical scheme.
Preferably, the volume-mass ratio of the fermentation liquid to the sodium alginate is 100ml: 45-55 mg; the volume mass ratio of the fermentation liquor to the fulvic acid is 100ml: 500-520 mg; the volume mass ratio of the fermentation liquor to the sugar alcohol calcium is 100ml: 200-210 mg; the volume-mass ratio of the fermentation liquor to the magnesium sulfate is 100ml: 90-110 mg; the volume mass ratio of the fermentation liquor to the zinc sulfate is 100ml: 100-120 mg.
Preferably, the viable count of bacillus bailii YTQ in the fermentation broth is not less than 1×10 6 CFU/mL.
Preferably, the fermentation medium for preparing the fermentation broth comprises the following components in concentration: beef extract 2-3 g/L, naCl-1.5 g/L, soybean protein 1.5-2.5 g/L and corn starch 1-2 g/L.
The invention provides bacillus bailii YTQ or application of the compound agent in improving low-temperature freeze injury resistance and/or preventing and controlling plant diseases.
Preferably, the plant comprises cherry.
Preferably, the pathogenic bacteria of the plant disease include one or more of Botrytis cinerea (Botryosphaeria dothidea), botrytis cinerea (Botrytis cinerea), fusarium oxysporum (Fusarium oxysporum) and phomopsis (Phomopsis spp.).
The invention provides a method for preventing low-temperature freezing injury of cherry in winter and/or preventing and controlling cherry diseases, which comprises the following steps: spraying cherry branches with the compound agent in the middle 12 months and the middle 1 month respectively; the compound agent is the compound agent according to the technical scheme.
The invention provides a method for preventing cherry early spring frost damage and/or preventing cherry diseases, which is characterized by comprising the following steps: spraying cherry flower buds with the compound agent and spraying cherry young fruits with the compound agent 3-5 days after flowers are removed in the red period of flower buds; the compound agent is the compound agent according to the technical scheme.
The beneficial effects are that:
The invention provides bacillus beleiensis (Bacillus velezensis) YTQ3, wherein the preservation number of bacillus beleiensis YTQ is CCTCC No: m20211465. The bacillus belicus YTQ is a strain separated, purified and screened from cherry orchard rhizosphere soil of the agricultural science institute of tobacco stand, shandong province, and antibacterial substances and biological stimulation hormone contained in fermentation bacteria liquid can improve the disease resistance and low-temperature freeze injury resistance of plants.
Furthermore, the compound agent formed by the bacillus belicus YTQ fermentation liquor, sodium alginate, fulvic acid, calcium sugar alcohol, magnesium sulfate and zinc sulfate according to a specific proportion can obviously improve the low-temperature freezing injury resistance of plants, has a control effect on the low-temperature freezing injury of the plants which is obviously better than that of the common Biyan powder, and provides a new technical support for cherry prevention and treatment.
Description of biological preservation
Bacillus belicus YTQ strain Bacillus velezensis, latin was deposited at China Center for Type Culture Collection (CCTCC) at 22 days 11 of 2021, with a deposit address of eight 299 ways in Wuchang district of Wuhan, hubei province, china center for type culture collection with a deposit number of CCTCC No: m20211465.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 shows colony morphology and gram microscopy of Bacillus bailii YTQ;
FIG. 2 is a Bacillus bailii YTQ phylogenetic tree;
FIG. 3 is a circle diagram of the genome of Bacillus bailii YTQ.
Detailed Description
The invention provides bacillus beleiensis (Bacillus velezensis) YTQ3, wherein the preservation number of bacillus beleiensis YTQ is CCTCC No: m20211465.
The bacillus belgium YTQ is a strain separated, purified and screened from cherry orchard rhizosphere soil of the agricultural science institute of tobacco, shandong province, and is determined to be bacillus belgium through morphological observation, physiological and biochemical identification and 16S rDNA molecular identification, and is sent to China center for type culture collection, wherein the collection number is CCTCC No: m20211465; the bacillus bailii YTQ and the antibacterial substances contained in the fermentation liquor thereof mainly comprise surface active substances, bacillus and ubiquitin, bacillus, macrocyclic lactons and difficile, can effectively prevent and control plant diseases caused by Botrytis cinerea, gray mold, fusarium oxysporum and phomopsis, and the plant growth regulator contained in the fermentation liquor can improve the low-temperature freezing injury resistance of plants.
In the invention, the formula of the separation and purification solid culture medium used for separation and purification is preferably as follows: 10g/L, naCl g/L peptone, 5g/L yeast extract and 18g/L agar powder, the pH value is preferably 7.0. The separation and purification culture medium is favorable for the growth and enrichment of bacillus bailii and improves the separation and purification efficiency.
The invention also provides a compound agent for improving the low-temperature freezing injury resistance of plants, which comprises the following components: fermentation liquor, sodium alginate, fulvic acid, calcium sugar alkoxide, magnesium sulfate and zinc sulfate; the fermentation liquor is the fermentation liquor of bacillus bailii YTQ according to the technical scheme. In the invention, the volume-mass ratio of the fermentation liquid to the sodium alginate is preferably 100ml: 45-55 mg, more preferably 100ml:50mg; the volume-mass ratio of the fermentation liquor to the fulvic acid is preferably 100ml:500 to 520mg, more preferably 100ml:500mg; the volume mass ratio of the fermentation liquor to the sugar alcohol calcium is preferably 100ml:200 to 210mg, more preferably 100ml:200mg; the volume-mass ratio of the fermentation liquor to the magnesium sulfate is preferably 100ml: 90-110 mg, more preferably 100ml:100mg; the volume-mass ratio of the fermentation liquor to the zinc sulfate is preferably 100ml:100 to 120mg, more preferably 100ml:120mg.
The present invention is not limited to the specific source of the components of the formulation, and may be commercially available products known to those skilled in the art, unless otherwise specified. In the present invention, the compounding agent preferably further comprises water.
The compound agent of the invention preferably comprises 100ml/L of fermentation liquor. In the invention, the viable count of bacillus bailii YTQ in the fermentation broth is preferably not less than 1 multiplied by 10 6 CFU/mL; the fermentation medium for preparing the fermentation broth preferably comprises the following concentrations of components: beef extract 2-3 g/L, naCl-1.5 g/L, soybean protein 1.5-2.5 g/L and corn starch 1 g-2 g/L, more preferably beef extract 2.5g/L, naCl 1g/L, soybean protein 2g/L and corn starch 1.5g/L; the pH value of the fermentation medium is preferably 7.0; the fermentation temperature for preparing the fermentation broth is preferably 28 ℃. The fermentation medium is favorable for rapid growth and propagation of bacillus beijerinckii YTQ, and improves the bacteria content and antibacterial activity of fermentation liquor. The antibacterial substances (surface active substances, bacillus and ubiquitin, bacillus, macrocyclic lactogen and difficile) and the plant growth regulator contained in the fermentation broth can improve the disease resistance and low-temperature freeze injury resistance of plants.
The compound agent of the invention preferably comprises 45-55 mg/L sodium alginate, preferably 50mg/L sodium alginate by taking water as a solvent. The sodium alginate has better biocompatibility, can enhance the adhesiveness of the compound agent, improve the control effect of plants on low-temperature freeze injury, and prolong the effective period of the compound agent.
The compound agent of the invention preferably comprises 500-520 mg/L of fulvic acid, preferably 500mg/L, calculated by taking water as a solvent. The molecular structure of the fulvic acid has stronger ion exchange and adsorption capacity, can be complexed with magnesium ions and zinc ions in the compound agent, enhances the physiological activity of mineral elements, and can also be used as biostimulant to enhance the response and adaptability of plants to adversity stress.
The compound agent of the invention preferably comprises 200-210 mg/L, preferably 200mg/L, of calcium sugar alkoxide based on water as a solvent. The sugar alcohol calcium provided by the invention is used as a calcium ion donor, the chelate state calcium ions move fast in a plant body and are not easy to react with other substances, and the calcium ions are used as extracellular signals and intracellular second messengers to participate in the growth and development regulation of the plant, so that the low-temperature freezing injury resistance of the plant is further improved.
The compound agent of the invention preferably comprises 90-110 mg/L magnesium sulfate, preferably 100mg/L, by taking water as a solvent. The magnesium sulfate provided by the invention is used as a magnesium ion donor, magnesium ion is an important component of chlorophyll and is also an activator for metabolism of a plurality of biochemical reaction enzymes, and the low-temperature freezing injury resistance of plants can be further improved.
The compound agent of the invention preferably comprises 110-120 mg/L zinc sulfate, preferably 120mg/L zinc sulfate, calculated by taking water as a solvent. The zinc sulfate provided by the invention is used as a zinc ion donor, and zinc ions are catalysts for oxidation reduction in plants, and can participate in synthesis of auxin in plants, so that the resistance of crops to low-temperature freeze injury is enhanced.
The invention also provides bacillus bailii YTQ or application of the compound agent in improving the low-temperature freezing injury resistance of plants and/or preventing and controlling plant diseases.
In the present invention, the plant preferably comprises cherry; the pathogenic bacteria of the plant diseases preferably comprise one or more of the group consisting of Botrytis cinerea, fusarium oxysporum and Phomopsis; the improvement of the low temperature freeze injury resistance of the plant comprises one or more of improving the superoxide dismutase content of the plant, improving the peroxidase content of the plant, improving the catalase content of the plant, reducing the malondialdehyde content of the plant and reducing the relative conductivity of the plant.
The invention also provides a method for preventing low-temperature freezing injury of winter cherry, which comprises the following steps: spraying cherry branches with the compound agent in the middle 12 months and the middle 1 month respectively; the compound agent is the compound agent according to the technical scheme. In the invention, the application amount of the compound agent is preferably 75 kg-100 kg/mu, more preferably 85 kg/mu. The ten days in 12 months are preferably 12 days to 16 days in 12 months, and the average air temperature is preferably 0.5 to 4 ℃; the average temperature is preferably-1 to-4 ℃ in the middle ten days of 1 month, preferably 1 month 15 days to 1 month 20 days.
The invention also provides a method for preventing the frost damage of the cherry in early spring and/or preventing and controlling the cherry diseases, which comprises the following steps: spraying cherry flower buds with the compound agent and spraying cherry young fruits with the compound agent 3-5 days after flowers are removed in the red period of flower buds; the compound agent is the compound agent according to the technical scheme. In the invention, the application amount of the compound agent is preferably 50 kg-75 kg/mu, more preferably 60 kg/mu.
For further explanation of the present invention, bacillus beijerinckii YTQ, a compound preparation and its application in improving the low temperature freeze injury resistance of plants provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Isolation, purification and Classification of antagonistic strains
1 Separation of bacterial strain purification
1.1 Main Medium
LB culture medium and separation and purification solid culture medium; the formula of the separation and purification solid culture medium comprises 10g of peptone, 8g of NaCl, 5g of yeast extract, 18g of agar powder, and distilled water with a constant volume of 1L and a pH value of 7.0.
1.2 Isolation and purification of strains
Rhizosphere soil collection: cherry rhizosphere soil with growing health is collected in cherry orchard of agricultural science institute of tobacco stage City, shandong in 5 ten days of 2021. And (3) during sampling, placing cherry rhizosphere soil into a sterile bag for cooling and preserving, and carrying the cherry rhizosphere soil back to a laboratory for endophytic bacteria separation.
And (3) separation and purification of endophyte: 10g of cherry rhizosphere soil is added with 90mL of sterile water to prepare a soil suspension with the concentration of 10 -1, and the suspension is diluted into 10 -2～10-8 for standby. 100 mu L of each soil suspension with different gradients is taken out from a culture medium flat plate, uniformly coated by a coater, naturally dried and placed in an incubator, and dark-cultured for 48 hours at the temperature of 28 ℃. After single colony is grown, dipping bacterial colonies with different forms by using a sterile gun head, streaking the bacterial colonies on a culture medium flat plate for culture, repeating the steps for several times until the bacterial colony forms are stable, numbering the obtained bacterial strains, and then storing the bacterial strains in a refrigerator at the temperature of minus 80 ℃.
1.3 Screening of highly antagonistic strains and plate counter experiments
The bacterial colony to be detected is dipped in a triangular flask filled with a certain amount of LB liquid medium by a sterile gun head, and is placed in a shaking table for culturing for 24 hours at a constant temperature of 28 ℃ and 300r/min, so as to prepare bacterial suspension for later use.
The inhibition of the isolated strain against the major pathogenic bacteria of cherry (the pathogenic bacteria in Table 1 are from the self-segregating and preserving laboratory of fruit tree biological breeding in the Proc. Of the tobacco station, inc.) was determined by a plate counter method. Firstly, placing a pathogen bacterial cake with the diameter of 6mm which is beaten in advance in the center of a PDA culture medium plate, and crisscross symmetrically inoculating 2 mu L of bacterial suspension of a strain to be tested at a position 3cm away from the center of the plate, inoculating sterile water in a control group in the same way, and repeating each treatment for 3 times. Placing in a temperature-controlled incubator, culturing at 28deg.C under constant temperature, measuring pathogen radius by crisscross method when the radius of pathogen in control group grows to 3/4 of the radius of plate, and calculating inhibition rate according to the following formula.
Inhibition ratio = (control colony radius-treatment colony radius)/(control colony radius) ×100%.
TABLE 1 screening results of highly antagonistic strains of major pathogenic bacteria of cherry
The strain 03 with the highest antagonistic activity is selected according to the results in the table 1 for further classification and identification.
1.4 Morphological characterization:
The purified strain was streaked on LB plate, cultured in a constant temperature incubator at 28℃for 48 hours, and then morphological characteristics such as shape and size, unevenness, color transparency, presence or absence of flagella and spores of single colony were observed.
As can be seen from FIG. 1, the colony of strain YTQ on LB medium is white, edge saw-tooth, round flat, dry and wrinkled; gram staining was positive.
1.5 Physiological and biochemical characterization:
The physiological and biochemical indexes such as the contact enzyme reaction, the starch hydrolysis, the V-P test, the maltose, the lactose, the D-glucose, the nitrate and the like are observed by referring to the Berger's bacteria identification manual and the common bacteria system identification manual.
TABLE 2 results of physiological and biochemical tests
| Detecting physiological and biochemical indicators | Results |
| Nitrate reduction test | Positive and negative |
| H 2 S gas production test | Positive and negative |
| Citrate test | Positive and negative |
| Contact enzyme test | Positive and negative |
| Oxidase test | Positive and negative |
| Starch hydrolysis test | Positive and negative |
| V-P test | Positive and negative |
| Methyl Red test | Negative of |
| Glucose | Positive and negative |
| Lactose and lactose | Positive and negative |
| Sucrose | Positive and negative |
| Fructose | Positive and negative |
| Maltose | Positive and negative |
1.6 Molecular biological identification
Extracting the total genome DNA of the strain as a template to amplify the 16S rDNA sequence. Sequencing was done by Shanghai bioengineering limited. The target fragment is amplified by adopting a universal primer 27F/1492R, and the amplified product is detected by 1% agarose gel electrophoresis and is subjected to gene sequencing by adopting a two-way sequencing method. Sequencing results after BLAST sequence homology alignment by GenBank database (phylogenetic tree, see FIG. 2, Q3 in FIG. 2 is Bacillus bailii YTQ 3), strain number 03 was identified as Bacillus bailii (Bacillus velezensis), designated Bacillus bailii YTQ3. Primer information and 16S rDNA sequence were as follows:
27F：5'-AGAGTTTGATCCTGGCTCAG-3'(SEQ ID NO.1)；
1492R：5'-TACGGYTACCTTGTTACGACTT-3'(SEQ ID NO.2)；
16S rDNA：5'-GGGGGTGCCTATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTAGGAGCCAGCCGCCGAAAGGGGG-3'(SEQ ID NO.3).
Full genome structural analysis of 1.7YTQ3 Strain
The sequencing results were functionally resolved with NR, swiss-Prot, pfam, eggNOG, GO and KEGG databases using a sequencing technique combining the second generation Illumina and the third generation PacBio, as shown in FIG. 3.
The analysis and sequencing result shows that the bacillus subtilis YTQ3 contains 1 chromosome with the size of 3973404bp, the GC content of the chromosome is 46.52 percent, 3790 encoding genes contain 27 rRNA, 86 tRNA genes and 94 sRNA; contains 99 tandem repeats.
Of the 3790 genes predicted, the construction of a genomic circle map (fig. 3) based on COG database annotation results was found to be:
(1) Category of genetic information: 1 chromatin structure-and power-related gene, 0 RNA processing-and modification-related genes, 159 ribosome structure, translation and biosynthesis-related genes, 238 transcription-related genes, 151 replication, recombination and repair-related genes;
(2) Cell-related class: 33 genes related to cell cycle control, chromosome segregation and cell division, 177 genes related to synthesis of cell membranes, cell membranes and cell walls, 0 genes related to cytoskeleton, 0 genes related to extracellular structure, 34 genes related to intracellular transport, extracellular secretion and vesicle transport, 38 genes related to cell movement, 51 genes related to defense mechanisms and 113 genes related to signal transduction mechanisms;
(3) Metabolism category: 98 posttranslational modification, chaperone and protein transport related genes, 174 energy production and conversion related genes, 287 amino acid transport and metabolism related genes, 221 carbohydrate transport and metabolism related genes, 105 lipid transport and metabolism related genes, 79 nucleotide transport and metabolism related genes, 107 coenzyme transport and metabolism related genes, 176 inorganic ion transport and metabolism related genes, 79 secondary metabolite synthesis, transport and metabolism related genes; unknown (Poorly) categories: only 0 genes predicted by conventional functions, 759 genes unknown in functions.
The secondary metabolites encoded by bacillus beijerinus YTQ were analyzed by ANTISMASH on-line prediction and NCBI BLAST comparison for non-ribosomal pathway of surfactants (surfactin), bacteriocins (bacillibactin) and ubiquitin (fengamycin); the polyketide synthase synthesis pathways are bacillus (bacillaene), macrocyclic lactons (macrolactin) and difficile (difficidin); a plantazolicin in the ribosomal pathway; the new cyclic lipopeptide antibiotic rocamycin (locillomycin) of the lanothiopeptides synthesis pathway. In the comparison result with the known gene clusters, the similarity of the thiopeptides butyrylcycline encoding gene cluster is only 7%, the similarity of the rocamycin encoding gene cluster is 35%, the similarity of the surfactant encoding gene cluster and the Plantazolicin encoding gene cluster is 91%, and the similarity of other gene clusters is 100%.
Table 3 identification of Bacillus bailii YTQ gene cluster of secondary metabolite
| Product(s) | Pathway | Similarity/% |
| Surfactant | NRPS | 91 |
| plantazolicin | LAP | 91 |
| Butyryl glycoside bacteriocin | PKS-like | 7 |
| Macrocyclic lactating substances | transAT-PKS | 100 |
| Bacillus bacteriocin | transAT-PKS | 100 |
| Ubiquitin | NRPS | 100 |
| Difficile extract | transAT-PKS | 100 |
| Bacitracin | NRPS | 100 |
Example 2
The field control effect of the compound agent for preventing cherry from low-temperature freeze injury in winter
1. Test tree: the 20-year-old cherry variety 'Mei Zao', the stock is all 'dyers woad' and the row spacing of cherry tree cultivation plants is 4 meters multiplied by 5 meters. Test site: cherry base of Long Guo vegetable professional co-workers in Laishan area of tobacco table city in Shandong province.
2. Bacillus bailii YTQ fermentation broth, wherein the formula of a fermentation medium during preparation of the fermentation broth is as follows: 25g of beef extract, 10g of NaCl, 20g of soybean protein, 15g of corn starch and clear water to 10L, wherein the fermentation temperature is 28 ℃ and the fermentation time is 48h.
3. Preparation of anti-low Wen Donghai compound agent: bacillus bailii YTQ fermentation broth volume ratio is 10%, and sodium alginate concentration is 50mg/L; the concentration of the fulvic acid is 500mg/L, the concentration of the calcium sugar alcohol is 200mg/L, the concentration of the magnesium sulfate is 100mg/L, the concentration of the zinc sulfate is 120mg/L, and the rest components are clear water.
4. And (3) a winter low-temperature freezing injury prevention test: spraying cherry branches with the compound agent as treatment 1 on 12/13/2021 and 1/12/2022 respectively; plants sprayed with 5000 times of diluent of Biyan powder with the equivalent total active ingredient mass percent of 0.136% are used as treatment 2, the Biyan powder is produced by Alafrican agriculture and forestry environment biotechnology Co., ltd, and the pesticide production license number is: pesticide production (robust) 0060; plants sprayed with an equal amount of clear water were used as treatment 3. 30 cherry trees with similar growth vigor are selected for each treatment, and the application amount of each cherry tree is 3kg. 3 treated current annual cherry shoots were collected at 10, 2, 2022 for testing analysis.
5. Test method
5.1 Determination of conductivity
Cutting the branches retrieved in the field into small sections with the length of 2cm by avoiding bud eyes, flushing the small sections with distilled water for 3 times, weighing 2g of branches, putting the branches into a triangular flask, adding 40mL of distilled water, and repeating the above operation for 3 times. The mixture was allowed to stand at room temperature for 12 hours, and the initial conductivity was measured by a conductivity meter. And sealing the triangular flask, opening the flask after boiling water bath for 20min, standing at room temperature for two hours to measure the final conductivity value, and calculating the relative conductivity.
5.2 Determination of physiological index
Washing the branches retrieved from the field with distilled water, avoiding bud eyes, reducing the branches into slices with the thickness of 3-5 mm, uniformly mixing, weighing 0.5g, fully grinding, and measuring the superoxide dismutase (SOD) content, the Peroxidase (POD) content, the Catalase (CAT) content and the Malondialdehyde (MDA) content. The content of superoxide dismutase (SOD) is measured by adopting a nitrogen blue tetrazolium photoreduction method, the content of Peroxidase (POD) is measured by adopting a guaiacol photoreduction method, the content of Catalase (CAT) is measured by adopting an ultraviolet absorption method, and the content of Malondialdehyde (MDA) is measured by adopting a thiobarbituric acid (TBA) method.
5.3 Statistical analysis
And quantitatively converting each index measurement value by using a membership function value method, and solving membership values corresponding to different indexes of each treatment. The formula is U (X i)＝(Xij-Xjmin)/(Xjmax-Xjmin),P＝1/n∑U(Xi), if the index and the cold resistance are in negative correlation, the inverse membership function is used for conversion, and the formula is calculated: u (X i)＝1-(Xij-Xjmin)/(Xjmax-Xjmin),P＝1/n∑U(Xi) wherein XIj is the ith treatment jth measurement index; p is the comprehensive evaluation result of each index measurement processed; n is the total number of measured indexes; x jmax,Xjmin is the maximum and minimum of the j-th index of each process.
6. Results and analysis
6.1 Comparison of Cold resistance indicators of cherry branches treated differently
As can be seen from Table 4, the SOD activity and CAT activity of treatment 1 were both highest, and they were 865.31U/(g.min), 0.64U/(g.min), respectively, and the malondialdehyde content was the lowest 3.45. Mu. Mol/g. The SOD activity, POD activity and CAT activity of treatment 3 were the lowest, namely 480.64U/(g.min), 280U/(g.min) and 0.48U/(g.min), respectively, whereas the relative conductivity of treatment 3 was the highest of 15.98%. The POD activity of treatment 2 was the same as that of treatment 1, and the other cold resistance index of treatment 2 was between treatment 1 and treatment 3.
TABLE 4 comparison of Cold resistance indicators for different treated cherry shoots
6.2 Comprehensive evaluation of membership means and Cold resistance of various indexes treated differently
The cold resistance of 3 kinds of treated cherry branches is scientifically and accurately evaluated by using a membership function method, and as can be seen from table 5, the comprehensive evaluation value of the treated cherry branch is highest, which indicates that the cold resistance of the treated cherry branch is strongest, and the comprehensive evaluation value of the treated cherry branch is lowest, which indicates that the cold resistance of the treated cherry branch is weakest.
TABLE 5 comprehensive evaluation of membership means and Cold resistance of various indexes by different treatments
| Experimental treatment | SOD Activity | POD Activity | CAT Activity | Relative conductivity | Malondialdehyde content | Comprehensive evaluation value | Ordering of |
| Process 1 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 1 |
| Process 2 | 0.8572 | 1.0000 | 0.5638 | 0.4932 | 0.7531 | 0.7335 | 2 |
| Process 3 | 0.0000 | 0.0000 | 0.0000 | 0.0000 | 0.0000 | 0.0000 | 3 |
Example 3
The field control effect of the compound agent for preventing cherry early spring frost
1. Test tree: the 8-year-old cherry varieties 'black pearl' and 'Mei Zao', the stock is 'Gisela No. 6', and the row spacing of the cherry tree cultivation plant is 2 m×4 m. Test site: cherry base in Shandong province tobacco stand city agricultural science institute.
2. Bacillus bailii YTQ fermentation broth, wherein the formula of a fermentation medium during preparation of the fermentation broth is as follows: 25g of beef extract, 10g of NaCl, 20g of soybean protein, 15g of corn starch and clear water to 10L, wherein the fermentation temperature is 28 ℃ and the fermentation time is 48h.
3. Preparation of anti-low Wen Donghai compound agent: bacillus bailii YTQ fermentation broth volume ratio is 10%, and sodium alginate concentration is 50mg/L; the concentration of the fulvic acid is 500mg/L, the concentration of the calcium sugar alcohol is 200mg/L, the concentration of the magnesium sulfate is 100mg/L, the concentration of the zinc sulfate is 120mg/L, and the rest components are clear water.
4. And (3) a winter low-temperature freezing injury prevention test: according to the past year agriculture experience, the Fushan region of the tobacco stand city is extremely vulnerable to frost from 3 months to 4 months old. Therefore, cherry buds are sprayed with the compound agent as treatment 1, plants sprayed with 5000 times of diluent with equivalent amount of 0.136% of Biqing powder as treatment 2, and plants sprayed with equivalent amount of clear water as treatment 3 on the 3 rd month of 2020 and the 10 th month of 2020. 30 cherry trees with similar growth vigor are selected for each treatment, and the application amount of each cherry tree is 1kg. And counting the freezing condition of cherry flower buds in the period of 4 months and 15 days in 2020, and testing the main frost-resistant physiological indexes of the flower buds.
5. Test method
5.1 Flower bud freezing injury investigation
Dissecting the processed sweet cherry flower buds, observing whether the flower primordium is browned and wilted or not by using a microscope, and determining the freezing injury level according to the browning position and the browning degree. 0 level-0% flower primordial browning, 1 level-25% flower primordial browning, 2 level-50% flower primordial browning, 3 level-75% flower primordial browning, 4 level-100% flower primordial browning. The base meteorological data is determined by a smoke counter national institute meteorological station.
The freeze injury index= [ Σ (number of freeze injury per stage×number of freeze injury stages)/(total number of surveys×number of highest freeze injury stages) ]×100.
Freeze rate (%) = number of brown stain shoots/total number of investigation shoots x 100.
5.2 Determination of conductivity
The flower buds retrieved in the field are washed 3 times by distilled water, bud scales are stripped, 2g of branches are weighed and put into a triangular flask, 40mL of distilled water is added, and the above operation is repeated 3 times. The mixture was allowed to stand at room temperature for 12 hours, and the initial conductivity was measured by a conductivity meter. And sealing the triangular flask, opening the flask after boiling water bath for 20min, standing at room temperature for two hours to measure the final conductivity value, and calculating the relative conductivity.
5.3 Determination of physiological index
Washing flower buds retrieved from the field with distilled water, removing bud scales, uniformly mixing, weighing 0.5g, fully grinding, and measuring the superoxide dismutase (SOD) content, the Peroxidase (POD) content, the Catalase (CAT) content and the Malondialdehyde (MDA) content. The content of superoxide dismutase (SOD) is measured by adopting a nitrogen blue tetrazolium photoreduction method, the content of Peroxidase (POD) is measured by adopting a guaiacol photoreduction method, the content of Catalase (CAT) is measured by adopting an ultraviolet absorption method, and the content of Malondialdehyde (MDA) is measured by adopting a thiobarbituric acid (TBA) method.
6. Results and analysis
6.1 Comparison of frost conditions of cherry flower buds treated differently
As can be seen from Table 6, 3 times of larger frost are recorded in the weather station of the national academy of sciences at 4 th month of the year 2020, and different degrees of frost damage are caused to cherry flower buds. As can be seen from table 7, both treatments 1 and 2 reduced the frost impact, with treatments 1 'black pearl' and 'early-in-the-united states' having freeze rates of 15.6% and 14.1%, respectively, and reduced the freeze rates by 55.3% and 53.8%, respectively, as compared to control treatment 3.
Table 6 frost weather record for 4 month 4 smoke table national academy of sciences in 2020
TABLE 7 comparison of the frost damage index and the frost damage rate of cherry flower buds treated differently
6.2 Comparison of Cold resistance index of cherry flower buds treated differently
The content of active oxygen in the plant is increased after the plant is frozen, so that the coordinated action of antioxidant enzymes (SOD, POD, CAT) is promoted to remove the active oxygen, and the damage of the active oxygen to cells is reduced. As can be seen from Table 8, the SOD activity, POD activity and CAT activity of treatment 1 were all highest and were 1235.2U/(g.min), 420U/(g.min) and 0.96U/(g.min), respectively. The SOD activity, POD activity and CAT activity of treatment 3 were the lowest, and they were 796.5U/(g.min), 330U/(g.min) and 0.64U/(g.min), respectively. Treatment 2 has antioxidant enzyme activity intermediate between treatment 1 and treatment 3.
After the plant is damaged by freezing, the cell membrane permeability is changed, resulting in an increase in conductivity and malondialdehyde content. As can be seen from table 8, both the relative conductivities and malondialdehyde content of treatments 1 and 2 were significantly different from the fresh water control, indicating that treatments 1 and 2 effectively reduced the damage to flower bud cell membrane permeability.
Table 8 comparison of Cold resistance indicators of cherry flower buds treated differently
In conclusion, the compound agent formed by the bacillus beijerinckii YTQ fermentation liquor, sodium alginate, fulvic acid, calcium sugar alcohol, magnesium sulfate and zinc sulfate according to a specific proportion can obviously improve the low-temperature freezing injury resistance of plants, has a control effect on the low-temperature freezing injury of the plants which is obviously better than that of the conventional Bikangfu powder, and provides a new technical support for cherry prevention and treatment.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (7)
1. Bacillus bailii (Bacillus velezensis) YTQ, wherein the Bacillus bailii YTQ has a preservation number of CCTCC No: m20211465.
2. The compound agent for improving the low-temperature freezing injury resistance of the cherries is characterized by comprising the following components in parts by weight: fermentation liquor, sodium alginate, fulvic acid, calcium sugar alkoxide, magnesium sulfate, zinc sulfate and the balance of water; the fermentation broth is the fermentation broth of bacillus bailii YTQ of claim 1; the viable count of bacillus bailii YTQ in the fermentation broth is more than or equal to 1 multiplied by 10 6 CFU/mL;
In the compound agent, the volume concentration of fermentation liquor is 10%, the concentration of sodium alginate is 50mg/L, the concentration of fulvic acid is 500mg/L, the concentration of calcium sugar alcohol is 200mg/L, the concentration of magnesium sulfate is 100mg/L, and the concentration of zinc sulfate is 120mg/L.
3. The compounding agent of claim 2, wherein the fermentation medium from which the fermentation broth is prepared comprises the following concentrations of components: beef extract 2-3 g/L, naCl-1.5 g/L, soy protein 1.5-2.5 g/L and corn starch 1-2 g/L.
4. Use of bacillus bailii YTQ according to claim 1 for controlling plant diseases whose pathogenic bacteria are one or more of plasmopara viticola (Botryosphaeria dothidea), botrytis cinerea (Botrytis cinerea), fusarium oxysporum (Fusarium oxysporum) and phomopsis (Phomopsis spp.).
5. Use of the compound of claim 2 or 3 for improving the low temperature freeze injury resistance of cherries.
6. A method for preventing low temperature freeze injury of winter cherry, comprising: spraying cherry branches with the compound agent in the middle 12 months and the middle 1 month respectively; the compound agent is the compound agent of claim 2 or 3.
7. A method of preventing frost damage to cherries in early spring comprising: spraying cherry flower buds with a compound agent in the red-colored period of the flower buds and spraying cherry young fruits with the compound agent 3-5 days after flowers are removed; the compound agent is the compound agent of claim 2 or 3.
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