CN116836873A - Biocontrol bacterium for preventing and treating grape black root disease and application thereof - Google Patents
Biocontrol bacterium for preventing and treating grape black root disease and application thereof Download PDFInfo
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Classifications
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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
- A01N63/27—Pseudomonas
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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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- C12N1/20—Bacteria; Culture media therefor
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- 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
- C12R2001/38—Pseudomonas
- C12R2001/385—Pseudomonas aeruginosa
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Abstract
The invention discloses a biocontrol strain for preventing and treating grape black root disease and application thereof, wherein the biocontrol strain is preserved in China general microbiological culture collection center (CGMCC) No.26646. The strain has good antagonism to pathogenic bacteria of various grape diseases, and particularly has remarkable inhibition to pathogenic bacteria of grape branch diseases. The strain is derived from grape rhizosphere soil, has the characteristics of high efficiency, low toxicity, no environmental pollution, difficult generation of drug resistance and the like, and has great biocontrol potential in the aspect of grape branch disease control, in particular to grape black root disease control.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to a biocontrol bacterium for preventing and treating grape black root disease and application thereof, and belongs to the technical field of biological prevention and treatment of plant diseases.
Background
Grape (grape vinifera l.) is one of four fruits in the world, and has high economic value. The planting area of the grapes in China is 79.79 ten thousand hm 2 The second place of the world, the yield reaches 1349 ten thousand t, and the first place of the world. While the global grape industry is developing at a high rate, grape diseases are increasingly serious, with grape branch diseases being considered the most damaging grape diseases over the past 30 years, and are widely distributed in major grape planting areas worldwide, with serious disease occurrence rates up to 100% (Pintos, c., redndo, v., costas, d., aguin, o., and Mansilla, p. (2019) Fungi associated with grapevine trunk diseases in nursery-produced Vitis vinifera plants. Phytopathological media disease 57, 407-424), it is counted that necrotic grape trees caused by branch diseases consume more than 15 billions of dollars (hofstater, v., bucck, b., croll, d., viret, o., couloux, a., and gindoro, k. (2012) What if esca disease of grapevine were not a fungal diseaseFungal Diversity, 51-67) per year in the global grape planting area. Grape branch diseases are fungal diseases, mainly comprising grape wilt disease (ESCA), grape canker disease (Botryosphaeria Dieback), grape top blight disease (Eutypa dieback), grape gummy stem disease (Diaporthe dieback) and grape black root disease (Black Foot Disease) 5, and the occurrence and development of grape branch diseases are related to root rot caused by fusarium at the root of grape, the pathogen and the disease condition are very complex, and the pathogenesis rule is not clear. There is no pesticide product for treating grape branch diseases and high disease resistance variety, and in actual production, once the fruit tree is ill, the fruit tree eventually dies gradually, and the grape branch diseases are controlled by biological control means and become hot spots for disease control research (Gramaje, D., urboz-Torres, J.R., and Soswski, M.R. (2018). Managing Grapevine Trunk Diseases With Respect to Etiology and Epidemiology: current Strategies and Future Proselect. PLANT DIS 102,12-39.).
Pseudomonas spp is a highly diverse microorganism population, is the most common microorganism population in plant rhizosphere, and the produced metabolite plays a significant role in crop disease control, and has the characteristics of high efficiency, low toxicity, easy degradation, no residue, environmental friendliness and the like. Typical reported Pseudomonas biocontrol bacteria include Pseudomonas fluorescens, pseudomonas aeruginosa and the like. In recent years, a series of rhizosphere-derived pseudomonas aeruginosa bacteria having a good biocontrol effect have been isolated and identified, which can secrete various metabolites against plant pathogens, and which can effectively inhibit various disease pathogens, such as fusarium (Durairaj, k., velcurgan, p., park, j., chang, w., park, y., senthilkumar, p., choi, k., lee, j., and Oh, b. (2018), an investigation of biocontrol activity Pseudomonas and Bacilus strains against Panax ginseng root rot fungal phytaphogens, biological Control 125, 138-146), pythium, rhizoctonia, etc. (Jiang Haixia, zhou Lian, he Yawen (2015), which are metabolic products of the biocontrol strain of pseudomonas aeruginosa bacteria, and the development of biocontrol study thereof, microbiology, and 1339, have not been studied in the aspect of controlling grape stem diseases, and thus the effect of controlling pseudomonas aeruginosa in the grape stem is expected to be effectively controlled by exploring new grape stem diseases.
Disclosure of Invention
The invention aims to provide a biocontrol bacterium for preventing and treating grape black root disease and application thereof. The strain has a strong inhibition effect on pathogenic bacteria causing grape branch diseases, and has a good control effect on grape branch diseases, so that the investment of chemical pesticides can be reduced, the environmental pollution is lightened, and the sustainable development of agriculture is realized.
The biocontrol bacteria for preventing and treating grape black root disease are collected and separated from grape rhizosphere soil of Yinchuan of Ningxia Hui nationality, and the grape rhizosphere soil is classified and named as pseudomonas aeruginosa Pseudomonas aeruginosa, O-C9, and is preserved in China general microbiological culture collection center (CGMCC, address is North Chen Xili No. 1, no. 3 in Beijing, chaoyang area) for 2 months and 21 days in 2023, and the preservation number is CGMCC No.26646.
The invention separates a strain with broad-spectrum inhibition effect on grape pathogenic fungi from grape rhizosphere soil collected from Yinchuan of Ningxia Hui nationality. Based on physiological characteristics and genome-wide molecular identification study, the strain is identified as pseudomonas aeruginosa (Pseudomonas aeruginosa) and named as pseudomonas aeruginosa (Pseudomonas aeruginosa) O-C9, and the physiological characteristics are as follows:
the pseudomonas aeruginosa (Pseudomonas aeruginosa) O-C9 of the present invention produced pale yellow to yellowish green pigment on LB, beef extract peptone and PDA media, and exhibited metallic luster on PDA media. The bacterial colony on the three culture mediums is yellow-green, opaque, round, smooth and moist in surface, regular in edge and fluorescent under an ultraviolet lamp.
The pseudomonas aeruginosa has antibacterial activity against grape pathogenic bacteria. The pathogenic fungi of the grape are cocoa hair color two spores (Lasiodiplodia theobromae), grape vine chamber bacteria (Botryosphaeria dothidea), sweet cherry seed shells (Diadorthe eres), grape anthracnose bacteria (Coletotrichum viniferum), botrytis cinerea (Botrytis cinerea), fusarium oxysporum (Fusarium oxysporum), fusarium equisetum (F.incarnatum-equiseti species complex), fusarium acuminatum (F.acuminatum), fusarium vine (F.fujikuroi), F.spartus, dactylonectria torresensis and Phaeoacremonium iranianum.
The application of the pseudomonas aeruginosa or the antibacterial active substance generated by the pseudomonas aeruginosa in preparing biological pesticides with antagonistic activity on plant pathogenic fungi belongs to the protection scope of the invention.
The invention also provides application of the pseudomonas aeruginosa in preventing and controlling plant pathogenic fungi.
The plant pathogenic fungi are grape pathogenic fungi, preferably one of cocoa bisporus (Lasiodiplodia theobromae), botrytis cinerea (Botryosphaeria dothidea), prunus cerasifera (Diadorthe eres), botrytis cinerea (Coletotrichum viniferum), fusarium oxysporum (Fusarium oxysporum), fusarium equisetum (F.incarnatum-equiseti species complex), fusarium acuminatum (F.acuminatum), fusarium canum (F.fujikuroi), F.spark, dactylonectria torresensis and Phaeoacremonium iranianum.
The pseudomonas aeruginosa (Pseudomonas aeruginosa) O-C9 has antibacterial activity on grape pathogenic fungi and has a wider antibacterial spectrum. The strain O-C9 has remarkable inhibition effect on the pathogenic bacteria Phaeoacremonium iranianum LN of the grape blight and the pathogenic bacteria diapason-cherokee 26-2SA of the grape blight, and the inhibition rate is 76.04% and 65.40%. The bacterial strain O-C9 has the worst antibacterial activity on grape root rot pathogen F.acuminatum JZB3110090, and the inhibition rate is 34.56%. The inhibition rate of the strain O-C9 to other 9 main grape disease pathogens is 36.27% -57.80%. The bacterial strain O-C9 fermentation liquor has a treatment effect of 47.44% on grape canker pathogenic bacteria L.theobromae CSS-01s artificially inoculated on isolated green branches of grapes, and can respectively reduce 76%, 78% and 43% of colonization amount of grape black root pathogenic bacteria in grape roots, branches and new-born branches. The strain O-C9 fermentation liquor has obvious control effect on black root disease on greenhouse planted grapes. The strain O-C9 has better biological potential for grape fungus diseases and has further development prospect.
Drawings
FIG. 1 is a colony characterization of strain O-C9;
wherein, left: LB medium, medium: beef extract peptone medium, right: PDA medium.
FIG. 2 is a phylogenetic tree of the genome-based of strain O-C9.
FIG. 3 is a phylogenetic tree of strain O-C9 based on the 16S rRNA gene.
FIG. 4 shows the results of the experiment of strain O-C9;
wherein the first group served as a control group, and the pathogenic fungi from left to right were CSS-01S, GXZY-02S, 26-2SA, BT-2, XY1, and 16B-2R-B1. The second behavioural treatment group, from left to right, was CSS-01S, GXZY-02S, 26-2SA, BT-2, XY1 and 16B-2R-B1. The third behavioral control group, from left to right pathogenic fungi: LN1, JZB3110103, JZB3110180, JZB3110090, JZB3110089, and JZB3110102. A fourth behavioral treatment group, from left to right pathogenic fungi: LN1, JZB3110103, JZB3110180, JZB3110090, JZB3110089, and JZB3110102.
FIG. 5 shows the control effect of O-C9 fermentation broth on grape canker;
wherein the values in the figures are mean ± standard deviation.
Description of biological Material preservation
Name: pseudomonas aeruginosa O-C9
Classification naming: pseudomonas aeruginosa Pseudomonas aeruginosa
Preservation unit: china general microbiological culture collection center (CGMCC)
Deposit unit address: beijing city, chaoyang area, north Chenxi Lu No. 1 and 3
Preservation number: CGMCC No.26646
Preservation time: 2023, 2, 21
Whether or not to survive: is the result.
Detailed Description
Example 1 obtaining Strain O-C9
1 sample Source
The strain O-C9 is separated from grape rhizosphere soil with grape branch diseases in Yinchuan of Ningxia Hui autonomous region. The specific separation method is as follows: 1g of grape rhizosphere soil was weighed into 9mL of sterile ddH 2 O, prepared as 10 -1 The soil suspension is subjected to continuous gradient dilution to prepare 10 -2 -10 -8 A soil suspension. 180 mu L of beef extract peptone liquid culture medium is added into a 96-well plate, and 20 mu L of 10 are respectively sucked -5 To 10- 8 Suspending the concentrated soil in the 96-well plates, culturing in a 28 deg.C incubator for 10-15 days, observing bacterial growth in each 96-well plate, and selecting<The bacteria in each well of the culture plate are preserved by the culture plate with a high probability that bacteria in each well are grown in pure culture in 20 wells.
Based on the method, the biocontrol bacteria are screened aiming at pathogenic bacteria of grape branch diseases, and the specific method is as follows: preparing 48-well PDA culture plate in advance, activating bacteria to be tested, shake culturing, sucking 10 μl of bacterial suspension into single culture well of 48-well plate, and shake culturing pathogenic fungi of grape branch to obtain spore suspension (10 6 spore/mL), and 10. Mu.L of the spore suspension was pipetted into a single culture well of a 48-well plate at 28 DEG CAfter culturing in an incubator for 5-7d, fungus growth is observed. The bacterial strain with antagonism to pathogenic bacteria is obtained by identification, and is named as O-C9, the bacterial colony of the bacterial strain O-C9 on an LB plate is shown in figure 1, and the bacterial colony is yellow green, opaque, round, smooth and moist in surface, regular in edge and fluorescent under an ultraviolet lamp.
Example 2 identification of Strain O-C9
And (3) accurately identifying the species of the strain based on the colony morphological characteristics and the whole genome sequence of the O-C9.
1 colony morphology characterization
Antagonistic bacteria O-C9 were inoculated on LB, beef extract peptone and PDA solid medium by three-zone streaking method, colony morphology was observed after 3d of cultivation in an incubator at 28℃and colony morphology on different media is detailed in Table 1.
TABLE 1 culture characteristics of strain O-C9
Molecular characterization of strain 2O-C9
2.1 extraction of genomic DNA of Strain O-C9
And (3) picking single colonies with good growth conditions into an LB liquid culture medium, culturing overnight at 28 ℃ to obtain a large amount of purified bacterial liquid, extracting genome DNA by an SDS method, detecting the genome DNA by 1% agarose gel electrophoresis, determining that the genome quality is qualified, and performing whole genome sequencing by using a Illumina Hiseq NovaSeq PE standard flow.
2.2 genome sequencing of Strain O-C9
Filtering original off-machine Data to obtain high-quality Clean Data, performing preliminary assembly on the Clean Data by using SOAP denovo software, SPades software and Abyss software, integrating assembly results of the three software with CISA software, and selecting an assembly result with minimum scaffoldes as a preliminary result; filling gap through gapcloser software, filtering reads with low sequencing depth (< 0.35) to obtain a final assembly result; fragments below 500bp are filtered, and the final result is subjected to gene prediction.
2.3 species identification based on the genomic sequence of Strain O-C9
Uploading the whole GENOME sequence of STRAIN O-C9 to TYPE (STRAIN) GENOME SERVERhttps:// tygs.dsmz.de/user_requests/new)The platform can be used for species identification based on the whole genome of the strain and the 16S rRNA gene sequence, and can replace the traditional classification standard technology such as DNA-DNA hybridization (DDH, DNA-DNA hybridization), G+C content determination, multi-site sequence analysis (MLSA, multilocus Sequence Analysis) and the like. The result shows that the strain O-C9 gathers together with pseudomonas aeruginosa (Pseudomonas aeruginosa) in the phylogenetic tree (figure 2) based on the whole genome sequence and the phylogenetic tree (figure 3) based on the whole 16S RNA gene sequence (sequence 1 in the sequence table) respectively based on the whole genome sequence and the 16S rRNA gene sequence, and when the whole genome sequence is compared with a reference strain Pseudomonas aeruginosa DSM 50071, the dDDH value is 94.6, and the bacterial species classification gold standard of more than 70% indicates that the strain O-C9 is pseudomonas aeruginosa (Pseudomonas aeruginosa).
The pseudomonas aeruginosa (Pseudomonas aeruginosa) O-C9 is preserved in China general microbiological culture collection center (CGMCC) with the address of North Chen West Lu No. 1 and No. 3 in the Korean area of Beijing city at 2023, and the preservation number is CGMCC No.26646.
Example 3 antibacterial Activity test of antagonistic bacterium O-C9
The antagonistic culture method is adopted, 12 strains (the strains in Table 2 are collected in the field, separated and purified and identified by conventional strains) of 12 grape pathogenic fungi are taken as target bacteria, and the antagonism of pseudomonas aeruginosa (Pseudomonas aeruginosa) strain O-C9 to grape pathogenic fungi is detected.
Activating strain O-C9 on LB solid medium, culturing at 37deg.C for 2-3d, picking single colony in LB liquid medium, shake culturing at 28deg.C at 200rpm for 1d, diluting with sterilized 1×PBS to obtain OD 600 ＝0.01(≈4×10 6 CFU/. Mu.L) was used for the counter experiment. Cutting bacterial cakes at the edges of bacterial colonies of pathogenic bacteria (Table 2) cultured at constant temperature of 25 ℃ for 3-7 days on PDA culture medium by using a puncher (5 mm), and placing the bacterial cakes on PDA levelAt the center of the plate, 4 mu L of bacterial suspension is respectively dripped on 4 points (with filter paper sheets with the diameter of 4.5mm placed) which are 1.0cm away from the edge of the culture dish by taking pathogenic bacteria as the center of the plate in a crisscross manner, so that pathogenic fungi and the filter paper sheets are inoculated at the center of the culture medium, and the same dosage of clear water is dripped on the filter paper sheets to serve as a control. 3 bacteria were cultured in parallel at constant temperature and observed at regular time, and after 5 days of counter culture, the width (D) of the zone of inhibition and the diameter (D) of the growth of the mycelia of the pathogenic bacteria in the O-C9 direction of the strain were measured by the crisscross method. Antibacterial ratio (%) = (colony growth diameter of control group-colony growth diameter of treatment group)/(colony growth diameter of control group-inoculation block diameter) ×100%. Will inhibit bacteria rate<25% of the antibacterial agent is marked as "+", and the antibacterial rate is 25% or less<50% is marked as "++", the bacteriostasis rate is more than or equal to 50% is marked as "++", "-" means no inhibition.
The experimental results are shown in table 3, the pseudomonas aeruginosa (Pseudomonas aeruginosa) O-C9 has different degrees of antibacterial activity on different grape fungal diseases, and the growth inhibition rate is between 34.56% and 76.04%. The strain O-C9 has remarkable inhibition effect on the pathogenic bacteria Phaeoacremonium iranianum LN of the grape blight and the pathogenic bacteria diapason-cherokee 26-2SA of the grape blight, and the inhibition rate is 76.04% and 65.40%. The bacterial strain O-C9 has the worst antibacterial activity on grape root rot pathogen Fusarium anatase Fusarium acuminatum JZB3110090, and the inhibition rate is 34.56%. The inhibition rate of the strain O-C9 on grape black root pathogenic bacteria Dactylonectria torresensis B-2R-B1, grape canker Botryosphaeria dothidea GXZY-02S, grape root rot pathogenic fungi F.spartum JZB3110102, F.fujikuroi JZB3110089, F.incarnatum-equiseti species complex JZB3110180, F.oxysporum JZB3110103 and grape gray mold Botrytis cinerea XY1 is more than 40%, and is between 42% and 58%. The inhibition rates of the strain O-C9 on the grape anthracnose bacteria Coletotrichum viniferum BT-2 and the grape canker bacteria Lasiodiplo diatheobromae CSS-01s are 38% and 36% respectively.
TABLE 2 12 grape pathogenic fungi tested
TABLE 3 bacteriostatic Activity of Strain O-C9 against 12 grape pathogenic fungi
EXAMPLE 4 prevention and treatment Effect of Strain O-C9 on grape canker
4.1 test methods
4.1.1 preparation of fermentation broth
Single colony of strain was inoculated with sterilized toothpick into a triangular flask containing 300mL of fermentation medium, and cultured with shaking at 25deg.C and 180rpm for 2d to obtain fermentation broth (1.38X10) of strain O-C9 9 CFU/mL)。
4.1.2 preparation of pathogenic fungi:
lasiodiplodia theobromae CSS-01s (Lt) were cultured at 28℃for 2-3d, the colonies were grown on plates, and a sterilized punch with a diameter of 4.0mm was used to uniformly punch a cake along 2cm inside the outer periphery of the colonies.
4.1.3 inoculation treatments
Collecting healthy, fresh and current half-lignified green branches of summer black grapes from a vineyard, cutting off leaves, cleaning the surfaces with clean water, wiping and sterilizing with 75% alcohol wet tissues, and flushing the surfaces with sterile water and airing for later use. Immersing the treated branches into fermentation liquor and sterile water respectively for 30min, then inserting the branches into a nutrition pot filled with vermiculite matrix for preservation, after 24h, taking wounds with the diameter of 4.0mm and the depth of 1.0mm at the middle part of the joints of the branches by using a sterilized puncher with the diameter of 4.0mm, peeling off the epidermis, uniformly beating the pathogenic bacterial strain cultured in advance into bacterial cakes by using the puncher with the diameter of 4.0mm, placing the bacterial cakes at the wound by using a sterilized toothpick, wrapping the positions of the bacterial cakes by using a sealing film cut into small sections, and inserting the bacterial cakes into the nutrition pot filled with the vermiculite matrix, wherein blank PDA plates are used as controls. Each treatment was inoculated with 20 shoots and 3 replicates were performed. Placing the treated branches into an inoculation chamber with temperature and humidity control after inoculation, carrying out humidification culture for 2d after inoculation, wherein the relative humidity is 90%, and then adjusting the temperature to 25 ℃ and the humidity to 60%; and measuring the length of the disease spots 7-14 days after inoculation, investigating the disease condition of each treatment, recording and measuring the length of the disease spots by photographing, and analyzing the control effect.
4.1.4 data statistics and analysis
After 7-14d inoculation, the disease condition and the disease spot length of each treatment are measured, and statistical analysis is carried out on the data by adopting GraphPad Prism 8.0.2 software, wherein the calculation formula is as follows:
control effect (%) = [ (control lesion length-treated lesion length)/(control lesion length-cake diameter) ]×100%
4.2 test results
Grape green branch warp 10 9 After the treatment of the O-C9 fermentation broth of CFU/mL, as shown in Table 4 and FIG. 5, the disease spot length is obviously lower than that of the water treatment, the control effect is 47.44%, which shows that 10 9 The O-C9 fermentation liquor of CFU/mL has better control effect on grape canker.
Table 4: in-vitro green branch disease spot length statistics of grape
| Treatment of | Disease spot length (mm) | Preventing and curing effect is% |
| Sterile water | 13.50±1.46b | - |
| 10 9 CFU/mL O-C9 fermentation broth | 10.38±0.83a | 47.44％ |
Note that: values are mean ± standard deviation.
EXAMPLE 5 inhibition of the amount of pathogenic bacteria of the black root disease of Vitis vinifera by Strain O-C9
5.1 materials and methods
5.1.1 preparation of Strain O-C9 fermentation broth
The procedure is as in example 4, 4.1.1. After the fermentation broth is obtained, the concentration of the fermentation broth is adjusted to OD 600 ＝2.0(8.19×10 9 CFU/mL)。
5.1.2 preparation of pathogenic fungi:
dactylonectria torresensis 16B-2R-B1 (Dt) was cultured at 28℃for 5-7 days, mycelia were picked up by a sterile inoculating rod and inoculated in PDA liquid medium, and cultured at 28℃for 2-3 days at 180rpm, and spore production was observed. Filtering the bacterial liquid by using sterile filter paper, and diluting by using sterile water to prepare the bacterial liquid with the concentration of 1 multiplied by 10 6 spore/mL spore suspension.
5.1.2 inhibition of the amount of pathogenic bacteria of the black root disease in grape by Strain O-C9
Ma Selan seedlings which are cultured in a greenhouse and have consistent growth conditions and no disease symptoms are selected as experimental materials, the roots of Ma Selan seedlings are cleaned by sterile water, 1cm far away from the lateral roots of the grape seedlings are cut off by scissors sterilized by 75% ethanol, root injury operation is carried out, the roots are immersed in Dt spore suspension, the seedlings are transplanted into a sterile flowerpot after 30min, sterilized nutrient soil and vermiculite (V: V=3:1) are selected as culture substrates, and the grape seedlings show obvious disease symptoms after 6 months of culture. And pouring the prepared O-C9 fermentation liquid at the main roots of the grapes, adding 150mL of fermentation liquid into each seedling, and pouring 150mL of sterile water by contrast treatment.
1 month after inoculation, collecting grape roots, branches and branch tissues which are treated differently, extracting tissue DNA by using a CTAB method, and carrying out absolute quantification of the black root disease pathogenic bacteria Dt by using a qPCR absolute quantification method.
First, a standard curve of Ct value and copy number of qPCR was drawn by constructing a standard:
1. amplification of the Dt single copy Gene fragment by PCR with primers Q6634F (5'-TGTCAACCTGGGTACCATCA-3') and Q6635R
(5'-TGCGTAGGTCGTGAGAACAG-3'). The PCR amplification reaction system was (50. Mu.L): 25. Mu.L 2 XSG PCR Master mix, 1. Mu.L gDNA template, 1. Mu. L Q6634F (10. Mu.M), 1. Mu. L Q6635R (10. Mu.M), 22. Mu.L ddH 2 O. The PCR amplification reaction procedure was: pre-denaturation at 94℃for 3min, denaturation at 94℃for 20s, annealing at 58℃for 20s, extension at 72℃for 30s, 35 cycles, and extension at 72℃for 10min. After the specific amplification product was recovered, it was ligated to a T carrier, and the reaction system was (10. Mu.L): 2. Mu.L of 5 Xreaction Buffer, 6.5. Mu.L of PCR product, 1. Mu.L of pTZ57R/T, 0.5. Mu. L T4 ligase. The ligation product was transformed into Top10 competent cells, and subjected to plasmid extraction and concentration measurement after screening, PCR colony identification and sequencing to prepare a standard.
2. Standard Curve establishment
And (3) carrying out qPCR experiments after the standard substance is subjected to gradient concentration dilution, and drawing a standard curve according to the copy number of the standard substance and the Ct value of qPCR. qPCR reaction System (20. Mu.L): 10. Mu.L 2X SG qPCR MasterMix, 1. Mu.L plasmid standard, 0.5. Mu.L Forward Primer (10. Mu.M), 0.5. Mu.L Reverse Primer (10. Mu.M), 8. Mu.L ddH 2 O. The reaction procedure: 94℃for 10min, (94℃for 20s,60℃for 30 s). Times.40 cycles. And finally, outputting data and constructing a standard curve.
3. Sample Ct value detection
According to the qPCR system and the reaction program, replacing the standard plasmid with a sample gDNA, performing qPCR reaction, finally outputting data, and calculating the absolute quantity of Dt in the sample according to a standard curve.
5.2 results
As shown in Table 5, after 1 month of treatment with O-C9 fermentation broth, the absolute amount of the pathogenic bacteria Dt of black root disease in the roots, the branches and the branches of grape was significantly reduced by 76%, 78% and 43% respectively compared with the control, and in addition, the young grape seedlings treated with O-C9 fermentation broth had new leaves, which indicated that O-C9 could effectively alleviate the symptoms of dry disease of grape Miao Zhi. The above results demonstrate that strain O-C9 has great potential for control of grape black root disease.
Table 5: absolute quantity of black root pathogen Dt in grape tissue
Note that: values are mean ± standard error.
The above examples illustrate only a few embodiments of the invention, which are described in detail, but are merely illustrative of the invention and not limiting. It will be apparent to those skilled in the art that many modifications, variations and improvements may be made therein without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (8)
1. A biocontrol bacterium for preventing and treating grape black root disease is characterized in that the biocontrol bacterium is named as pseudomonas aeruginosa (Pseudomonas aeruginosa) O-C9 and is stored in China general microbiological culture Collection center with the preservation number of CGMCC No.26646.
2. The biocontrol bacterium of claim 1, wherein said pseudomonas aeruginosa O-C9 has antagonistic activity against grape pathogenic fungi.
3. The biocontrol bacterium according to claim 2, wherein the grape pathogenic fungus is one of the group consisting of conidiobolus (Lasiodiplodia theobromae), stropharia rugosa (Botryosphaeria dothidea), primrose-forming (Diaporthe eres), colletotrichum vitis (Coletotrichum viniferum), botrytis cinerea (Botrytis cinerea), fusarium oxysporum (Fusarium oxysporum), fusarium equiseti complex species (f.incarnatum-equiseti species complex), fusarium acuminatum (f.acuminatum), fusarium binghanii (f.fujikuroi), f.spartum, dactylonectria torresensis and Phaeoacremonium iranianum.
4. The use of the biocontrol bacterium according to claim 1 for controlling phytopathogenic fungal diseases.
5. Use according to claim 4, wherein the phytopathogenic fungi is a Botrytis fungi, preferably one of the species fusarium lanuginosum (Lasiodiplodia theobromae), fusarium viticola (Botryosphaeria dothidea), sakura glabra (Diaporthe eres), fusarium vitis (Coletotrichum viniferum), botrytis cinerea (Botrytis cinerea), fusarium oxysporum (Fusarium oxysporum), fusarium equisetum (f.incarnatum-equiseti species complex), fusarium acuminatum (f.acuminatum), fusarium binghanii (f.fujikuroi), f.spartum, dactylonectria torresensis and Phaeoacremonium iranianum.
6. The use of the biocontrol bacterium of claim 1 in the preparation of a biocontrol agent or microbial fertilizer for inhibiting plant pathogenic fungi.
7. A biocontrol microbial agent for plant pathogenic fungi, characterized in that the active ingredient of the biocontrol microbial agent is the biocontrol microbial agent of claim 1.
8. A microbial fertilizer for plant diseases, characterized in that the active ingredient of the microbial fertilizer is the biocontrol bacterium of claim 1.
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