CN116536207A - Bacillus atrophaeus WLKYSY-4, biological microbial inoculum and application thereof - Google Patents
Bacillus atrophaeus WLKYSY-4, biological microbial inoculum and application thereof Download PDFInfo
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- CN116536207A CN116536207A CN202310525514.8A CN202310525514A CN116536207A CN 116536207 A CN116536207 A CN 116536207A CN 202310525514 A CN202310525514 A CN 202310525514A CN 116536207 A CN116536207 A CN 116536207A
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- China
- Prior art keywords
- wlkysy
- bacillus
- bacillus atrophaeus
- fusarium
- culture
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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
- A01P3/00—Fungicides
-
- 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
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
Abstract
The invention discloses bacillus atrophaeus WLKYSY-4, a biological agent and application thereof, wherein the bacillus atrophaeus WLKYSY-4 is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center), and the preservation number is: CGMCC No.26815. The bacillus atrophaeus WLKYSY-4 provided by the application has good control effect on various plant diseases, whether prevention and control or treatment and control, and has very good application prospect in the aspect of biological pesticides.
Description
Technical Field
The invention belongs to the technical fields of microbiology, biotechnology and biological control, and in particular relates to bacillus atrophaeus WLKYSY-4, a biological microbial inoculum and application thereof.
Background
Bacillus is a class of aerobic or facultative anaerobic, gram-positive bacteria that produce spores. The bacillus is characterized in that the bacillus can generate the spores with strong stress resistance in a severe environment to pass through bad environmental conditions, is ubiquitous in nature, harmless to people and livestock and non-pathogenic to plants, and most of the bacillus can inhibit the growth of some plant pathogenic bacteria. Therefore, bacillus is one of the most widely used plant biocontrol bacteria in the world.
At present, the prevention and control of plant diseases are mainly chemical prevention and control, and long-term use of chemical pesticides not only causes plant pathogenic bacteria to generate drug resistance, but also causes pesticide residues, kills natural enemies, pollutes the environment, threatens human health and destroys ecological balance. The antagonistic microbe is utilized to prevent and treat plant diseases, so that the plant disease inhibitor has the advantages of no toxicity to human beings and animals, no environmental pollution and no residue, can keep the good quality of farm products, is safe to natural enemies of pests and beneficial organisms, and is beneficial to keeping ecological balance.
However, interaction often exists between the biocontrol microorganism and a microorganism community in a soil environment, and the introduction of exogenous antagonistic microorganisms can cause the composition change of the crop rhizosphere soil microorganism community, so that the use of the biocontrol microbial agent has great regional adaptability. At present, when some commercial biocontrol bacteria preparations at home and abroad are used for preventing and controlling some soil-borne diseases of local crops due to the reasons, the effect is not ideal, so that the special microorganism resources of the local are urgently needed to be erected, and some biocontrol bacteria suitable for the soil and the climate conditions of the local are screened out for preventing and controlling the crop diseases, which has great significance in the aspects of technical invention and practical application. The biocontrol strain with antagonism to various pathogenic bacteria is obtained by separating and screening from soil in a cistanche deserticola planting area of Minqiu QingTu lake, and has very important significance for preventing and controlling plant diseases.
Disclosure of Invention
In order to solve the problems, the invention aims to provide broad-spectrum disease-resistant bacillus atrophaeus WLKYSY-4, a biological microbial inoculum and application thereof, in particular to application of the bacteria in biological control of plant diseases.
In order to achieve the above purpose, the present invention is realized by the following technical scheme:
a bacillus atrophaeus WLKYSY-4 deposited in the China general microbiological culture Collection center, having a deposit number: CGMCC No.26815, classification naming: bacillus atrophaeus Bacillus atrophaeus, date of preservation: 2023, 3, 14, deposit address: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
A biological agent comprising bacillus atrophaeus wlky-4 as described above.
The biological microbial inoculum is prepared by fermenting the bacillus atrophaeus WLKYSY-4.
The preparation method of the biological agent comprises the following steps: inoculating the bacillus atrophaeus WLKYSY-4 into an LB solid culture medium for activation, inoculating the activated bacillus atrophaeus WLKYSY-4 into an LB liquid culture medium for fermentation culture to prepare seed liquid, and inoculating the seed liquid into the LB liquid culture medium for fermentation culture to obtain the biological microbial inoculum.
Preferably, the culture temperature is 26-30 ℃; the inoculation amount of the seed liquid is 4-6%, and the fermentation culture time of the seed liquid is 2-4 d.
The bacillus atrophaeus WLKYSY-4 or the biological bacterial agent is applied to resisting pathogenic bacteria or related diseases and plant diseases caused by the pathogenic bacteria.
Preferably, the pathogenic bacteria are at least one of cistanche stem rot pathogen, botrytis cinerea and Lycium barbarum bacterial leaf blight pathogen; the plant disease is at least one of cistanche stem rot, grape gray mold and medlar leaf blight.
Preferably, the cistanche stem rot germ is at least one of fusarium oxysporum, fusarium layering and fusarium acuminatum; the botrytis cinerea is botrytis cinerea; the bacterial strain of Lycium barbarum is Alternaria.
The application method comprises the step of irrigating roots or spraying the plants with the biological microbial inoculum containing the bacillus atrophaeus WLKYSY-4.
Preferably, the effective viable count of the bacillus atrophaeus WLKYSY-4 diluted by the biological bacterial agent is 1.0x10 6 ～1.0×10 8 cfu/mL.
The bacillus atrophaeus WLKYSY-4 provided by the application has good control effect on various plant diseases, whether prevention and control or treatment and control, and has very good application prospect in the aspect of biological pesticides.
Drawings
FIG. 1 shows colony morphology of antagonistic bacillus WLKYSY-4 according to the invention.
FIG. 2 is a gram stain of an antagonistic bacillus WLKYSY-4 according to the invention.
FIG. 3 is a phylogenetic tree of the invention antagonizing the strain of Bacillus WLKYSY-4.
FIG. 4 shows the ability of the invention to antagonize the cellulase production of Bacillus WLKYSY-4.
FIG. 5 shows the ability of the invention to antagonize the protease produced by Bacillus WLKYSY-4.
FIG. 6 shows the ability of the invention to antagonize the amylase production of Bacillus WLKYSY-4.
FIG. 7 is a bacteriostasis spectrum of the antagonistic bacillus WLKYSY-4 of the present invention.
Note that: a. fusarium oxysporum, wherein the left part of the figure is Fusarium oxysporum influenced by antagonistic bacillus WLKYSY-4 strain, and the right part of the figure is Fusarium oxysporum which grows normally; b. fusarium layering, wherein the left part of the figure is Fusarium layering influenced by antagonistic bacillus WLKYSY-4 strain, and the right part of the figure is Fusarium layering which grows normally; c. fusarium anatase, the left part of the figure is Fusarium anatase influenced by antagonistic bacillus WLKYSY-4 strain, and the right part of the figure is Fusarium anatase which grows normally; d. botrytis cinerea, wherein the left part of the figure is Botrytis cinerea influenced by antagonistic bacillus WLKYSY-4 strain, and the right part of the figure is Botrytis cinerea which grows normally; e. matrimony vine leaf blight bacteria: the left graph shows the bacterial strain of the medlar leaf blight influenced by antagonistic bacillus WLKYSY-4 strain, and the right graph shows the bacterial strain of the medlar leaf blight which grows normally;
FIG. 8 shows the effect of antagonizing the control of Botrytis cinerea on grape leaves by Bacillus WLKYSY-4 of the present invention; wherein, from left to right in proper order is: wlky-4 bacterial agent, positive agent control, negative control.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available. The pathogenic bacteria sources involved in the examples are shown in Table 1.
TABLE 1
| Pathogenic bacteria | Source |
| Fusarium oxysporum (Fusarium oxysporum) | Wuwei Forestry Research Institute |
| Fusarium layering (Fusarium prolifertum) | Wuwei Forestry Research Institute |
| Fusarium anatase (Fusarium acuminatum) | Wuwei Forestry Research Institute |
| Botrytis cinerea (Botrytis cinerea) | Wuwei Forestry Research Institute |
| Matrimony vine leaf blight fungus (Alternaria alternata) | Wuwei Forestry Research Institute |
Example 1
The isolation and screening method of the bacillus atrophaeus WLKYSY-4 of the present example and the identification thereof are as follows. The strain 2020 of the embodiment is obtained from soil in a cistanche salsa planting area in QingTu lake of Minn county, gansu province Wu Wei.
1. Screening for antagonizing bacillus WLKYSY-4, the screening process comprising the steps of:
1) The collected soil of the cistanche deserticola planting area is weighed 10g, added into a 250mL conical flask which is filled with 90mL of sterile water and is provided with glass beads, water-bath is carried out for 30min at 80 ℃, then the mixture is fully oscillated for 30min, diluted by 10 times, and the mixture is coated on an LB culture medium flat plate by using a sterile coating rod, and sterile water is used as a reference. The purified bacteria were gram-stained and spore-stained, showing that the bacillus was a rod-shaped, spore-producing, g+ isolate of bacillus. The bacillus isolated by the method is numbered, and the colony morphology is observed, wherein each 1000mL of LB culture medium contains 10g of tryptone, 5g of yeast extract, 10g of NaCl, 15g of agar powder and pH of 7.0-7.2.
2) Punching or inoculating a small amount of mycelia with a 5mm puncher for 3d of fusarium oxysporum (Fusarium oxysporum), fusarium venenatum (Fusarium prolifertum) and fusarium acuminatum (Fusarium acuminatum) (which are obtained by applicant separating and identifying cistanche deserticola plants in the cistanche deserticola planting area of QingTu lake, minn, gansu province), wherein the diameter of fusarium oxysporum (Fusarium oxysporum), fusarium venenatum (Fusarium prolifertum) and fusarium acuminatum (Fusarium acuminatum) is 5mm, transferring the cakes to the center of a potato agar medium (PDA), inoculating bacillus to be detected around the fungi equidistantly, placing in an incubator, reversely and oppositely culturing at a constant temperature of 28 ℃, and repeating each opposite experiment for 3 times in parallel.
3) After 3-5 d, observing and recording the existence and the size of the antibacterial zone, selecting 1 strain with the strongest inhibition effect on fusarium oxysporum (Fusarium oxysporum), fusarium layering (Fusarium prolifertum) and fusarium acuminatum (Fusarium acuminatum), and naming the strain as WLKYSY-4.
2. Identification of cistanche stem rot germ antagonizing bacillus WLKYSY-4 strain
The bacterial strain WLKYSY-4 is identified by adopting colony morphology observation and conventional physiological biochemical and molecular biological methods, and the bacterial strain is identified to belong to bacillus atrophaeus.
1) Antagonistic bacillus WLKYSY-4 strain is milky white, opaque, smooth, central raised, wrinkled and complete in edge on LB medium (1000 mL LB medium contains tryptone 10g, yeast extract 5g, naCl 10g, agar 15g, pH 7.0-7.2) plate (shown in figure 1).
2) Microscopic observation shows that the antagonistic bacillus WLKYSY-4 thallus is about 0.5-0.8 mu m multiplied by 1.5-2.5 mu m, has no capsule, has spores and periphyton, can move and has positive gram staining (shown in figure 2).
3) Biochemical experiment results show that the antagonistic bacillus WLKYSY-4 has mobility, is aerobic, has positive V-P reaction, positive methyl red reaction, positive contact enzyme reaction and positive oxidase reaction, can reduce nitrate, hydrolyze starch and casein, liquefy gelatin, can oxidize glucose, L-arabinose and mannitol to produce acid, cannot oxidize D-xylose to produce acid, and oxidizes glucose but does not produce gas. Can grow in LB culture solution (10 g of tryptone, 5g of yeast extract, 10g of NaCl, 15g of agar powder and pH of 7.0-7.2) with NaCl concentration of 0.5-10 percent. The biological properties of the antagonistic bacillus WLKYSY-4 strain of this example are shown in Table 2.
TABLE 2 antagonism of basic biological Properties of Bacillus WLKYSY-4 Strain
| Gram staining | + | Methyl red reaction | + |
| Starch hydrolysis | + | Contact enzyme | + |
| Determination of aerobics | + | 10％NaCl | + |
| V-P test | + | PH 5.7 | + |
| Glucose | + | Temperature growth range | 15～45℃ |
| Arabinose (Arabic sugar) | + | Litmus-milk | Reddening Red |
| Indole formation | ﹣ | Lecithase assay | + |
| Tyrosine hydrolysis | ﹣ | Casein hydrolysis | + |
| Mannitol (mannitol) | + | Hydrogen sulfide production | + |
| Nitrate reduction | + | Acid production using carbohydrates | + |
| Gelatin | + | pH after V-P reaction | 6.77～7.33 |
| Citric acid utilization | + | L-arabinose | + |
| Optimal growth pH | 7 | Optimum growth temperature | 28℃ |
| Oxidase test | + | Gas production by glucose | ﹣ |
| Malonate utilization | + | pH growth range | 4～10 |
| D-xylose acidogenesis | ﹣ |
Note that: "+" indicates positive and "-" indicates negative.
4) Molecular biological identification of antagonistic bacillus WLKYSY-4 strain
And (3) determining the sequence of the 16S rDNA gene and constructing a phylogenetic tree. Bacterial genomic DNA was extracted as a template, and 16S rDNA of the strain was amplified using 7f (5'-CAGAGTTTGATCCTGGCT-3') and 1540r (5'-AGGAGGTGAT CCAGCCGCA-3') (synthesized by Biotechnology (Shanghai) Co., ltd.) as the upstream and downstream primers. The PCR products were detected by 1% agarose gel electrophoresis and sent to the company for sequencing. The length of the 16SrDNA nucleotide sequence of the strain is 1460bp (shown as SEQ ID NO: 1), and the GenBank accession number is OQ345824. By performing similarity analysis on the sequence and related data in GenBank, analysis and phylogenetic tree construction (shown in FIG. 3) by alignment with the 16S rDNA sequence of the strain in NCBI can be found: the strain WLKYSY-4 and the bacillus atrophaeus Bacillus atrophaeus (GenBank accession number is NR 024689.1) are clustered in the same large branch (the supporting rate is 100%), which shows that the strain has the highest similarity with the bacillus atrophaeus strain.
And combining the culture characteristics, morphology, physiological and biochemical characteristics and the 16S rDNA sequence analysis result of the strain to finally determine that the WLKYSY-4 strain is bacillus atrophaeus Bacillus atrophaeus.
3. The invention provides a preparation method of a bacillus atrophaeus WLKYSY-4 biological bacterial agent, which comprises the following steps:
firstly inoculating bacillus atrophaeus WLKYSY-4 into an LB solid culture medium for activation, then inoculating the activated bacillus atrophaeus WLKYSY-4 into an LB liquid culture medium for fermentation culture to prepare seed liquid, and then inoculating the seed liquid into the LB liquid culture medium for fermentation culture to obtain the biological microbial inoculum.
Wherein, the culture temperature is preferably 26-30 ℃; the seed liquid inoculation amount is 4-6%, and the seed liquid fermentation culture time is 2-4 d.
Further, this embodiment provides a preferred preparation method, which specifically includes the following steps:
1) Preparing seed liquid: activating antagonistic bacillus by an LB solid culture medium, culturing for 36h at 28 ℃, inoculating the activated antagonistic bacillus WLKYSY-4 strain into an LB liquid culture medium, and culturing for 24h at 150r/min at 28 ℃ in an oscillating way to prepare seed liquid;
wherein, the composition of LB solid medium and liquid medium is: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of NaCl and pH7.4; the solid culture medium also comprises 15g/L of agar powder;
2) Fermentation culture: inoculating the seed solution into 250mL triangular flask containing 100mL LB liquid medium according to 5% inoculum size, shake culturing at 28deg.C at 150r/min, fermenting at constant temperature for 3d, and diluting with sterile water to active ingredient content of 1.0X10% 6 ～1.0×10 8 After cfu/mL, the biological microbial inoculum is obtained.
Example 2
Inoculating antagonistic bacillus WLKYSY-4 strain cultured for 1d on cellulose culture medium, casein culture medium and starch culture medium, inoculating 3 strains on each dish, culturing for 2-7 d at 30 ℃, taking blank as control, and setting 3 repeats; observing whether transparent rings are generated around colonies, wherein a plurality of drops of iodine solution are added into a starch culture medium before observation, measuring cellulase activity, namely, using 1g/L Congo red to dye for 10-15 min, pouring out the dye solution, then using 1mol/L NaCl to soak for 15min, and checking whether the transparent rings are present or not, wherein the detection is that: the antagonistic bacillus WLKYSY-4 strain can normally grow on cellulase, protease culture medium and amylase culture medium and has obvious hydrolysis circle around the colony, and is obvious from figures 4, 5 and 6, the antagonistic bacillus WLKYSY-4 strain is shown to secrete and produce cellulase, protease and amylase in the growth metabolism process, so that cellulose, protein and starch in the culture medium are hydrolyzed, transparent degradation circle appears around the colony, and the antagonistic bacillus WLKYSY-4 is shown to produce a series of enzymes in the process of inhibiting the growth of plant pathogenic bacteria and preventing and controlling plant diseases.
In this example, the cellulase-producing medium is carboxymethyl cellulose medium (1000 mL): CMC-Na 5.0g, mgSO 4 ·7H 2 O 0.1g，(NH 4 ) 2 SO 4 0.5g，K 2 HPO 4 0.25g, agar 16g, water l000mL.
In this example, the protease-producing medium is casein medium (1000 mL): 20g of skimmed milk powder, 5g of yeast extract, 5g of glucose, 16g of agar, 1000mL of water and pH7.0.
In this example, the amylase-producing medium is a starch medium (1000 mL): 20g of soluble starch, 10g of peptone, 5g of glucose, 5g of NaCl, 5g of beef extract, 16g of agar, 1000mL of water and pH7.0.
Example 3
Inhibition of plant pathogenic fungi by the Strain WLKYSY-4
The inhibition effect of the fungus bacteriostasis spectrum of WLKYSY-4 on 3 fusarium strains causing cistanche stem rot is detected by adopting a counter culture method, namely fusarium oxysporum (Fusarium oxysporum), fusarium venenatum (Fusarium prolifertum) and fusarium acuminatum (Fusarium acuminatum), botrytis cinerea (Botrytis cinerea) and medlar leaf blight bacteria (Alternaria alternata) is respectively measured, firstly, the pathogenic bacteria are activated on a PDA plate, a pathogenic bacteria cake is manufactured by using a 5mm puncher or a few mycelia are picked up, the mycelia are inoculated in the center of the PDA plate, the WLKYSY-4 point is connected with the position 2cm away from the bacterial cake by using a toothpick, the culture is carried out for 4-7 days at 25 ℃, the control diameter and the inhibition diameter are respectively measured, and the inhibition rate is calculated according to the formula I, and the results are shown in figure 7 and table 3.
Table 3 antibacterial spectrum of WLKYSY-4
| Pathogenic bacteria | Inhibition ratio (%) |
| Fusarium oxysporum (Fusarium oxysporum) | 57.93±1.21 |
| Fusarium layering (Fusarium prolifertum) | 45.60±1.78 |
| Fusarium anatase (Fusarium acuminatum) | 45.20±1.34 |
| Botrytis cinerea (Botrytis cinerea) | 76.67±0.92 |
| Matrimony vine leaf blight fungus (Alternaria alternata) | 54.55±0.45 |
Example 4
Effect of preventing and treating cistanche stem rot in field test
2021, selecting a cistanche deserticola planting area of Minqiu lake as an experimental area, picking just-outdated cistanche deserticola, digging out and exposing cistanche deserticola inoculation parts (the inoculation parts are parts for parasitic growth of cistanche deserticola seeds and the roots of the haloxylon ammodendron, cistanche deserticola grows out from about 1-2 cistanche deserticola inoculation parts per plant of haloxylon, about 1-20 cistanche deserticola grow on each inoculation part per year), treating 1 as a CK blank control, irrigating roots with 200mL of each inoculation part (about 200 in each field under a 10-time mirror) of cistanche deserticola stem rot 3 types of fusarium spores, and then irrigating roots with 200mL of blank fermentation liquor without inoculating bacillus WLKYSY-4 after 24 hours of complete absorption; treatment 2 is to irrigate roots with 200mL (the same as the following) of a microbial inoculum (prepared in example 1, the same as the following) for antagonizing bacillus WLKYSY-4, irrigate roots with 3 fusarium spore solutions of cistanche stem rot after 24 hours of complete absorption, and backfill sand after complete absorption; and (3) root irrigation is carried out on the treatment group 3 by using fusarium spore liquid of 3 types of cistanche stem rot, root irrigation is carried out on the cistanche stem rot by using a microbial inoculum for antagonizing bacillus WLKYSY-4 after 24 hours of absorption, sand backfilling is carried out after complete absorption, and the cistanche morbidity in all inoculation positions of each group of clostridium is investigated. The incidence of cistanche stem rot was compared with 6 Clostridium haloxylon plants for each treatment group, and as a result, the incidence of cistanche stem rot was found to be significantly lower in the plants administered with the antagonistic bacillus WLKYSY-4 bacteria (treatment group 2, treatment group 3) than in the control group not administered with the bacterial liquid (Table 4). And the incidence rate of cistanche stem rot of which the bacterial agent antagonizes the bacillus WLKYSY-4 is firstly applied in the treatment 2 is obviously lower than that of cistanche stem rot of which the bacterial agent antagonizes the bacillus WLKYSY-4 is applied after the treatment 3, so that the prevention and the control are proved to be carried out when the bacterial agent antagonizes the bacillus WLKYSY-4 is applied to the cistanche stem rot, preferably before the onset of the disease.
TABLE 4 morbidity of different treatment groups
| Experimental group | Incidence of cistanche stem rot (%) |
| Process 1 | 91±5.64a |
| Process 2 | 17±4.78c |
| Process 3 | 29±8.32b |
Note that: the data in the table are mean ± standard deviation. The different letters after the same column of data indicate significant differences at P <0.05 levels as tested by Duncan's new complex polar error method.
Stem rot of cistanche deserticola caused by fusarium often causes the meat stem of cistanche deserticola to rot, and is a main disease of cistanche deserticola. Because cistanche is directly eaten or used for medicine, most of the production processes are mainly organic production, and any chemical fertilizer and pesticide cannot be used, so that producers can hardly take any control measures when the diseases occur, and the healthy development of the industry is seriously hindered. The bacillus atrophaeus WLKYSY-4 provided by the application is proved by laboratory bacteriostasis and field experiments, and can effectively prevent and treat the incidence of cistanche stem rot.
Example 5
Example WLKYSY-4 effect of controlling Botrytis cinerea on grape leaves
The control effect is measured by an in-vitro leaf method: healthy grape leaves (grape variety is wine grape Gangnong No. 6) with consistent growth are selected, washed clean by sterile water, soaked in 200mL of WLKYSY-4 microbial inoculum prepared in example 1 (same as above) for 30 minutes, covered with absorbent cotton with petioles, the leaf backs upward, placed in culture dishes (diameter of 200 mm) filled with sterile wet gauze, placed one leaf in each culture dish, sealed by preservative film, and cultured for 1d at 25 ℃. Then inoculating cultured Botrytis cinerea cake with diameter of 5mm at the main vein of the leaf back, sealing again, culturing at 25deg.C, taking 50% carbendazim 500 times solution as positive agent control, and sterile water as negative control, and processing method and dosage are the same as WLKYSY-4 microbial inoculum. And (3) observing every day, measuring the diameter of the lesion by using a cross multiplication method after the contrast is fully developed, and calculating the area of the lesion. A blank control was set and each treatment was repeated 6 times (table 5).
Area of lesion (mm) 2 ) =lesion length (mm) ×lesion width (mm) ×3.14/4
Control effect (%) = (control lesion area-treated lesion area)/control lesion area×100
TABLE 5 control effect of WLKYSY-4 on Botrytis cinerea on grape leaves
| Treatment of | Area of lesion | Preventing effect (%) |
| Blank control | ＝208.81±3.56a | - |
| WLKYSY-4 liquid preparation | ＝50.24±4.53b | 75.94±3.24b |
| Carbendazim | ＝32.97±7.81c | 84.21±5.63a |
Note that: the data in the table are mean ± standard deviation. The different letters after the same column of data indicate significant differences at P <0.05 levels as tested by Duncan's new complex polar error method.
Example 6
Effect of field test for preventing and treating wolfberry leaf blight
The field control test is carried out in 2021 in a medlar cultivation garden of the national institute of forestry, wu Wei in Gansu province, and the medlar variety is Ningxia medlar. The field efficacy test is carried out on the screened antagonistic bacillus WLKYSY-4 strain, the WLKYSY-4 microbial inoculum prepared in the example 1 (the same as above) is adopted, and 80% mancozeb WP 800 times liquid and clear water are used as contrast. The specific design is as follows:
1) The prevention and control effect is to spray medicines and then inoculate pathogenic bacteria of the leaf blight of the medlar. Selecting branches of about 20cm, performing needling treatment on the upper green fruits (about 20), spraying about 10mL of each treatment agent, spraying about 10mL of spore suspension (about 200 spores per field under a 10-time mirror) after 24 hours, sleeving a fresh-keeping bag for preserving moisture for 12 hours, taking clear water as a control, randomly selecting medlar plants, and repeating 10 branches per treatment every time for 3 times. After 10d, the disease conditions were examined, and the disease index and the control effect were calculated, and the results are shown in Table 6.
Disease index = Σ (number of disease fruits at each stage×value of disease grade)/(total fruits investigated×highest value) ×100%;
control effect = [ (control disease index-treatment disease index/control disease index) ]×100%;
the medlar leaf blight grading standard, grade 0: wound healing on the surface of the fruit is achieved, and the fruit is not expanded; stage 1: the surface (0-1/8) of the fruit is blackened; 2 stages: the surface (1/8-1/4) of the fruit is blackened; 3 stages: the surface (1/4-1/2) of the fruit is blackened; 4 stages: the fruit surface turns black by more than 1/2.
The results in Table 6 show that the disease index of the antagonistic bacillus WLKYSY-4 microbial inoculum after spraying and inoculating the bacterial strain of the medlar plant is 34.32, and the control effect is 64.78%; the disease index of the control medicament 80% mancozeb wettable powder subjected to 800 times liquid treatment is 31.35, and the control effect is 67.83%.
Table 6 antagonizes the effect of Bacillus WLKYSY-4 on the field prevention and control of bacterial blight of wolfberry
| Treatment of | Index of disease condition | Control effect (%) |
| WLKYSY-4 | 34.32±0.56b | 64.78b |
| 80% mancozeb wettable powder 800 times liquid | 31.35±0.21c | 67.83a |
| Fresh water control CK | 97.45±0.86a | — |
Note that: the data in the table are mean ± standard deviation. The different letters after the same column of data indicate significant differences at P <0.05 levels as tested by Duncan's new complex polar error method.
2) The treatment and prevention effects are that firstly, the pathogenic bacteria of the medlar leaf blight are inoculated, then the medicine is sprayed, branches with the length of about 20cm are selected, the needling treatment is carried out on the fruits (about 20), 10mL of the spore suspension of the pathogenic bacteria is sprayed (about 200 in each field under a 10-time mirror), the fresh-keeping bag is sleeved for 12 hours for moisturizing, clear water is used as a contrast, then about 10mL of each treatment agent is sprayed (same as above), medlar plants are randomly selected, 10 branches are treated each time, and 3 repetitions are carried out each time. And after 10d, respectively investigating the disease conditions, and calculating the disease index and the control effect. The disease index, the prevention and treatment effect and the grading standard of the medlar leaf blight are the same.
The result (Table 7) shows that the disease index of the antagonistic bacillus WLKYSY-4 strain microbial inoculum after inoculating medlar bacterial blight bacteria on medlar plants is 35.42, and the control effect is 63.54%; the disease index of the control medicament 80% mancozeb wettable powder subjected to 800 times liquid treatment is 29.45, and the control effect is 69.69%.
Table 7 antagonizes the effect of Bacillus WLKYSY-4 on the field treatment and control of bacterial blight of wolfberry
| Treatment of | Index of disease condition | Control effect (%) |
| WLKYSY-4 | 35.42±0.38b | 63.54b |
| 80% mancozeb wettable powder 800 times liquid | 29.45±0.98c | 69.69a |
| Fresh water control CK | 97.15±0.43a | — |
Note that: the data in the table are mean ± standard deviation. The different letters after the same column of data indicate significant differences at P <0.05 levels as tested by Duncan's new complex polar error method.
Through field experiments, the antagonistic bacillus WLKYSY-4 strain has better control effect on various plant diseases, whether prevention and control or treatment and control, and has a certain development and application prospect as microbial pesticide.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention. It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
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Claims (10)
1. A bacillus atrophaeus WLKYSY-4, which is deposited in the China general microbiological culture Collection center with deposit number: cgmccno.26815.
2. A biological agent comprising the bacillus atrophaeus WLKYSY-4 as claimed in claim 1.
3. The biological agent according to claim 2, which is obtained by fermentation of bacillus atrophaeus WLKYSY-4.
4. A method for preparing a biological agent according to claims 2 to 3, comprising the steps of: inoculating the bacillus atrophaeus WLKYSY-4 into an LB solid culture medium for activation, inoculating the activated bacillus atrophaeus WLKYSY-4 into an LB liquid culture medium for fermentation culture to prepare seed liquid, and inoculating the seed liquid into the LB liquid culture medium for fermentation culture to obtain the biological microbial inoculum.
5. The method according to claim 4, wherein the culture temperature is 26 to 30 ℃; the inoculation amount of the seed liquid is 4-6%, and the fermentation culture time of the seed liquid is 2-4 d.
6. Use of bacillus atrophaeus wlky-4 as claimed in claim 1 or of a biological agent as claimed in any one of claims 2 to 3 for combating pathogenic bacteria, or related diseases and plant diseases caused by pathogenic bacteria.
7. The use according to claim 6, wherein the pathogenic bacteria are at least one of the species phoma cistanches, botrytis cinerea and lycium barbarum; the plant disease is at least one of cistanche stem rot, grape gray mold and medlar leaf blight.
8. The use according to claim 7, wherein the cistanche stem rot germ is at least one of fusarium oxysporum, fusarium layering and fusarium acuminatum; the botrytis cinerea is botrytis cinerea; the bacterial strain of Lycium barbarum is Alternaria.
9. The method of use according to any one of claims 6 to 8, wherein the plant is root irrigated or sprayed with a biological agent comprising said bacillus atrophaeus WLKYSY-4.
10. The method of claim 9, wherein the raw materials are selected from the group consisting ofThe effective viable count of the bacillus atrophaeus WLKYSY-4 diluted by the microbial inoculum is 1.0x10 6 ～1.0×10 8 cfu/mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN117229985A (en) * | 2023-11-16 | 2023-12-15 | 北京嘉博文生物科技有限公司 | Biocontrol strain bacillus atrophaeus BGB-98R and application thereof |
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| CN117229985A (en) * | 2023-11-16 | 2023-12-15 | 北京嘉博文生物科技有限公司 | Biocontrol strain bacillus atrophaeus BGB-98R and application thereof |
| CN117229985B (en) * | 2023-11-16 | 2024-01-30 | 北京嘉博文生物科技有限公司 | Biocontrol strain bacillus atrophaeus BGB-98R and application thereof |
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