CN113355262A - Burkholderia, bacterial agent comprising Burkholderia, bacterial fertilizer, preparation method and application - Google Patents
Burkholderia, bacterial agent comprising Burkholderia, bacterial fertilizer, preparation method and application Download PDFInfo
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Images
Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
-
- 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
Abstract
The invention provides Burkholderia, a microbial inoculum containing Burkholderia, a bacterial fertilizer, a preparation method and application, and belongs to the technical field of microbial preparations. Burkholderia sp K2b of the present invention has a deposit number of: CGMCC NO.22002, which can inhibit the growth of phytophthora capsici by generating active substances, shows better control effect on phytophthora capsici, and has the maximum inhibition rate of 88.53 percent. Meanwhile, the method can effectively colonize roots, stems and leaves of the peppers, and improve the micro-ecological environment of crops. The pesticide effect result of the indoor pot culture shows that the K2b fungicide has the control effect of 85.64 percent on pepper diseases and is not easy to generate drug resistance. When in use, the root irrigation is carried out at the seedling stage or the adult stage of the pepper. The microbial inoculum can also be mixed with organic fertilizer to prepare bacterial fertilizer for use.
Description
Technical Field
The invention relates to the technical field of microbial preparations, in particular to Burkholderia, a microbial inoculum comprising the Burkholderia, a bacterial fertilizer, a preparation method and application.
Background
The pepper phytophthora blight is a destructive soil-borne disease, and pepper can be diseased at any time and any part in the growth process. In recent years, the dead seedling rate of a heavily diseased field with pepper phytophthora blight reaches 30-100%. At present, the pepper phytophthora blight is one of the key factors which prevent the stable yield and the yield increase of pepper. The chemical bactericide plays a vital role in preventing and treating phytophthora capsici, for example, mandipropamid has a strong inhibition effect on the growth of phytophthora capsici hyphae. However, with the large amount and unscientific use of chemical bactericides, many drawbacks are increasingly highlighted, such as drug resistance of pathogenic bacteria, environmental pollution, ecological system destruction and the like, which become major problems of the currently applied chemical bactericides. The biocontrol microbial inoculum has good compatibility with the environment, high efficiency, low toxicity, low residue and small selection pressure on pathogenic bacteria, and is one of the popular areas in the research and development of pesticides at present.
The core of microbial control is screening effective biocontrol bacteria. At present, no record or report about the control of phytophthora capsici by using Burkholderia is available.
Disclosure of Invention
The invention aims to provide a burkholderia, a bactericide comprising the burkholderia, a bacterial fertilizer, a preparation method and application.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides Burkholderia (Burkholderia sp.) K2b with the preservation number as follows: CGMCC NO. 22002.
The invention also provides a bacterial agent containing the Burkholderia in the scheme.
Preferably, the effective viable count of the Burkholderia in the microbial inoculum is 1 multiplied by 108～8×1010CFU/ml。
The invention also provides a preparation method of the microbial inoculum in the scheme, which comprises the following steps:
and inoculating the burkholderia to an LB liquid culture solution, and performing fermentation culture to obtain the microbial inoculum.
Preferably, the conditions of the fermentation culture include: the OD value of the seed liquid of the Burkholderia is 1.2, the inoculation amount of the Burkholderia is 5% -10% of the volume percentage of the LB liquid culture liquid, the pH value of the LB liquid culture liquid is 7.0, the culture temperature is 28-30 ℃, the shaking table oscillation rate is 180-200 rpm, and the culture time is 60-80 h.
The invention also provides a bacterial fertilizer which comprises an organic fertilizer and the microbial inoculum of the scheme or the microbial inoculum obtained by the preparation method.
The invention also provides application of the burkholderia, the microbial inoculum obtained by the preparation method or the bacterial manure in preventing and treating pepper phytophthora blight and/or promoting pepper growth.
Preferably, the application comprises the following steps:
and applying a microbial inoculum or a bacterial fertilizer containing the Burkholderia during the seedling stage and/or the adult stage of the pepper.
Preferably, when the microbial inoculum is applied, the application mode comprises root irrigation, the application amount of each pepper is 50-100 mL of the microbial inoculum, the application times are 2-3 times, and the interval time between every two applications is 8-12 days.
Preferably, the microbial inoculum is applied for 2-3 times in the seedling stage and the adult stage of the pepper.
The invention has the beneficial effects that: the invention provides Burkholderia (Burkholderia sp.) K2b with the preservation number as follows: CGMCC NO. 22002. The Burkholderia K2b has a good control effect on phytophthora capsici by generating active substances to inhibit the growth of phytophthora capsici, and the inhibition rate can reach 88.53% at most. Meanwhile, the method can effectively colonize roots, stems and leaves of the peppers, improve the micro-ecological environment of crops and promote the growth of the peppers. The pesticide effect result of the indoor pot culture shows that the K2b fungicide has the control effect of 85.64 percent on pepper diseases and is not easy to generate drug resistance. When in use, the root irrigation is carried out at the seedling stage or the adult stage of the pepper. The microbial inoculum can also be mixed with organic fertilizer to prepare bacterial fertilizer for use. The strain K2b is obtained from the soil around the roots of plants, is harmonious and compatible with the soil ecology, is nontoxic and non-pathogenic, and therefore has good practical application value. The Burkholderia K2b has simple culture conditions, is easy for industrial production, and has good development and application prospects.
Biological preservation Instructions
Burkholderia (Burkholderia sp.) K2b was deposited at the China general microbiological culture Collection center on 12.03.12.2021, with the address of the institute of microbiology, China academy of sciences, No. 3, North West Lu 1 institute of Western province, North Cheng, the area of facing Yang, in Beijing, and the deposit numbers are: CGMCC NO. 22002.
Drawings
FIG. 1 shows the colony morphology of Burkholderia K2 b;
FIG. 2 shows the microstructure of Burkholderia K2 b;
FIG. 3 is a phylogenetic tree of Burkholderia K2b and the gyrB sequence of related species; adopting MEGA5.0 software, adopting an ortho-position connection method to display a systematic development tree of K2b and a 16S rDNA sequence of a related species, carrying out similarity repeated calculation for 1000 times, wherein the development tree nodes only display Bootstrap values larger than 50% numerical values, and the superscript 'T' represents a model strain;
FIG. 4 shows the inhibition of P.capsici growth by B.berghei K2 b; note: wherein A is CK, B is K2B fermentation liquor treatment;
FIG. 5 shows the colonization of Burkholderia K2b on the pepper roots;
FIG. 6 shows that Burkholderia K2B significantly promoted pepper growth, where A was Burkholderia K2B treatment and B was CK fermentation medium treatment.
Detailed Description
The invention provides Burkholderia (Burkholderia sp.) K2b with the preservation number as follows: CGMCC NO. 22002.
In the invention, the Burkholderia K2b is cultured for 15h at 36 ℃ in a TSA culture medium, and the colony is milky, round, opaque, wet in surface and neat in edge; when the TSA culture medium is cultured for 18 hours at 36 ℃, the thalli are in short rod shapes, 0.5-0.8 mu m multiplied by 0.8-1.8 mu m, are arranged singly or in pairs and are gram-negative.
The burkholderia K2b is separated from the root and stem junction of the green soy beans planted in suburbs of Zhangzhou dragon, Fujian, China, and is harmoniously compatible with soil ecology, thereby being beneficial to fully exerting the advantages of the strains.
The invention also provides a bacterial agent containing the Burkholderia in the scheme. In the present invention, the effective viable count of Burkholderia in the microbial agent is preferably 1X 108～8×1010CFU/ml。
The invention also provides a preparation method of the microbial inoculum in the scheme, which comprises the following steps:
and inoculating the burkholderia to an LB liquid culture solution, and performing fermentation culture to obtain the microbial inoculum.
In the present invention, the conditions of the fermentation culture preferably include: the pH value of the LB liquid culture solution is 7.0, the culture temperature is 28-30 ℃, the shaking speed of a shaking table is 180-200 rpm, and the culture time is 60-80 h. In the present invention, the time for the culture is preferably 72 hours. In the invention, the inoculation amount of the Burkholderia is preferably 5-10% of the volume fraction of the LB liquid culture solution; the OD value of the seed liquid containing burkholderia was 1.2.
The invention also provides a bacterial fertilizer which comprises an organic fertilizer and the microbial inoculum of the scheme or the microbial inoculum obtained by the preparation method.
In the invention, the organic fertilizer preferably comprises an agricultural waste organic fertilizer and/or a livestock and poultry manure organic fertilizer; the mass ratio of the microbial inoculum to the organic fertilizer is preferably (5-10): 1000, more preferably 8: 1000. the bacterial fertilizer is obtained by mixing an organic fertilizer and a microbial inoculum, and the invention has no special requirement on the mixing mode and takes uniform mixing as the standard.
The invention also provides application of the burkholderia, the microbial inoculum obtained by the preparation method or the bacterial manure in preventing and treating pepper phytophthora blight and/or promoting pepper growth.
In the present invention, the preferred application comprises the following steps:
and applying a microbial inoculum or a bacterial fertilizer containing the Burkholderia during the seedling stage and/or the adult stage of the pepper.
In the invention, when the microbial inoculum is applied, the application mode comprises root irrigation, the application amount of the microbial inoculum of each pepper is 50-100 mL, the application frequency is 2-3 times, and the microbial inoculum is preferably applied for 2-3 times in the seedling stage and the adult stage of the pepper. The interval time between every two applications is 8-12 days, and further preferably 10 days.
In the invention, when bacterial manure is applied, the application mode of the bacterial manure is preferably hole application, the application amount of the bacterial manure per capsicum annuum is 1-1.5 jin per capsicum annuum, the application frequency is preferably 2 times, and the bacterial manure is further preferably applied once in each seedling stage and adult stage of the capsicum annuum.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without any inventive step, are within the scope of the present invention.
Example 1 isolation, screening and characterization of Burkholderia K2b Strain
Isolation of the Strain
Test materials: collected from the root-stem junction of green soy beans planted in suburbs of Zhangzhou dragon sea city, Fujian, China.
Soaking the joint of the roots and stems of the green soybean in 75% alcohol for 30s on a clean bench, rinsing with sterile water for 3 times, soaking with 0.1% mercuric chloride for 1min, soaking in 75% alcohol for 30s, and rinsing with sterile water for 5 times. Weighing 1g of tissue block with a sterilized surface, soaking in L0mL phosphate buffer solution, grinding in a sterile mortar, standing for 5min, taking supernatant, performing gradient dilution by 10 times with sterile water, respectively taking 100 mu L of stock solution and diluent, coating the stock solution and the diluent on an LB culture medium plate, and culturing at 28 ℃ for 2-3 d until a single colony grows out. After the colony grows out, strain purification is carried out on an LB culture medium plate by a line drawing method.
II, identification of the strains
(I) morphological characteristics of the Strain
Colony morphology feature observation (see fig. 1 and 2): culturing in a TSA culture medium at 36 deg.C for 15 hr to obtain milky, round, opaque, wet surface and regular edge colony; when the TSA culture medium is cultured for 18 hours at 36 ℃, the thalli are in a short rod shape, 0.5-0.8 mu m multiplied by 0.8-1.8 mu m, are arranged singly or in pairs and are gram negative.
(II) analysis of gyrB sequence of Strain
gyrB sequence analysis of Strain K2 b: using gyrB primer gyrB-F
(GAAGTCATCATGACCGTTCTGCA, shown in SEQ ID NO: 1) and gyrB-R (AGCAGGGTACGGATGTGCGAGCC, shown in SEQ ID NO: 2) amplified the gyrB gene of the bacteria. The PCR reaction program is: pre-denaturation at 94 ℃ for 3min, followed by 94 ℃, 55s, 50 ℃, 50s, 72 ℃, 1min, 35 cycles, and finally 72 ℃, l0 min. The PCR amplification product was detected by 1.2% agarose gel electrophoresis. After the PCR product is recovered, cloning the PCR product to a T vector, selecting positive clone and sending the positive clone to Shanghai biological engineering company for sequencing, and logging and comparing and analyzing sequencing results in GenBank.
The experimental result shows that the gyrB gene segment of the strain K2b is 1087bp, and the BLAST ratio shows that the result has more than 99 percent of homology with the gyrB sequence of most Burkholderia strains. Therefore, the strain of the invention is identified as Burkholderia (Burkholderia sp.) through the analysis of colony morphology, gyrB sequence analysis and the like of the strain, the strain is numbered as K2b, and the strain is preserved as Burkholderia sp (Burkholderia sp.) CGMCC No.22002, the preservation unit is as follows: china general microbiological culture Collection center, preservation Address: the institute of microbiology, national academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, Beijing.
The nucleotide sequence of the gyrB segment is shown as SEQ ID NO: 3, specifically:
example 2 preparation of biocontrol Burkholderia K2b microbial inoculum
Activating Burkholderia K2b preserved at 4 ℃ on an LB plate culture medium, and culturing at 28 ℃ for 24h to obtain the activated strain. The activated strain K2b was selected with an inoculating loop and inoculated into 100mL of LB medium, and cultured at 28 ℃ and 220rpm for 24 hours to prepare a seed solution. Inoculating Burkholderia K2b seed solution (OD value of the Burkholderia K2b seed solution is 1.2) into LB liquid culture solution according to the inoculation amount of 5% by volume fraction, culturing at 28 ℃, and culturing at 180rpm for 72h to obtain biocontrol bacteria fermentation liquid, namely biocontrol bacteria agent, wherein the total viable bacteria concentration of the bacteria agent obtained by fermentation is 1 × 108～8×1010CFU/ml。
Example 3 biocontrol Burkholderia K2b antagonistic assay
A plate confronting culture method is adopted to perform antagonistic determination on the phytophthora capsici by the strain K2b, firstly, a phytophthora capsici mycelium block is connected to the center of a PDA culture medium plate, when the colony of the phytophthora capsici grows to 3cm, the strain K2b is connected to 4 weeks of the colony, the width of a bacteriostatic band of the strain K2b on the phytophthora capsici is measured after the colony is cultured for 5 days at the temperature of 28 ℃, the experimental result is shown in figure 4, and the result shows that the strain K2b has better antagonistic effect on the phytophthora capsici and the suppression rate reaches 90.00%.
Example 4 potted control of Phytophthora capsici by biocontrol Burkholderia K2b fermentation broth
The pepper variety is sweet pepper, and the experimental place is the perennial pepper epidemic disease occurrence place. After the seeds are sowed and the seedlings are grown for 7-10 days,each seedling is watered with 50ml of K2b strain fermentation liquor (8 multiplied by 10)8CFU/ml), watering once more 100ml strain fermentation liquor in the flowering period. A50% anc wettable powder 800-fold liquid (purchased from Hanbang plant protection agent, Limited liability company, Tianjin) is used for root irrigation treatment as a pesticide control, and a fermentation medium and a clear water treated plant are used as a blank control. Each treatment was 10 seedlings, 3 replicates per treatment. After the disease occurs, the disease condition is investigated every day, the disease incidence and the disease index are counted, and the experiment is ended until the disease incidence of the clear water contrast reaches more than 80%.
The disease grading criteria are as follows:
level 0: the inoculated part is unchanged or slightly browned;
level 1: the inoculation part generates scabs, and the area of the scabs accounts for less than 1/4 of the area of the stem;
and 3, level: the lesion area of the inoculation part accounts for about 1/4-1/2 of the total area of the stem part;
and 5, stage: the lesion at the inoculation part occupies more than 1/2 of the area of the stem part;
and 7, stage: the disease spots of the inoculated part are connected and form a stem winding phenomenon, but the plant does not wither or die;
and 9, stage: the plant will wither or die.
Disease index (%) [. sigma (number of disease plants at each stage x disease value)/(number of total investigated plants x highest value) ]. times.100%
The control effect (%) is [ (control disease index-treatment disease index)/control disease index ] × 100%
(control refers to clear water control treatment).
See fig. 5, fig. 6 and table 1 for experimental results. FIG. 5 shows the colonization of the capsicum root by Burkholderia K2 b; FIG. 6 shows that Burkholderia K2b significantly promoted pepper growth. Table 1 shows that the fermentation liquor of the actinomycete K2b to be tested can obviously reduce the disease index of the pepper phytophthora blight, the control effect is up to 85.64 percent, and the control effect is 80.81 percent higher than that of 800 times of chemical pesticide 50 percent Anke wettable powder. In addition, as can be seen from table 1, the disease index of the fermentation medium treatment is slightly lower than that of the clear water control treatment, but the difference between the two is not significant. The greenhouse pot experiment result shows that the K2b strain can effectively prevent and treat pepper phytophthora blight and shows good application potential.
TABLE 1 potted plant control of phytophthora capsici by biocontrol Burkholderia K2b fermentation broth
| Treatment of | Disease index% | The control effect is% |
| K2b fermentation broth | 11.27±2.18c | 85.64％ |
| CK1 (50% Anke wettable powder 800 times liquid) | 16.56±3.21b | 80.81％ |
| CK2 (fermentation medium) | 80.23±2.12a | -- |
| CK3 (Water) | 81.25％±2.25a | -- |
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> institute of plant protection of academy of agricultural sciences of Fujian province
<120> Burkholderia, microbial inoculum comprising Burkholderia, bacterial fertilizer, preparation method and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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gaagtcatca tgaccgttct gca 23
<210> 2
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
agcagggtac ggatgtgcga gcc 23
<210> 3
<211> 1087
<212> DNA
<213> Klebsiella pneumoniae (Klebsiella pneumoniae)
<400> 3
cgtctcggtg gttaacgccc tgtcacagaa gctggagctg gttatccagc gcgataacaa 60
agttcacaag cagatgtacg aacacggtgt gccgcaggca ccgctggccg tgactggcga 120
aaccgacaaa accggcacca tggtgcgttt ctggccaagc ctggaaacct tcaccaatgt 180
caccgaattt gaatatgaca tcctggcgaa acgcctgcgc gagctgtcgt tcctgaactc 240
cggcgtctct atccgcctgc gcgataagcg cgatggcaaa gaagaccatt tccactacga 300
aggcggcatc aaggcgttcg ttgagtatct caacaagaac aaaacgccga tccacccgaa 360
tattttctat ttctccaccg aaaaagacgg tatcggcgtg gaagtggccc tgcagtggaa 420
cgacggcttc caggaaaata tctactgctt taccaacaac attccgcagc gtgacggcgg 480
tactcacctc gccggcttcc gcgcggcgat gacccgtacc cttaacgcct atatggacaa 540
agagggctac agcaaaaaag cgaaagtcag cgccaccggc gacgatgcgc gtgaaggcct 600
gattgccgtg gtatccgtga aggtgccgga tccgaaattc tcctcgcaga ccaaagacaa 660
gctggtctcc tccgaggtga aatcggcggt ggagcagcag atgaacgaac tgctgagcga 720
atacctgctg gaaaacccat ccgatgcgaa aatcgtggtc ggcaaaatta ttgacgccgc 780
gcgcgcccgt gaggccgcac gtcgcgctcg tgagatgact cgtcgtaaag gcgcgctgga 840
tctggcaggc ctgccgggca aactggcgga ttgccaggag cgcgacccgg cgctgtccga 900
actgtacctg gtggaagggg actccgcggg cggctctgct aaacagggtc gtaaccgcaa 960
gaaccaggcg atcctgccgc tgaagggtaa aatccttaac gttgagaaag cccgcttcga 1020
taaaatgctc tcttcccagg aagtggcgac gctgatcacc gcgctgggct gcggtatcgg 1080
tcgcgat 1087
Claims (10)
1. Burkholderia (Burkholderia sp.) K2b with the collection number: CGMCC NO. 22002.
2. A bacterial agent comprising the Burkholderia bacterium according to claim 1.
3. The microbial preparation according to claim 2, wherein the effective viable count of Burkholderia in the microbial preparation is 1X 108～8×1010CFU/ml。
4. A method for producing the microbial agent according to claim 2 or 3, comprising the steps of:
and inoculating the burkholderia to an LB liquid culture solution, and performing fermentation culture to obtain the microbial inoculum.
5. The method of claim 4, wherein the conditions of the fermentation culture comprise: the OD value of the seed liquid of the Burkholderia is 1.2, the inoculation amount of the Burkholderia is 5% -10% of the volume percentage of the LB liquid culture liquid, the pH value of the LB liquid culture liquid is 7.0, the culture temperature is 28-30 ℃, the shaking table oscillation rate is 180-200 rpm, and the culture time is 60-80 h.
6. A bacterial fertilizer, which comprises an organic fertilizer and the microbial inoculum according to claim 2 or 3 or the microbial inoculum prepared by the preparation method of any one of claims 4 to 5.
7. Use of the burkholderia according to claim 1, the microbial inoculum according to claim 2 or 3, the microbial inoculum obtained by the preparation method according to any one of claims 4 to 5 or the bacterial manure according to claim 6 for controlling pepper phytophthora blight and/or promoting pepper growth.
8. The application according to claim 7, characterized in that it comprises the following steps:
and applying a microbial inoculum or a bacterial fertilizer containing the Burkholderia during the seedling stage and/or the adult stage of the pepper.
9. The use of claim 8, wherein when the microbial inoculum is applied, the application mode comprises root irrigation, the application amount of each pepper is 50-100 mL microbial inoculum, the application times are 2-3 times, and the time interval between every two applications is 8-12 d.
10. The use as claimed in claim 9, wherein the fungicide is applied 2-3 times in a seedling stage and an adult stage of pepper.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014065589A1 (en) * | 2012-10-23 | 2014-05-01 | 한국화학연구원 | Lysinibacillus sphaericus tc1 strain, microbial preparation for controlling plant diseases comprising said strain, and plant disease control method using same |
| CN107151641A (en) * | 2017-07-04 | 2017-09-12 | 沈阳农业大学 | One plant suppression Rhizoctonia solani Kuhn Brevibacillus laterosporus and its application |
| US20180092363A1 (en) * | 2016-10-05 | 2018-04-05 | Fmc Corporation | Bacillus thuringiensis rti545 compositions and methods of use for benefiting plant growth and controlling plant pests |
| US20210112815A1 (en) * | 2018-02-12 | 2021-04-22 | Boost Biomes, Inc. | Microbial compositions for the prevention or reduction of growth of fungal pathogens on plants |
-
2021
- 2021-05-20 CN CN202110554014.8A patent/CN113355262A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014065589A1 (en) * | 2012-10-23 | 2014-05-01 | 한국화학연구원 | Lysinibacillus sphaericus tc1 strain, microbial preparation for controlling plant diseases comprising said strain, and plant disease control method using same |
| US20180092363A1 (en) * | 2016-10-05 | 2018-04-05 | Fmc Corporation | Bacillus thuringiensis rti545 compositions and methods of use for benefiting plant growth and controlling plant pests |
| CN107151641A (en) * | 2017-07-04 | 2017-09-12 | 沈阳农业大学 | One plant suppression Rhizoctonia solani Kuhn Brevibacillus laterosporus and its application |
| US20210112815A1 (en) * | 2018-02-12 | 2021-04-22 | Boost Biomes, Inc. | Microbial compositions for the prevention or reduction of growth of fungal pathogens on plants |
Non-Patent Citations (7)
| Title |
|---|
| MAO SOPHEARETH等: "Biocontrol of Late Blight (Phytophthora capsici) Disease and Growth Promotion of Pepper by Burkholderia cepacia MPC-7", 《PLANT PATHOL. J.》 * |
| 任家琰等著: "《动物病原微生物学》", 31 March 2001, 中国农业科技出版社 * |
| 无: "NCBI Reference Sequence: MBC7176011.1", 《GENBANK》 * |
| 朱德旋等: "一株寒地高效解无机磷细菌的分离鉴定及拮抗作用", 《微生物学报》 * |
| 胡晓峰等: "一株溶磷抑病细菌的筛选及其溶磷特性", 《中国农业科学》 * |
| 葛红莲等: "根围细菌防治植物土传病害的研究进展", 《河南农业科学》 * |
| 郑作峰著: "《临床微生物实用技术》", 31 May 2019, 科学技术出版社 * |
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