CN116286526A - Mulberry endophytic antagonistic bacterium RSTC-Q6 strain and application thereof - Google Patents
Mulberry endophytic antagonistic bacterium RSTC-Q6 strain and application thereof Download PDFInfo
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- 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
- 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
- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
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- 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
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- 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
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- 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
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- C12R2001/07—Bacillus
Abstract
The invention discloses a mulberry endophytic antagonistic bacterium RSTC-Q6 strain and application thereof. The invention separates an endophytic antagonistic bacterium Bacillus amyloliquefaciens (Bacillus sp.) RSTC-Q6 strain from mulberry, and the strain has the functions of resisting various plant pathogenic bacteria: sang Xi Arthrobacter, sang Xin Fusarium brown spot, fusarium equisetum, fusarium oxysporum, fusarium solanacearum, siamese anthrax, laurella of Solanaceae, klebsiella oxytoca, pseudomonas syringae, etc. have good antagonism; meanwhile, the plant disease caused by the plant pathogenic bacteria has good prevention and control effects. The invention adopts RSTC-Q6 strain fermentation liquor to biologically prevent and control plant diseases caused by the infection of plants by the plant pathogenic bacteria, is safe and effective, provides a new method and thought for preventing and controlling the mulberry diseases, and provides excellent strain resources with good prevention and control effects.
Description
Technical Field
The invention belongs to the technical field of agricultural microbial control. More particularly relates to a mulberry endophytic antagonistic bacterium RSTC-Q6 strain and application thereof.
Background
The mulberry (Morus alba L.) is a shrub or shrub of Moraceae, morus, and is a perennial woody economic plant widely distributed in areas such as sub-European continents, america, and Africa. The mulberry leaf has rich nutritive value, is not only used as the sole food source of silkworms, but also has important medicinal value of polysaccharide, flavone and the like. However, the mulberry disease always affects the development of the mulberry industry, and the trend of aggravation is presented at present.
Mulberry diseases are classified into fungal diseases and bacterial diseases. The mulberry fungal diseases have various types and various hazard positions, and the mulberry diseases comprise Sang Heban diseases, mulberry leaf ring diseases, sang Tanju diseases, sang Chi rust diseases, sang Li powdery mildew, sang Wushe diseases, mulberry leaf curl diseases and the like; the mulberry disease is a hypertrophic sclerotium disease of mulberry, a shrinking sclerotium disease of mulberry and a small particle disease of mulberry; the diseases of the mulberry tree trunk include dry rot, mulberry quasi-dry rot and mulberry bud-dry disease; the Sang Genbu disease has mulberry purple streak feather. Bacterial diseases of mulberry mainly include bacterial wilt of mulberry, sang Qing blight, sang Yibing and the like, and the bacterial diseases of mulberry have a plurality of pathogenic bacteria incidence periods and strong infectivity, are frequently exploded in a plurality of mulberry producing areas, and generally cause the wilt death of the whole plant.
Meanwhile, a plurality of diseases of the mulberry are often existed at the same time, and some diseases are not caused by one pathogenic bacterium, for example, sang Xijun pathogenic bacteria of wilt have diversity, and are caused by pathogenic bacteria of enterobacter cloacae, klebsiella of klebsiella, klebsiella oxytoca and the like, and possibly other pathogenic bacteria of a plurality of species. Therefore, it is difficult to control the above diseases or pathogenic bacteria.
Traditional agricultural control and chemical control are the main control measures for the fungal diseases of the mulberry at present, but the disease-resistant mulberry varieties are still lacking in production due to the diversity and pathogenic instability of pathogens, and the breeding of the resistant varieties is a time-consuming process and is difficult to cope with the increase of the diseases. However, excessive use of chemical pesticides not only can enhance pathogen resistance, but also can negatively affect the environment and human health, and can easily cause silkworm poisoning or pesticide residue exceeding of agricultural products due to improper operation. With the research and development of microorganisms, a new approach is provided for preventing and controlling mulberry diseases, such as the prior art discloses that bacillus thuringiensis (Bacillus thuringiensis) can inhibit sclerotinia sclerotiorum (Sclerotinia sclerotiorum) of mulberry hypertrophic sclerotinia; bacillus belicus (Bacillus velezensis) can inhibit Mortierella sclerotiella (Scleromitrula shiraiana), callicarpa suis (Ciboria carunculoides) and Callicarpa multocida (Ciboria shiraiana) which are main pathogenic bacteria of Morous sclerotinia; pseudomonas aeruginosa (Pseudomonas aeruginosa) can prevent and treat mulberry wilt and the like.
However, the microbial strains capable of being applied to the prevention and treatment of the mulberry diseases are few, and the strains capable of inhibiting other different pathogenic bacteria are not found. Therefore, more antagonistic bacteria with excellent performance and stronger control effect on various plant pathogenic bacteria are needed to be developed, safer and more efficient control means and ways are provided for common disease control in the silkworm industry, and the method has important significance for more effective, long-term and safe control of mulberry diseases.
Disclosure of Invention
The invention aims to overcome the defects and the shortcomings of the prior art and provide a mulberry endophytic antagonistic bacterium RSTC-Q6 strain and application thereof.
The first object of the present invention is to provide a strain of the endophytic antagonistic bacterium Bacillus amyloliquefaciens RSTC-Q6 of mulberry.
A second object of the present invention is to provide the use of the Bacillus amyloliquefaciens RSTC-Q6 strain.
It is a third object of the present invention to provide a plant pathogenic bacteria inhibitor.
A fourth object of the present invention is to provide a method for inhibiting plant pathogenic bacteria and/or controlling plant diseases.
The above object of the present invention is achieved by the following technical scheme:
the invention separates an endophytic antagonistic bacterium Bacillus amyloliquefaciens (Bacillus sp.) RSTC-Q6 strain from mulberry, wherein the strain is preserved in the microorganism strain collection of Guangdong province at 2022, 12 months and 15 days, and the preservation number is GDMCC No:63052. the study of the invention shows that RSTC-Q6 strain (hereinafter called Q6 for short) has the following effects on various plant pathogenic bacteria: sang Xi Arthrobacter, sang Xin Fusarium brown spot, fusarium equisetum, fusarium oxysporum, fusarium solanacearum, siamese anthrax, laurella of Solanaceae, klebsiella oxytoca, pseudomonas syringae, etc. have good antagonism; meanwhile, the plant disease caused by the plant pathogenic bacteria, such as Sang Heban disease, mulberry leaf scald disease, sang Tanju disease, sang Tan maggot disease, mulberry bacterial wilt or mulberry epidemic disease, has good prevention and control effects.
Therefore, the invention provides application of a mulberry endophytic antagonistic bacterium Bacillus amyloliquefaciens (Bacillus sp.) RSTC-Q6 strain or bacterial liquid thereof in inhibiting plant pathogenic bacteria or preparing a plant pathogenic bacteria inhibitor and preventing and controlling plant diseases caused by the plant pathogenic bacteria.
Preferably, the plant pathogenic bacteria are plant pathogenic fungi, specifically: sang Xi, arthrospira (Gonatophragmium mori), sang Xin Fusarium brown spot (Neophloeospora maculans), fusarium equisetum (Fusarium equiseti), fusarium oxysporum (Fusarium oxysporum), fusarium solani (Fusarium solani), and Siamese anthrax (Colletotrichum siamense).
Preferably, the plant pathogenic bacteria are plant pathogenic bacteria, specifically: one or more of Lauraceae (Ralstonia solanacearum), klebsiella oxytoca (Klebsiella oxytoca) and Pseudomonas syringae (Pseudomonas syringae).
In particular, the present inventors have studied to show that the Q6 strain can inhibit the above-mentioned various plant pathogenic bacteria, and similarly that the Q6 strain can be used for plant diseases caused by the infection of plants with the above-mentioned plant pathogenic bacteria, and have studied to show that the application of Q6 to plants can prevent and treat plant diseases by the inhibition of pathogenic bacteria. Therefore, the Q6 strain can be used for control of plant diseases caused by infection of plants with the above-mentioned plant pathogenic fungi and/or pathogenic bacteria.
Preferably, the plant disease is one or more of Sang Heban disease, mulberry leaf scald disease, mulberry bacterial wilt, sang Tan maggot disease, sang Qing blight and mulberry epidemic disease.
The invention provides a plant pathogenic bacteria inhibitor which contains bacillus amyloliquefaciens RSTC-Q6 strain or bacterial liquid thereof.
Preferably, the bacterial liquid is fermentation liquid.
Preferably, the concentration of the fermentation broth is not less than 10 8 cfu/mL。
The invention also provides a method for inhibiting plant pathogenic bacteria and/or preventing and controlling agricultural diseases, which adopts bacillus amyloliquefaciens RSTC-Q6 strain or the plant pathogenic bacteria inhibitor to treat samples.
The invention has the following beneficial effects:
the invention separates from mulberry to obtain an endophytic antagonistic Bacillus amyloliquefaciens (Bacillus sp.) RSTC-Q6 strain which has good antagonism to a plurality of plant pathogenic bacteria Sang Xi festival mould, sang Xin brown spot fusarium, fusarium equiseti, fusarium oxysporum, fusarium solani, siamese anthrax, solanaceae Layer, klebsiella, clove pseudomonas and the like, wherein the inhibition effect to Sang Xin brown spot fusarium, sang Xi festival mould, fusarium equiseti and Solanaceae Layer is most obvious; meanwhile, the plant disease caused by the infection of the plant pathogenic bacteria on the plants, such as Sang Heban disease, mulberry leaf scald disease, mulberry bacterial wilt, sang Tanju disease, sang Qing blight, sang Yibing and the like, has good prevention and control effects. The bacillus amyloliquefaciens RSTC-Q6 strain provided by the invention is used for biological prevention and control of plant diseases caused by plant pathogen infection, is green, safe and effective, does not cause drug resistance of pathogens, does not negatively affect the environment and human health, can also avoid the problems of soil environment deterioration and the like caused by chemical pesticides, provides a new method and thinking for preventing and controlling mulberry diseases, provides excellent strain resources with good prevention and control effects, and has good application prospects in prevention and control of plant diseases or preparation of preparations for preventing and controlling plant diseases.
Drawings
FIG. 1 is a graph showing the results of screening an endophyte antagonistic bacterium optimal fermentation medium (panel inhibition zone graph on the left and data on the right).
FIG. 2 is a graph showing the results of antagonistic activity of lipopeptides extracted from endophytic antagonistic bacteria on Sang Xi Arthrobacter, sang Xin Eichhornia crassipes, fusarium equisetum, fusarium oxysporum, fusarium solanacearum, siamese anthracis, lauraceae, klebsiella oxytoca and Pseudomonas syringae (a, b, c: all endophytic antagonistic lipopeptides; d: methanol).
FIG. 3 is a diagram showing the growth of endophytic antagonistic strains on LB medium.
FIG. 4 is a graph of the results of gram staining of endophytic antagonistic strains.
FIG. 5 is a graph showing the results of scanning electron microscopy (7000X) on endophytic antagonistic bacteria strain.
FIG. 6 is a phylogenetic tree diagram of the construction of endogenous antagonistic gyrB sequences.
FIG. 7 is a graph showing the results of potted plant control experiments of endophytic antagonistic bacteria (A group is mulberry inoculated with a strain solution of L.solanaceae, B group is mulberry treated with Q6 after inoculation with L.solanaceae).
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The bacterial DNA extraction kit and PCR reagents used in the following examples were purchased from Bio-engineering (Shanghai) Co., ltd; potato dextrose agar medium, beef extract peptone agar medium, physiological and biochemical medium, LB broth were purchased from Guangdong Cryptographic microorganism Co., ltd.
Pathogenic bacteria adopted by the invention: sang Xi Arthrospira (Gonatophragmium mori), sang Xin Fusarium brown spot (Neophloeospora maculans), fusarium equisetum (Fusarium equiseti), fusarium oxysporum (Fusarium oxysporum), fusarium solani (Fusarium solani) and Siamese anthrax (Colletotrichum siamense) were isolated by the following prior art techniques:
gao Qiaoxing Ji, templo Cumin, hu Xingping, etc. the life history and classification of Phellinus linteus (Gonatophragmium mori) [ J ]. Foreign agronomic-silkworm industry, 1988 (01): 27-30.
Soylu S,Kurt S,Soylu E M.First report of phloeospora leaf spot on mulberry caused by phloeospora maculans(＝cylindrosporium maculans)in the east mediterranean region of turkey[J].Plant Pathology,2003,52(3):415.
Han Zhongming, sun Zhuo, wang Yan, etc. screening and identification of wind-proof root rot antagonistic fungi and research on biocontrol effect [ J ]. Chinese journal of biological control, 2022,38 (05): 1288-1295.
Zhao Zhixiang, ling, yan Wanrong, et al isolation and identification of pepper fusarium wilt and analysis of evolution [ J ]. Molecular plant breeding, 2019,17 (19): 6383-6389.
Shen Baoyu, sun Wensong, zhang Tianjing, et al. Isolation and identification of root rot pathogen of Dictamni 1-14. Molecular plant breeding.
Yang Xiaoqi infection pathogenic study of Siamese anthrax (Colletotrichum siamense) on the basal part of strawberry stems [ D ] university of agriculture and forestry Zhejiang, 2022.
The pathogenic bacteria adopted by the invention are all common pathogenic bacteria in the research of the prior art, and are stored and researched in the laboratory of the applicant after being separated by the market or the prior art.
EXAMPLE 1 isolation of endogenous antagonistic bacterial Strain
1. Isolation and purification of strains
Collecting healthy strong mulberry 1 mulberry leaves from a mulberry training center in the asia-tai area of agricultural university in south China, taking newly collected mulberry leaf tissues, washing the fresh mulberry leaf tissues with sterile water, shearing the fresh mulberry leaf tissues into long strips on an ultra-clean workbench, adding 75% alcohol into a sterile glass dish, soaking for about 15 seconds, draining the alcohol, treating for 5 minutes by using mercuric chloride in the same method, and washing the fresh mulberry leaf tissues with sterile water for 3 times; placing leaf tissue in a sterile mortarGrinding juice with grinding rod, and diluting with sterile gun head 3 After that, 25. Mu.L of the dilution was pipetted and spread evenly on LB medium (yeast extract 5g/L, peptone 20g/L, naCl 20 g/L), and the plating was repeated 3 times for each dilution. After 24 hours, single colony is picked up on LB culture medium for streak expansion culture, single colony is picked up every other day and inoculated on LB inclined plane and flat culture medium, and stored in glycerol and placed in a refrigerator at-80 ℃ for one portion.
2. Screening of endophytic antagonistic bacterial strains
Using Sang Heban disease bacteria Sang Xin brown aschersonia aleyrodis N.maculons as indicator bacteria, testing antibacterial activity by adopting an agar diffusion method, culturing the indicator bacteria on a potato dextrose agar medium for 15d, then placing the indicator bacteria in a sterile mortar, removing redundant agar, adding 10mL of sterile water, grinding the indicator bacteria by using a sterile grinding rod, uniformly smearing 500 mu L of the indicator bacteria on the potato dextrose agar medium, uniformly punching 5mm small holes in a culture dish coated with a tested strain by using a puncher, transferring 10 mu L of fermentation liquor, adding the indicator bacteria into the punched small holes, standing and culturing for 5d in a culture box at 25 ℃, detecting whether a bacteria inhibition ring exists or not, and measuring the diameter of the bacteria inhibition ring. Screening to obtain an antagonistic bacterium with inhibition effect on indicator bacterium Sang Xin brown spot aschersonia aleyrodis N.maculons, and carrying out the next experiment.
3. Screening of optimal fermentation medium for endophytes
Picking single colony of the screened antagonistic bacteria, inoculating to NYD culture medium (8 g/L of beef extract, 3g/L of yeast extract, 1g/L of glucose), YPG culture medium (10 g/L of yeast extract, 20g/L of peptone, 20g/L of glucose), YPD culture medium (10 g/L of yeast extract, 20g/L of tryptone, 20g/L of glucose), YSB culture medium (20 g/L of sucrose, 20g/L of yeast extract, 15g/L of beef extract, mgSO) 4 ·7H 2 O 0.06g/L，FeSO 4 ·7H 2 O0.009 g/L), BPY medium (beef extract 5g/L, peptone 10g/L, yeast extract 5g/L, glucose 10g/L, naCl 5 g/L) and LB medium 6 kinds of medium, at 28deg.C at 180r/min for 48h, each concentration was adjusted to 10 8 cfu/mL. Centrifuging the antagonistic fermentation liquor at 10000r/min for 15min, removing precipitate, collecting supernatant, filtering with 0.2 μm bacterial filter, and testing by agar diffusion methodBacteriostatic activity was repeated 3 times for each medium, and the diameter of the zone of inhibition was measured.
4. Extraction and activity determination of lipopeptides secreted by bacterial strain
According to the prior study, the endophyte can produce the antibacterial lipopeptide with extremely strong antibacterial activity, so the embodiment provides technical support for preventing and controlling the mulberry diseases or other plant diseases by extracting lipopeptid substances in the endophyte of the mulberry and measuring the antibacterial activity of the lipopeptid substances. The specific experimental operation is as follows: selecting the single colony of the screened antagonistic bacteria, inoculating into an optimal fermentation culture medium, culturing at 28 ℃ for 72 hours at 180r/min, centrifuging at 8000r/min for 15min, discarding the precipitate, and collecting the supernatant; HCl was slowly added thereto, and allowed to stand overnight with pH adjusted to 2.0,4 ℃. Centrifuging at 8000r/min for 15min, discarding supernatant, collecting precipitate, adding 5 times of acetone, mixing, suspending, centrifuging at 8000r/min for 15min, collecting acetone organic phase, and repeating the collection twice. Mixing the acetone extract for 2 times, placing in a fume hood for air drying, dissolving with methanol, sterilizing with a 0.2 μm organic filter membrane filter to obtain an antagonistic lipopeptide crude extract solution, and measuring the mass concentration with an Abbkine BCA protein quantitative kit.
5. Antagonism experiment of endophytic antagonistic lipopeptides on pathogenic bacteria
The agar diffusion method is adopted to test the pathogen bacteria by the endogenous antagonistic lipopeptides substances: the accession number of the Lauraceae bacteria (Ralstonia solanacearum) is GDMCC No:1.1616 Klebsiella oxytoca (Klebsiella oxytoca) accession number GDMCC No:1.1603 Pseudomonas syringae (Pseudomonas syringae) accession number GDMCC No:61844 (the above pathogenic bacteria are commercially available and stored in the laboratory). Respectively inoculating pathogenic bacteria into LB liquid culture medium, shake culturing at 28deg.C and 180r/min to D 600 About 0.3, at 1:30 volume ratio of pathogenic bacteria bacterial liquid is added into sterile LB agar medium at about 40 ℃, after mixing evenly, 15mL of culture is poured into each culture dish, 5mm holes are evenly punched in the culture dish after solidification, 10 mu L of antagonistic lipopeptide substance is removed, added into the punched holes, and static culture is carried out at 25 ℃ for 24 hours, whether a bacteriostasis ring exists is detected, and the diameter of the bacteriostasis ring is measuredSize of the product.
6. Antagonism experiment of endophytic antagonistic lipopeptides on pathogenic fungi
The method adopts a plate counter method to measure the pathogenic fungi of endophytic antagonistic bacteria: antagonistic activity against Sang Xi Arthrobacter (Gonatophragmimmori), sang Xin Fusarium brown spot (Neophloeospora maculans), fusarium equisetum (Fusarium equiseti), fusarium oxysporum (Fusarium oxysporum), fusarium solani (Fusarium solani), siamese anthrax (Colletotrichum siamense) (the above strains were obtained by the prior art and isolated and stored in the present laboratory). Sang Xin the antagonism test method of the aschersonia aleyrodis and the Sang Xi festival mould is the same as above, and 10 mu L of antagonism lipopeptides substance is added into the punched small holes. The antibacterial activity of other fungi is measured by adopting a flat plate counter method, a fungus cake is placed at the center of a potato dextrose agar medium, 4 small holes with the thickness of 5mm are drilled at equal intervals on the periphery of the fungus cake by using a puncher, and 10 mu L of lipopeptid substances are added.
7. Experimental results
According to the invention, the endophytic antagonistic bacteria with inhibition effect on the indicator bacteria N.macus is obtained through preliminary screening, and the optimal fermentation culture medium of the endophytic bacteria is screened, as shown in figure 1, the size of the bacteriostasis circle of the indicator bacteria N.macus in the sterile culture solutions of 6 common culture mediums can be shown, the bacteriostasis circle of the sterile culture solution of the endophytic antagonistic bacteria after fermentation in the YPG culture medium is the largest, so that the YPG culture medium is selected as a standby culture medium for extracting antibacterial active substances from the endophytic antagonistic bacteria.
Further, through the antagonism experiment of lipopeptid substances of endophytic antagonistic bacteria on pathogenic bacteria, the strain has antagonism on pathogenic fungi such as Sang Xin brown spot aschersonia, sang Xi section mould, fusarium equiseti, fusarium oxysporum, fusarium solani, siamese anthrax and the like, and on pathogenic bacteria such as Solanaceae Laurella, klebsiella, pseudomonas syringae and the like, and is named as Q6; the results of the inhibition zone are shown in the following table 1, and show that Q6 has good antagonistic effect on Sang Xin brown spot aschersonia, sang Xi festival mould, fusarium equiseti, solanaceae Laurella, klebsiella and pseudomonas syringae.
TABLE 1 results of zone of inhibition for endogenous antagonistic strains
The antagonistic effect of the endophytic antagonistic bacteria Q6 lipopeptides on pathogenic bacteria is shown as a graph in fig. 2, and shows that Q6 has good antibacterial effects on fungal pathogens such as Sang Xin brown spot aschersonia, sang Xi festival mould, fusarium equiseti, fusarium oxysporum, eggplant disease fusarium and Siamese anthracnose, wherein the antagonistic effect on Sang Xin brown spot aschersonia, sang Xi festival mould and fusarium equiseti is most obvious, so that Q6 can be suitable for preventing and treating brown spot and ring spot of mulberry. And Q6 has better antibacterial effect on bacterial pathogenic bacteria such as Laurella of Solanaceae, klebsiella oxytoca and Pseudomonas syringae, wherein the effect on the Laurella of Solanaceae is most obvious, so Q6 can be suitable for preventing and treating bacterial wilt of mulberry.
EXAMPLE 2 identification of endogenous antagonistic bacterial strains
And preliminarily judging the classification of bacterial strains according to morphological characteristics, gram staining, scanning electron microscope observation and physiological and biochemical index tests of bacterial colonies on a culture medium, and performing evolution analysis through gyrB gene sequences.
The endophytic antagonistic strain Q6 obtained by screening in example 1 was inoculated into LB agar medium, cultured at 28℃for 24 hours, and the colony morphology, growth status and the like were observed and recorded. And the physical and chemical parameters related to the activities of the contact enzyme and the oxidase are measured through gram staining, morphological observation (scanning electron microscope), and the specific methods refer to the common bacteria System identification Manual and the Berger bacteria identification Manual.
And then carrying out gene sequencing on the endophytic antagonistic bacterial strain, carrying out multi-sequence comparison on the sequencing result and the sequence of a target species related species on NCBI, adopting MUSCLE v.3.8.31 (http:// www.drive5.com/MUSCLE /) software for analysis, and constructing a phylogenetic tree by using Mega6 software and a maximum likelihood method (ML method: maximum Likelihood method), wherein a Bootstrap value is set to be 1000.
The results show that: as shown in FIG. 3, the growth of the endophytic antagonistic strain Q6 on the LB medium is shown, and it can be seen that the endophytic antagonistic strain Q6 is white, opaque, dry on the surface, rough and wrinkled on the LB medium, and uneven on the edge. The gram staining results of the strain are shown in FIG. 4, and it can be seen that the endophytic antagonistic strain Q6 is gram positive, rod-shaped and sporulated. The scanning electron microscope results of the strain are shown in FIG. 5, and it can be seen that the endophytic antagonistic strain Q6 is a rod-shaped strain with rounded ends and no flagella.
The physiological and biochemical characteristic results of the endophytic antagonistic bacteria Q6 strain show that the endophytic antagonistic bacteria Q6 strain is positive in contact with enzyme, V-P test and nitrate reduction reaction, indole test, hydrogen sulfide test, methyl red test, nitrate reduction, phenylalanine deaminase test and oxidase reaction, and can produce acid from glucose and degrade starch and gelatin.
The phylogenetic tree is constructed based on the full length of the endophytic antagonistic gyrB sequence, and the result is shown in figure 6, and the endophytic antagonistic bacteria Q6 strain and the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) are found to be one, and the confidence coefficient is close to 100%.
In summary, the endogenous antagonistic strain Q6 was identified as Bacillus (Bacillus) -Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) by combining morphological features with physiological and biochemical results, and the taxonomic name was: bacillus sp.sp.designated RSTC-Q6 strain and deposited at the Cantonese collection of microorganisms and cell cultures at 12.15 2022 under the accession number GDMCC No:63052. the preservation address is Guangzhou City first China No. 100 college 59 building 5 Guangdong province microbiological institute.
EXAMPLE 3 experiment of Engineer antagonistic bacterial Strain Sang Penzai
The results of the plate bacteriostasis experiments according to example 1 show that Q6 can inhibit Lauraceae and then verify the control effect of Bacillus amyloliquefaciens Q6 on Sang Qing blight on mulberry.
Taking 6 mulberry seedlings (90 d) with the same growth vigor, equally dividing into A, B groups, and applying 10mL of 10-concentration solution after root injury treatment 8 CFU/mL Solanaceae familyThe Laurella bacteria liquid is subjected to tieback experiment, after 3d culture in artificial climatic box, the A group is applied with clear water as blank control group, the B group is applied with fermentation liquor of Bacillus amyloliquefaciens Q6 as treatment group, and the concentration is 10 8 CFU/mL, 100mL was applied, and 6 replicates were set. After further culturing for 7d, the number of diseased plants was counted.
The experimental results of potted plant prevention and control are shown in fig. 7, obvious withering phenomenon (100%) appears on all the mulberry groups A tiebased with the Laurella multocida bacterial liquid, and all the mulberry groups B treated by Q6 grow normally, and Q6 can prevent and control diseases by inhibiting the Laurella multocida, and the bacillus amyloliquefaciens Q6 obtained by separation has good prevention and control effects when applied to plants, and can prevent and control Sang Qing blight caused by the Laurella multocida.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. An endophytic antagonistic Bacillus amyloliquefaciens (Bacillus sp.) RSTC-Q6 strain of mulberry, characterized in that said strain has been deposited at the microorganism strain deposit center in guangdong province at 12-15 of 2022 under the accession number GDMCC No:63052.
2. the use of the bacillus amyloliquefaciens RSTC-Q6 strain or the bacterial liquid thereof according to claim 1 for inhibiting plant pathogenic bacteria or preparing a plant pathogenic bacteria inhibitor.
3. Use of the bacillus amyloliquefaciens RSTC-Q6 strain or the bacterial liquid thereof according to claim 1 for controlling plant diseases caused by plant pathogenic bacteria.
4. Use according to claim 2 or 3, characterized in that the phytopathogenic fungi are phytopathogenic fungi, in particular: sang Xi, arthrospira (Gonatophragmium mori), sang Xin Fusarium brown spot (Neophloeospora maculans), fusarium equisetum (Fusarium equiseti), fusarium oxysporum (Fusarium oxysporum), fusarium solani (Fusarium solani), and Siamese anthrax (Colletotrichum siamense).
5. Use according to claim 2 or 3, characterized in that the phytopathogenic bacteria are phytopathogenic bacteria, in particular: one or more of Lauraceae (Ralstonia solanacearum), klebsiella oxytoca (Klebsiella oxytoca) and Pseudomonas syringae (Pseudomonas syringae).
6. The use according to claim 3, wherein the plant disease is a mulberry disease.
7. A plant pathogenic bacteria inhibitor comprising the Bacillus amyloliquefaciens RSTC-Q6 strain or a bacterial liquid thereof according to claim 1.
8. The inhibitor according to claim 7, wherein the bacterial liquid is a fermentation liquid.
9. The inhibitor according to claim 8, wherein the concentration of the fermentation broth is not less than 10 8 cfu/mL。
10. A method for inhibiting plant pathogenic bacteria and/or controlling plant diseases, characterized in that a sample is treated with the bacillus amyloliquefaciens RSTC-Q6 strain according to claim 1 or the inhibitor according to any one of claims 7 to 9.
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