CN110564651B - Bacillus siamensis and application thereof - Google Patents

Bacillus siamensis and application thereof Download PDF

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Publication number
CN110564651B
CN110564651B CN201910943040.2A CN201910943040A CN110564651B CN 110564651 B CN110564651 B CN 110564651B CN 201910943040 A CN201910943040 A CN 201910943040A CN 110564651 B CN110564651 B CN 110564651B
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tobacco
bacillus
disease
fusarium
pathogenic bacteria
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CN110564651A (en
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王晓强
王凤龙
任广伟
沈宏
雷强
谢中玉
孙光军
李斌
李光雷
杨举田
冯超
徐传涛
余佳敏
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CHINA NATIONAL TOBACCO Corp SICHUAN
China Tobacco Corp Guizhou Provincial Co
Tobacco Research Institute of CAAS
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CHINA NATIONAL TOBACCO Corp SICHUAN
China Tobacco Corp Guizhou Provincial Co
Tobacco Research Institute of CAAS
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION 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
    • A01N63/00Biocides, 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
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, 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
    • C12N1/20Bacteria; Culture media therefor
    • C12N1/205Bacterial isolates
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12RINDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00Microorganisms ; Processes using microorganisms
    • C12R2001/01Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07Bacillus

Abstract

The invention provides a Siamese bacillus and application thereof, belonging to the technical field of microbial control. Researches show that the Siamese Bacillus (Bacillus siamensis) LZ88 provided by the invention has very good effects on biological control of plant diseases such as tobacco brown spot, tobacco black shank, wheat scab, grape anthracnose, cucumber fusarium wilt, peanut fusarium and the like, induction of disease resistance of tobacco plants and the like, and provides new strain resources for biological control of main diseases such as the brown spot of the tobacco and the like, so that the Siamese Bacillus (Bacillus siamensis) LZ88 has good practical application value.

Description

Bacillus siamensis and application thereof
Technical Field
The invention belongs to the technical field of microbial control, and particularly relates to a Siamese bacillus and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Alternaria Alternata is a disease of tobacco plant in the late mature period caused by Alternaria Alternata. The disease was first discovered in the united states in 1892. Since the report, the harm of the tobacco brown spot is increasingly serious, the red spot is commonly generated in all tobacco regions of the world, and the serious economic loss is caused to the tobacco leaf production of the world for a few times. The outbreak started in 1956 in north carolina, causing a loss of 390 to 2100 million dollars per year for north carolina tobacco growers. In 2000, tobacco red spot was massively fulminated in both Connecticut and Massachusetts in the United states, resulting in a reduction of 75% and 89% in tobacco production in both states, respectively. The tobacco brown spot is found in Beijing area for the first time in 1916 years in China, and each smoke area in China has increasingly serious trend since 80 years. In 1992, the tobacco is extremely popular, the loss of Huang-Huai-tobacco areas is large, the disease area of the tobacco areas in Henan is 70 ten thousand mu, 10 ten thousand mu is destructive, the yield is reduced by 5000 thousand kilograms, and the yield loss and the RMB are about 1 hundred million yuan. Is one of the main fungal diseases which restrict tobacco production in China. Therefore, the prevention and treatment of the tobacco brown spot are not slow.
At present, in the actual production of tobacco leaves, the cultivation and selection of the variety resisting the alternaria alternate is the most economic and effective method and is an important prevention and treatment measure for fundamentally solving the alternaria alternate diseases of the tobacco, however, no suitable excellent variety resisting the alternaria alternate exists so far, and the alternaria alternate is common in fields. Chemical control methods are mostly adopted for control. Chemical control, while an ideal control means, many chemicals used to control plant fungal and bacterial diseases are harmful to animal and human health. Meanwhile, chemical pesticide residues and drug resistance enhancement caused by single drug use cannot be used as the most appropriate means for chemical control, so that safer and more environment-friendly biological control agents are required to replace the chemical agents.
At present, antagonistic bacteria for preventing and treating alternaria alternate have been reported, such as bacillus subtilis (CN 201110093415.4), streptomyces (CN 201710280079.1) pseudomonas defensive (CN 201810438730.8), beauveria bassiana (CN 201811059565.1) and the like, however, due to the limitations of a single strain, the synchronous evolution of pathogenic bacteria and the like, a new strain needs to be found for preventing and treating related plant diseases such as alternaria alternate and the like.
Disclosure of Invention
Based on the defects of the prior art, the invention provides a Siamese Bacillus (Bacillus siemensis) LZ88 capable of inhibiting various pathogenic bacteria such as tobacco red spot, and the like, and researches show that the Siamese Bacillus LZ88 has very good effects on biological control of plant diseases such as tobacco red spot and the like, induction of tobacco plant resistance and the like, provides new strain resources for biological control of main diseases such as tobacco red spot and the like, and has good practical application value.
In order to achieve the technical purpose, the invention relates to the following technical scheme:
in one aspect of the invention, a Siamese Bacillus (Bacillus siamensis) LZ88 strain is provided, the strain is preserved in China general microbiological culture Collection center (address: No. 3 of No.1 Siro Sebah. of the south China area of Beijing city) in 2019, 9 and 4 days, and the biological preservation number of the strain is CGMCC No. 18466.
Metabolites of the siamese bacillus LZ88 are also within the scope of the invention.
In a second aspect of the invention, a microbial inoculant is provided, which comprises the Siamese bacillus LZ88 and/or a metabolite containing the Siamese bacillus LZ 88.
The microbial agent may specifically be a pathogenic bacteria inhibitor or a disease inhibitor.
The active ingredient of the pathogenic bacteria inhibitor can be metabolites of Siamese bacillus LZ88 and/or Siamese bacillus LZ88, the active ingredient of the pathogenic bacteria inhibitor can also contain other biological ingredients or non-biological ingredients, and the other active ingredients of the pathogenic bacteria inhibitor can be determined by the technicians in the field according to the inhibiting effect on pathogenic bacteria.
The active ingredient of the disease inhibitor can be metabolites of Siamese bacillus LZ88 and/or Siamese bacillus LZ88, the active ingredient of the disease inhibitor can also contain other biological ingredients or non-biological ingredients, and the other active ingredients of the disease inhibitor can be determined by the technicians in the field according to the effect of inhibiting diseases.
In a third aspect of the present invention, the use of the above mentioned metabolite of the siamese bacillus LZ88 and/or siamese bacillus LZ88 in all or part of the following 1) -4) is also within the scope of the present invention:
1) the application in inhibiting pathogenic bacteria;
2) the application in the preparation of pathogenic bacteria inhibitor;
3) the application in preparing disease inhibitors;
4) the application in disease inhibition.
As hereinbefore, the pathogenic bacteria may be all or part of the following: alternaria alternata (alternaria alternata), Phytophthora nicotianae (Phytophthora parasitica var. nicotianae), Fusarium graminearum (Fusarium graminearum Sehw), botrytis cinerea (Colletotrichum gloeosporioides Penz), Fusarium oxysporum (Fusarium oxysporum sp. culmorum Owen), and Fusarium arachidicola (Fusarium oxysporum Link).
Above, the disease may be all or part of the following diseases: tobacco brown spot, tobacco black shank, wheat scab, grape anthracnose, cucumber fusarium wilt and peanut sickle disease.
In a fourth aspect of the present invention, the use of the above mentioned metabolite of the siamese bacillus LZ88 and/or the siamese bacillus LZ88 for promoting plant growth and/or inducing plant to generate disease resistance also belongs to the protection scope of the present invention.
Further, inducing disease resistance in plants includes increasing the levels of peroxidase and polyphenol oxidase in plants (e.g., plant leaves).
In the above application, the plant may be any one of the following plants:
p1) seed plants;
p2) dicotyledonous plants;
p3) solanaceae plants;
p4) tobacco.
In a fifth aspect of the present invention, there is provided a method for promoting the growth of tobacco, said method comprising spraying onto the surface of and/or surrounding soil of a tobacco plant a metabolite and/or microbial inoculant of the aforementioned siamese bacillus LZ88, siamese bacillus LZ 88.
The invention has the beneficial technical effects that:
the Siamese bacillus LZ88 obtained by screening for the first time has obvious antagonistic action on six plant pathogenic bacteria with serious occurrence in agricultural production, has a wide antibacterial spectrum, particularly has a strong prevention and control effect on tobacco red spot, can induce tobacco plants to generate red spot disease resistance and promote the growth of the tobacco plants, and shows that the strain has wide application prospect in the field of biological prevention and control of plant diseases.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a phylogenetic tree of the rDNA gene sequence of Siamese Bacillus (Bacillus siamensis) LZ 8816S in example 1 of the present invention.
FIG. 2 is a spectrum test of the inhibition by Siamese Bacillus (Bacillus siamensis) LZ88 in example 2 of the present invention, wherein (A) Alternaria alternata, (B) tobacco black shank (Phytophthora parastica var. nicotianae), (C) Fusarium graminearum (Fusarium graminearum Sehw), (D) Staphylococcus aureus (Colletobacterium globosum Penz), (E) Fusarium oxysporum (Fusarium oxysporum Owen), (F) Fusarium roseum (Fusarium roseum Link). A
FIG. 3 is a graph showing the measurement of the bacteriostatic activity of volatile substances in example 3 of the present invention, wherein (A) is a control CK; (B) strain LZ 88.
FIG. 4 is a graph showing that the bacterial strain LZ88 sterile filtrate of example 4 of the present invention can effectively inhibit the hyphal growth and sporulation of Alternaria alternata, wherein (A) the growth of Alternaria alternata is observed on a PDA medium plate; (B) microscopic observation of the hyphal growth and sporulation of Alternaria alternata; all experiments were performed in triplicate and results were similarly obtained.
FIG. 5 is a graph showing the bacteriostatic action of crude protein in fermentation broth of strain LZ88 in example 5 of the present invention.
FIG. 6 is a graph showing the bacteriostatic activity of crude protein extract after treatment at different temperatures in example 5 of the present invention; wherein, (A) the growth of alternaria alternate on the plate; (B) the bacteriostasis rate of the crude extract after different temperature treatments.
FIG. 7 is a graph showing changes in Peroxidase (POD) activity of tobacco leaves in example 6 of the present invention.
FIG. 8 is a graph showing the change in the polyphenol oxidase (PPO) activity of tobacco leaves in example 6 of the present invention.
FIG. 9 is a graph showing the control effect of the strain LZ88 on alternaria alternate in example 7 of the present invention; wherein, (A) the disease condition of the plants after 7d of treatment; (B) the tobacco leaves develop symptoms.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As described above, although antagonistic bacteria for preventing and treating alternaria alternate have been reported, it is necessary to find new strains for preventing and treating related diseases such as alternaria alternate due to limitations of single strains and synchronous evolution of pathogenic bacteria.
In view of the above, in one embodiment of the present invention, there is provided a Bacillus siamensis (Bacillus siamensis) LZ88, which has been deposited in the general microbiological center of the chinese committee for culture collection of microorganisms (address: western No.1 hospital No. 3 in north chen yang district, beijing, china) in 2019 and 4 days, and the biological preservation number thereof is CGMCC No. 18466.
In another embodiment of the present invention, the metabolite of the siamese bacillus LZ88 also belongs to the protection scope of the present invention.
In yet another embodiment of the present invention, the metabolite of siamese Bacillus LZ88 can be obtained from a fermentation broth of siamese Bacillus (Bacillus siamensis) LZ 88. The metabolite of Siamese bacillus LZ88 can be prepared according to the following method: inoculating the Siamese Bacillus (Bacillus siamensis) LZ88 into a liquid fermentation culture medium for fermentation culture, and removing the Siamese Bacillus (Bacillus siamensis) LZ88 in a liquid culture (fermentation liquid) to obtain a metabolite of the Siamese Bacillus LZ 88.
Wherein the liquid fermentation medium is preferably NB medium.
The fermentation culture conditions are specifically as follows: culturing at 26-30 ℃ (preferably 28 ℃) for 20-30 h (preferably 24h), rotating speed: 120-180rpm (preferably 150 rpm).
The NB culture medium formula (g/L) is as follows: 10g of peptone, 5g of beef extract, 5g of sodium chloride and 1000mL of distilled water, and sterilizing at 121 ℃ for 15 min.
In yet another embodiment of the present invention, there is provided a microbial inoculant comprising said bacillus siamensis LZ88 and/or a metabolite comprising bacillus siamensis LZ 88.
In another embodiment of the present invention, the microbial agent may be a pathogenic bacteria inhibitor or a disease inhibitor.
In still another embodiment of the present invention, the active ingredient of the pathogen inhibitor may be a metabolite of bacillus siamensis LZ88 and/or bacillus siamensis LZ88, the active ingredient of the pathogen inhibitor may further comprise other biological or non-biological components, and the other active ingredients of the pathogen inhibitor may be determined by one skilled in the art based on the inhibitory effect on pathogens.
In still another embodiment of the present invention, the active ingredient of the disease inhibitor may be a metabolite of siamese bacillus LZ88 and/or siamese bacillus LZ88, the active ingredient of the disease inhibitor may further contain other biological or non-biological components, and the other active ingredients of the disease inhibitor may be determined by one skilled in the art according to the effect of inhibiting diseases.
In another embodiment of the present invention, the use of the metabolites of the aforementioned siamese bacillus LZ88 and/or siamese bacillus LZ88 in all or part of the following 1) -4) is also within the scope of the present invention:
1) the application in inhibiting pathogenic bacteria;
2) the application in the preparation of pathogenic bacteria inhibitor;
3) the application in preparing disease inhibitors;
4) the application in disease inhibition.
In still another embodiment of the present invention, the pathogenic bacteria may be all or part of the following pathogenic bacteria: alternaria alternata (alternaria alternata), Phytophthora nicotianae (Phytophthora parasitica var. nicotianae), Fusarium graminearum (Fusarium graminearum Sehw), botrytis cinerea (Colletotrichum gloeosporioides Penz), Fusarium oxysporum (Fusarium oxysporum sp. culmorum Owen), and Fusarium arachidicola (Fusarium oxysporum Link).
In another embodiment of the present invention, the disease may be all or part of the following diseases: tobacco brown spot, tobacco black shank, wheat scab, grape anthracnose, cucumber fusarium wilt and peanut sickle disease.
In another embodiment of the present invention, the microbial agent further contains a carrier in addition to the active ingredient. The carrier may be one that is commonly used in the pesticide art and is biologically inert.
The carrier can be a solid carrier or a liquid carrier;
the solid carrier can be a mineral material, a plant material or a high molecular compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material may be at least one of corn flour, bean flour and starch; the high molecular compound can be polyvinyl alcohol or/and polyglycol;
the liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent may be decane or/and dodecane.
The preparation formulation of the microbial inoculum can be various preparation formulations, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules.
According to the requirement, the microbial inoculum can also be added with a surfactant (such as Tween 20, Tween 80 and the like), a binder, a stabilizer (such as an antioxidant), a pH regulator and the like.
In another embodiment of the present invention, the use of the metabolites of the siamese bacillus LZ88 and/or the siamese bacillus LZ88 in promoting plant growth and/or inducing plant disease resistance is also within the scope of the present invention.
In yet another embodiment of the present invention, said inducing disease resistance in a plant comprises increasing the levels of peroxidase and polyphenol oxidase in a plant (e.g., plant leaves).
In another embodiment of the present invention, in the above application, the plant may be any one of the following plants:
p1) seed plants;
p2) dicotyledonous plants;
p3) solanaceae plants;
p4) tobacco.
In yet another embodiment of the present invention, there is provided a method for promoting the growth of tobacco, said method comprising spraying onto the surface of and/or into the surrounding soil of a tobacco plant a metabolite and/or microbial inoculant of the aforementioned siamese bacillus LZ88, siamese bacillus LZ 88.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1 identification of Strain LZ88
16S rDNA sequence was amplified using bacterial 16S rDNA universal primers 27F (5 '-AGAGTTTGATCMTGGCTCAG-3', SEQ ID NO.1) and 1492R (5 '-TACGGYTACCTTG TTACGACTT-3', SEQ ID NO.2) using strain LZ88 genomic DNA as template. Amplification system and procedure: 25 μ L of the PCR system contained 5.0 μ L of 5 XPCR buffer and DNA template (100ng μ L)-1) 1.0. mu.L, dNTPs (10mM) 0.75. mu.L, forward primer (10. mu.M) 0.75. mu.L, reverse primer (10. mu.M) 0.75. mu.L, MgCl22.5. mu.L (25mM), Taq DNA polymerase (5U. mu.L)-1Promega) 0.25. mu.L, remainder ddH2And (4) supplementing and finishing. The PCR amplification program is pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 1min for 30s, for 30 cycles. Finally, extension is carried out at 72 ℃ for 10min, and storage is carried out at 4 ℃. After the reaction is finished, the PCR product is subjected toAnd (4) performing agarose gel electrophoresis analysis. The fragments obtained were subjected to sequencing analysis. Phylogenetic trees were constructed using MEGA7.0 software.
As shown in FIG. 1, the NJ phylogenetic tree showed that LZ88 polymerized on the same branch as Bacillus siamensis, with 99.72% gene sequence similarity. Bacterial isolate LZ88 was identified as Bacillus siamensis based on sequence similarity and phylogenetic analysis, combined with its physiological and biochemical indices.
EXAMPLE 2 measurement of bacteriostatic ability of Bacillus siamensis LZ88
The LZ88 strain is separated from the tobacco rhizosphere soil, the bacteriostatic activity of the strain on the alternaria alternata is measured by adopting a confronting culture method, the activated pathogenic bacteria cake is punched by a sterilization puncher, the pathogenic bacteria cake is firstly connected to the center of a fresh PDA flat plate, then the antagonistic bacteria is connected at the position 2.5cm away from the bacteria cake, and the flat plate only connected with the bacteria cake is used as a contrast. Putting into a constant temperature incubator at 28 ℃ for inverted culture. After 5d, the colony diameters of the treated and control were measured and the inhibition rate was calculated. The results show that strain LZ88 has very significant antagonistic effects on tobacco brown spot (alternaria alternata), tobacco black shank (Phytophthora parasitica var. nicotianae), wheat scab (FusaHum graminearum Sehw), grape anthracnose (Colletotrichum gloeosporioides Penz), cucumber Fusarium wilt (Fusarium oxysporum sp. cumulus Owen) and peanut sickle disease (Fusarium roseum Link) (fig. 2).
EXAMPLE 3 determination of the bacteriostatic Activity of a volatile bacteriostatic substance
Volatile substance activity was determined by the plate-to-plate method. Inoculating Alternaria alternata fungus cakes in the center of a PDA plate, simultaneously streaking and inoculating a strain LZ88 on an NA plate, buckling the openings of two culture dishes, sealing the culture dishes by using a sealing film, setting a plate which is not connected with antagonistic bacteria LZ88 as a control, placing the plate in a constant-temperature incubator at 28 ℃ for culture, and observing the growth conditions of the control and treatment after 7 days. During the cultivation, it should be noted that a plate inoculated with pathogenic bacteria was placed on the top and a plate inoculated with antagonist bacteria LZ88 was placed on the bottom.
Experimental results show that volatile substances of the antagonistic strain LZ88 can effectively inhibit the growth of alternaria alternate on the flat plate, the alternaria alternate in a control overgrows the whole flat plate, the growth of the alternaria alternate in an experimental group is obviously inhibited, and the bacteriostatic effect is obvious. (FIG. 3).
EXAMPLE 4 bacterial Strain LZ88 sterile filtrate bacteriostatic Activity assay
Centrifuging the fermentation liquor of the strain LZ88 at 8000r/min for 10min, collecting the supernatant, filtering the supernatant with 0.22 μm bacterial microporous membrane under aseptic condition, and sterilizing the filtrate for metabolite activity determination. Adopting a growth rate method, taking 1mL of filtrate and 15m of PDA culture medium which is sterilized and cooled to 60 ℃ to be uniformly mixed to prepare a flat plate, taking the flat plate added with the same amount of sterile water to replace the filtrate as a control, and inoculating the phytopathogen fungus cake which grows vigorously and has the diameter of 0.5cm on the flat plate. Each treatment was repeated 3 times. Culturing at 28 deg.C, measuring the diameters of control and treated colony after the control grows, and calculating the antibacterial rate. The result is shown in fig. 4, the aseptic filtrate can obviously inhibit the growth of pathogenic bacteria, and the spore growth condition is observed under a microscope, so that the spore amount on the culture medium added with the aseptic filtrate is obviously reduced, and the antibacterial substance generated by the aseptic filtrate can effectively inhibit the formation of spores.
EXAMPLE 5 determination of bacteriostatic Activity of crude extract of Strain LZ88 protein
Fermenting and culturing strain LZ88 in NB culture medium for 3d, centrifuging at 8500r/min for 15min, collecting supernatant, and filtering with bacteria filterRemoving thallus to obtain sterile fermentation filtrate. Adding 51.6g ammonium sulfate into 100mL fermentation liquid, slowly adding ammonium sulfate into ice water bath, standing at 4 deg.C overnight, centrifuging at 4 deg.C for 15min, removing supernatant, and collecting precipitate. The precipitate was dissolved in 0.02mol/L phosphate buffer (pH 7.2) and desalted by dialysis at 20mmol/L of LTris-HCl buffer (pH9.0) overnight at 4 ℃ (molecular weight cut-off of the dialysis bag 14000 Da). Bacteria filter for dialysateFiltering to remove impurities to obtain the crude protein extract. Then the bacteriostatic activity of the crude protein extract is determined.
The experimental result shows that the crude protein extract can effectively inhibit the growth of alternaria alternata. As shown in FIG. 5, the growth of Alternaria alternata was significantly inhibited on the medium mixed with the crude protein extract, and neither the PBS-treated plate nor the blank control had any bacteriostatic effect, indicating that the substance having the primary bacteriostatic effect was a protein substance. As shown in FIG. 6, the crude extract after different temperature treatments still has bacteriostatic activity, and the protein is not denatured even at 120 ℃. The crude protein extract has better stability and is less influenced by temperature.
Example 6 Strain LZ88 induces systemic resistance in tobacco
In order to evaluate the systemic resistance of tobacco leaves induced by the strain LZ88, the activities of the resistance-associated enzymes POD and PPO were examined. After the strain LZ88 is fermented in NB culture solution, the concentration of bacterial suspension is adjusted to OD600The value 0.3, the changes in Peroxidase (POD) and polyphenol oxidase (PPO) activities on the day of spraying and after 1, 3, 5, 7, 9 and 12 days, respectively, were measured on tobacco plants grown from 10 leaves, as compared with the treatment with NB broth.
Preparing a tobacco leaf crude extract: weighing 0.1g of each tobacco leaf treated by fermentation liquor of the strain LZ88 and blank control tobacco leaves, removing the main veins from the tobacco leaves, putting the tobacco leaves into a mortar, adding a small amount of distilled water and quartz sand into the mortar, and fixing the volume to 10mL by using the distilled water after fully grinding. Then centrifuged at 4 ℃ and the collected supernatant was used to detect the enzyme activity.
6.1 determination of Peroxidase (POD) Activity 1mL of the crude extract, 1mL of 0.2mol/L acetate buffer (pH5.0), and 1mL of 1% o-methoxyphenol were used. Shaking, adding 1mL of hydrogen peroxide solution in a water bath at 30 deg.C for 5min, reacting for 2min, and measuring OD with ultraviolet spectrophotometer470The value is obtained. Per gram fresh tissue per minute OD470The change in value of 0.01 is one enzyme activity unit.
6.2 measurement of Polyphenol oxidase (PPO) Activity 1mL of the enzyme solution was added with 1.5mL of 0.02mol/L catechol solution and 1.5mL of 0.05mol/L phosphate buffer solution (pH 6.8). Shaking the mixed solution, placing in30 deg.C water bath, measuring OD with ultraviolet spectrophotometer after 2min398The value is obtained.
The experimental result shows that the contents of peroxidase and polyphenol oxidase in the tobacco leaves treated by the fermentation liquor of the strain LZ88 are higher than those of the control group, as shown in figures 7 and 8, the content of Peroxidase (POD) in the tobacco leaves treated by the fermentation liquor is rapidly increased within 3d and then begins to decrease; whereas the polyphenol oxidase (PPO) content reaches a maximum within 5d and then decreases rapidly. In a blank control, the activity change of the two enzymes is not obvious along with the change of time, which shows that the fermentation liquor of the strain LZ88 can improve the activity of enzymes POD and PPO related to tobacco resistance in a period of time, and shows that the fermentation liquor of the strain LZ88 can possibly induce the resistance of tobacco plants to the tobacco brown spot.
Example 7 biocontrol Effect of Strain LZ88 in greenhouse
For the greenhouse test, tobacco variety K326 was used as test plant. And spraying the tobacco to 5-6 leaves after the tobacco grows. Adjusting the concentration of the fermentation liquor of the strain Bacillus siamensis LZ88 to OD600Is 0.1. Uniformly spraying tobacco leaves for three times, spraying once every three days, and spraying NB culture solution as a control. Each treated 10 tobacco seedlings. Spraying for 24h for the third time, and inoculating tobacco brown spot by injection. Each tobacco seedling is inoculated with two tobacco leaves, and each tobacco leaf is injected with 6 inoculated positions. After inoculation, the cells are first treated in the dark for 8h, and then cultured in the light for 16h and in the dark for 8 h. The disease condition is investigated on the 5 th day after receiving the disease, and the disease index and the disease prevention effect are calculated. The investigation is carried out according to the GB/T23222-2008 national tobacco brown spot disease classification standard (taking leaves as a unit). Grading according to 0-9 grades, wherein the grading standards are as follows (taking the blades as units):
level 0: the leaves have no disease spots;
level 1: the area of the lesion spots accounts for less than 1% of the area of the leaves;
and 3, level: the area of the scab accounts for 2 to 5 percent of the area of the leaf;
and 5, stage: the area of the scab accounts for 6 to 10 percent of the area of the leaf;
and 7, stage: the lesion area accounts for 11% -20% of the leaf area;
and 9, stage: the area of the lesion spots accounts for more than 21 percent of the area of the leaves.
The disease index and the biological control effect of antagonistic bacteria were calculated as follows:
disease index [ ∑ (number of diseased leaves at each stage × disease grade value)/(total number of investigated leaves × highest grade value) ] × 100
The control effect is [ (control disease index-each treatment disease index)/control disease index ] × 100%
The greenhouse pot experiment result shows that Bacillus siamensis LZ88 has obvious inhibition effect on alternaria alternata. Compared with the control group, the disease index of the LZ 88-treated plants is 9.45 lower than that of the control group, and the control effect reaches 80.98 percent (Table 1). As shown in FIG. 9, the tobacco seedlings treated with Bacillus siamensis LZ88 fermentation broth showed a lower degree of disease development than the control. In contrast, the diseased leaves had significant symptoms and large lesions. Plants treated with LZ88 had little disease and developed well. The test results show that Bacillus siamensis LZ88 can be used as a biological control agent for effectively controlling the alternaria alternata.
TABLE 1 disease prevention Effect of Strain LZ88 on tobacco Alternaria alternata
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. 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. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
SEQUENCE LISTING
<110> Guizhou province company, China tobacco general company, China institute of tobacco research, China academy of agricultural sciences
Sichuan Branch of China Tobacco Corp.
<120> Siamese bacillus and application thereof
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 1
agagtttgat cmtggctcag 20
<210> 2
<211> 22
<212> DNA
<213> Artificial Synthesis
<400> 2
tacggytacc ttgttacgac tt 22

Claims (7)

1. Bacillus siamensis: (Bacillus siamensis) LZ88, wherein the strain is preserved in China general microbiological culture Collection center (CGMCC) on 9/4 in 2019, and the biological preservation number is CGMCC No. 18466.
2. A microbial agent, a pathogenic bacteria inhibitor or a disease inhibitor comprising the siamese bacillus LZ88 of claim 1 or/and the siamese bacillus LZ88 fermentation broth of claim 1.
3. The use of a pathogen inhibitor according to claim 2, characterized in that: the application has an inhibiting effect on all or part of the following pathogenic bacteria: alternaria alternate, phytophthora parasitica, fusarium graminearum, colletotrichum, fusarium oxysporum and fusarium arachidis.
4. The use of the disease inhibitor according to claim 2, characterized in that: the diseases are all or part of the following diseases: tobacco brown spot, tobacco black shank, wheat scab, grape anthracnose, cucumber fusarium wilt and peanut sickle disease.
5. Use of the Siamese Bacillus LZ88 of claim 1 or/and the Siamese Bacillus LZ88 fermentation broth of claim 1 for inhibiting pathogenic bacteria or/and for preparing a pathogenic bacteria inhibitor;
the pathogenic bacteria are all or part of the following pathogenic bacteria: alternaria alternate, phytophthora parasitica, fusarium graminearum, colletotrichum, fusarium oxysporum and fusarium arachidis.
6. The use of the Siamese Bacillus LZ88 of claim 1 or/and the Siamese Bacillus LZ88 fermentation broth of claim 1 to induce disease resistance in tobacco, wherein said inducing disease resistance in tobacco comprises increasing the levels of peroxidase and polyphenol oxidase in tobacco.
7. A method for controlling tobacco brown spot comprising spraying the siamese bacillus LZ88 of claim 1, the fermentation broth of the siamese bacillus LZ88 of claim 1, or/and the microbial inoculant of claim 2 onto the surface of a tobacco plant and/or onto the surrounding soil.
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