CN115369064A - Development and application of multifunctional composite microbial inoculum for enhancing rhizosphere colonization and control effect of biocontrol bacillus belgii - Google Patents
Development and application of multifunctional composite microbial inoculum for enhancing rhizosphere colonization and control effect of biocontrol bacillus belgii Download PDFInfo
- Publication number
- CN115369064A CN115369064A CN202211218878.3A CN202211218878A CN115369064A CN 115369064 A CN115369064 A CN 115369064A CN 202211218878 A CN202211218878 A CN 202211218878A CN 115369064 A CN115369064 A CN 115369064A
- Authority
- CN
- China
- Prior art keywords
- hnu6
- hnu24
- bacillus
- microbial inoculum
- enhancing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/05—Fruit crops, e.g. strawberries, tomatoes or cucumbers
-
- 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
-
- 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
-
- 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
- A01P21/00—Plant growth regulators
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Environmental Sciences (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Botany (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Forests & Forestry (AREA)
- Virology (AREA)
- Ecology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Mycology (AREA)
- Medicinal Chemistry (AREA)
- Toxicology (AREA)
- Dentistry (AREA)
- General Engineering & Computer Science (AREA)
- Agronomy & Crop Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to development and application of a multifunctional compound microbial inoculum for enhancing rhizosphere colonization and control effect of biocontrol Bacillus belgii. The composite microbial inoculum consists of Bacillus belgii HNU24 and Bacillus frenkensis HNU6-13. The two strains are separated from rhizosphere soil of eggplant stocks, a metabolic cross-feeding relationship exists between the two strains, the Bacillus flexneri HNU6-13 can obviously enhance the biofilm formation of the Bacillus flexneri HNU24 and enhance the antagonistic effect of the Bacillus flexneri on the solanaceous bacteria, and the double-strain combination can stably colonize at the rhizosphere of tomatoes and can reduce the incidence rate of bacterial wilt under a synergistic effect. The composite flora can also obviously enhance the antagonistic effect on fusarium and phomopsis, and has certain biocontrol prospect in preventing and treating banana wilt and areca coconut leaf spot.
Description
Technical Field
The invention belongs to the field of agricultural microorganisms, and relates to development of a plant growth-promoting composite microbial inoculum based on rhizosphere biomembrane co-colonization of mixed flora metabolic intercropping and resistance excitation, which has application prospects in prevention and treatment of tomato bacterial wilt, banana wilt and areca coconut leaf spot.
Background
Crop bacterial wilt caused by Ralstonia solanacearum (Ralstonia solanacearum, called as Ralstonia solanacearum for short) is a worldwide plant disease, and new diseases caused by Ralstonia solanacearum are continuously reported, and statistics show that the bacterial wilt becomes the second most plant bacterial disease in the world. Bacterial wilt is one of the most serious diseases in agricultural production in China, and is reported in at least 30 provinces in China according to statistics, and especially when 90 kinds of crops such as tomatoes, eggplants, hot peppers, tobaccos, peanuts, gingers and the like exist in the provinces of the south of Yangtze river basin, serious losses are caused to economic crops such as the tomatoes, the eggplants, the hot peppers, the tobaccos and the like. Therefore, the deep research on the ralstonia solanacearum infection mechanism and the pathogenic regulation and control have important significance for the prevention and control of diseases.
The microorganism is a natural abundant resource, and in the aspect of agriculture, the microbial agent has the advantages of greenness, high efficiency, no pollution, no action, strong pertinence and the like. In recent years, synthetic biology has become a research focus in related fields, and two or more microorganisms are artificially combined for a certain purpose from natural microorganism resource banks to become a synthetic microorganism community having a certain specific function, following a certain design principle. Compared with a single microbial agent, the synthetic microbial community has high controllability and good stability. The use of synthetic microbial communities for agricultural purposes is at an initial stage and will become the main research direction for synthetic biology related aspects in the future.
Currently, in the agricultural field, bacillus is the most studied and developed microbial species, and more than 70% of microbial fertilizers registered in China belong to the same type. Most of the bacillus species are beneficial microorganisms, capable of secreting large amounts of different types of secondary metabolites, and which are capable of producing spores that more readily maintain activity in complex environments. Uses bacillus as a center, searches an excitant of the bacillus in a wide microbial resource library, amplifies the characteristics of the bacillus in the aspect of biocontrol, increases the colonization ability of the bacillus in plant rhizosphere, fully exerts strong growth-promoting and antibacterial activities of the bacillus, and has great significance for fully utilizing microbial preparations in the aspects of disease resistance and growth promotion.
In order to solve the problem of green prevention and control of bacterial wilt of solanaceae crops, the invention provides a bacterial liquid for preventing and controlling tomato bacterial wilt by applying a double-strain combination and an application method thereof. In addition, the combined flora is also found to have better antagonistic effect on pathogenic bacteria such as fusarium oxysporum causing banana wilt, phomopsis verticillata causing areca coconut leaf spot and the like. In view of the serious harm of banana wilt, bananas are important in economic crops and food crops, and meanwhile, betel nuts and coconuts are also important economic crops in Hainan areas, so that the invention has important application value.
Disclosure of Invention
The invention provides a Franke bacillus HNU6-13, characterized by that its strain preservation information: the name of the depository: guangdong province microbial strain preservation center; the address of the depository: "Zhou 59 building 5" of the institute for microbiology, guangzhou, ministry of sciences, guangdong province, junior 100, guangzhou; the preservation date is as follows: 30 months at 2022; the preservation number is: GDMCC No.62498; and (3) classification and naming: franconicobacter pumps.
Another embodiment of the invention provides the application of the Francisella HNU6-13 in preventing and treating tomato plant bacterial wilt and/or banana wilt. Enhancing the application of plant rhizosphere colonization.
Another embodiment of the invention provides the application of the Francisella HNU6-13 in the synergistic enhancement of the Bacillus belgii HNU24 in the prevention and treatment of tomato plant bacterial wilt and/or banana wilt. Enhancing the application of plant rhizosphere colonization.
Another embodiment of the invention provides a complex microbial inoculum, which is characterized in that the effective components of the complex microbial inoculum comprise Bacillus belgii HNU24 and Bacillus frenkensis HNU6-13. The bacterial quantity ratio is preferably HNU24: HNU6-13= 2.
Another embodiment of the present invention provides a complex microbial inoculum, which is characterized in that the preparation method of the complex microbial inoculum comprises the following steps:
re-suspending the cultured and obtained matrix-removed thallus HNU24 and HNU6-13 to bacterial liquid OD respectively with sterile water 600 And (3) when the temperature is 1.0-2.0, uniformly mixing according to the proportion of HNU24: HNU6-13= 2.
Another embodiment of the present invention provides a method for preparing the complex microbial inoculum, which is characterized by comprising the following steps:
re-suspending the cultured and obtained matrix-removed thallus HNU24 and HNU6-13 to bacterial liquid OD respectively with sterile water 600 And (3) mixing uniformly according to the volume ratio of HNU24 to HNU6-13=2 of 1 to 10, and thus obtaining the composite microbial inoculum.
The other embodiment of the invention provides application of the compound microbial inoculum in preventing and treating tomato plant bacterial wilt and/or banana wilt.
Another embodiment of the invention provides application of the complex microbial inoculum to enhancing plant rhizosphere colonization.
The "medium-removed cells HNU24 and HNU6-13 obtained by culture" according to the present invention are obtained by a conventional seed liquid culture method in the art, and can be obtained, for example, by the following method: respectively inoculating the seed liquid of the activated strains HNU24 and HNU6-13 to an LB liquid culture medium, and culturing for 24h at 28 ℃ and 200 rpm; centrifuging and washing to obtain the matrix-removed thallus. The preparation method of the LB liquid culture medium takes the preparation of 1L of culture medium as an example: 10g of peptone, 5g of yeast extract, 10g of sodium chloride, 15g of agar and the balance of water. Sterilizing at 121 deg.C for 20min.
The strain preservation information of the Bacillus belgii HNU24 is as follows: the name of the depository: guangdong province microbial strain preservation center; the address of the depository: "Zhou 59 building 5" of the institute for microbiology, guangzhou, ministry of sciences, guangdong province, junior 100, guangzhou; the preservation date is as follows: 10 and 15 days in 2020; the preservation number is: GDMCC No.61231; and (3) classification and naming: bacillus sp.
Compared with the prior art, the invention has the advantages that: according to the invention, the bacterial strain HNU24 screened from the rhizosphere of healthy tomato plants and the bacterial strain HNU6-13 screened from the rhizosphere of a rootstock with high resistance to bacterial wilt, namely Haima stock No. 3, find that the combination formed by the bacterial strains HNU6-13 and HNU24 can obviously reduce the incidence rate of the tomato bacterial wilt, and can jointly form a biomembrane structure with higher biomass and stably colonize the rhizosphere of tomatoes.
Drawings
FIG. 1 is a phylogenetic tree diagram of HNU24 and HNU 6-13;
FIG. 2 is a confocal drawing of a co-culture of HNU24 and HNU6-13, wherein red represents HNU6-13 and green represents HNU24;
FIG. 3 is a phenotype graph of the co-culture of HNU24 and HNU6-13 biofilms, MIX representing the results of the equal ratio mixing of the two strains;
FIG. 4 is a confocal drawing of a laser beam formed by mixed culture of HNU24 and HNU6-13, wherein red represents HNU6-13 and green represents HNU24;
FIG. 5 is a graph of crystal violet staining method for detecting biofilm formation in tomato rhizosphere;
FIG. 6 is a diagram of biocontrol experiments of tomato bacterial wilt antagonism by composite microbial inoculum, wherein group A represents clear water treatment; group B represents inoculation of forced pathogenic Serratia solani EP1; group C indicates inoculation of HNU24 followed by EP1; group D shows inoculation of HNU6-13 followed by EP1; group E indicates that HNU6-13 is inoculated after HNU24 is inoculated, and EP1 is inoculated at the same time;
FIG. 7 statistics of tomato seedling bacterial wilt incidence under different treatments;
FIG. 8 is a diagram showing the antagonistic effect of the complex microbial inoculum on banana wilt bacteria (the intermediate fungus is banana wilt bacteria strain FOC 4).
Detailed Description
EXAMPLE 1 isolation and identification of the Strain
The biocontrol strain is separated and purified from rhizosphere soil of a stock variety (Haima stock No. 3) with high resistance to the solanacearum EP1. Carefully taking out the rootstock seedling from the soil completely, shaking off the soil, cutting the root system into 1cm stem segments by using sterilized scissors, adding 10mL PBS buffer solution, grinding into homogenate, oscillating for 20min, and diluting into 10 -2 、10 -3 、10 -4 、10 -5 Different gradient suspensions are coated on LB solid medium and cultured for 48h at 28 ℃. Selecting strains with different forms, and purifying on a new LB solid culture medium until obtaining a pure culture, and storing in 20% glycerol at-80 ℃.
The obtained bacteria are named as HNU6-13, become faint yellow colonies and gram-negative bacteria on an LB plate, and are preserved in Guangdong province microbial strain preservation center with the preservation date of 2022 years, 5 months and 30 days, and the preservation number is GDMCC No:62498. 16S rDNA sequencing and sequence comparison are carried out, the strain is preliminarily identified as a strain of the genus Frankbacter, and the nucleic acid consistency rate is 99.39 percent in an comparison result table in an NCBI database. The results of the sequence clustering analysis are shown in FIG. 1.
The obtained bacterium is named as HNU24, forms a faint yellow colony on an LB plate, is a gram-positive bacterium, is currently preserved in Guangdong province microbial strain preservation center, has the preservation date of 2020, 10 and 15 days, and has the preservation number of GDMCC No:61231. and finally identifying the bacillus beleisis through whole genome sequencing and Sequence comparison, wherein the original Sequence of the genome sequencing is submitted to a Sequence Read Archive database with an authorization number: SRR 162165016500. The results of the sequence clustering analysis are shown in FIG. 1.
The preparation method of the PBS buffer solution comprises the following steps: weighing 8.0g NaCl, 0.2g KCl and 1.44g Na 2 HPO 4 、0.24g KH 2 PO 4 Dissolved in 800mL of distilled water, the solution was adjusted to pH =7.4 with HCl, and finally distilled water was added to a volume of 1L at a concentration of 0.01M.
The preparation method of the LB culture medium takes the preparation of 1L culture medium as an example: 10g of peptone, 5g of yeast extract, 10g of sodium chloride and 15g of agar. Sterilizing at 121 deg.C for 20min.
Example 2 Co-culture of Complex microbial inoculum biofilm
Construction of fluorescent-labeled Strain preparation of 1-1.5mL HNU24 bacterial solution (OD) 600 = 1.0), ice-cooling for 20min, centrifuging at 6000rpm at 4 ℃ for 5min, collecting the thallus, suspending with 20% glycerol solution, repeating for 3-5 times, and resuspending the thallus with 100 μ L of 20% to prepare competent cells. Adding 10 mu L of GFP fluorescent plasmid (green) into 100 mu L of competent cells, fully and uniformly mixing, adding mixed bacterial liquid into an electric shock cup with the diameter of 2mm, carrying out electric shock under the condition of 2.5KV, adding 500mL of LB liquid culture medium, culturing for 24 hours under the condition of 28 ℃ and 200rpm, uniformly coating the bacterial liquid on an LB flat plate containing Kan and Amp, carrying out static culture for 24 hours at 28 ℃, picking single colony shake bacteria, and detecting whether plasmid introduction is successful or not under a fluorescence microscope. The preparation method of the Mcherry fluorescent plasmid (red) transferred HNU6-13 fluorescent labeling strain is the same as the above.
Experiment setup 3 experimental groups: an HNU24 group (2. Mu.L of HNU24 bacterial liquid is added into the selected culture medium), an HNU6-13 group (2. Mu.L of HNU6-13 bacterial liquid is added into the selected culture medium), and a Mix group (2. Mu.L of double-bacterium mixed culture liquid is added into the selected culture medium, wherein HNU24: HNU6-13= 2. Repeating each group for 3 times, repeating each group for 3 holes, standing and culturing at 28 ℃ for 24 hours, photographing and recording, collecting the gas-liquid interface biomembrane, drying the biomembrane at 60 ℃, and measuring the dry weight of the biomembrane.
The fluorescence labeling strain biomembrane of the two bacteria is subjected to laser confocal observation, and the result shows that the two bacteria jointly form the biomembrane (figure 2). At the gas-liquid interface of the SOBG broth, bacillus belgii HNU24 and lactobacillus freeckii HNU6-13 formed a more robust biofilm with a twelve-fold increase in biomass compared to single bacteria (fig. 3, fig. 4). Indicating that the double bacteria can exist closely in the form of a biological membrane.
The preparation method of the SOBG culture medium comprises the following steps: peptone 20g, yeast extract 5g, mgSO 4 ·7H 2 O2.4g, naCl 0.5g, KCl 0.186g, glycerin 20mL.
Example 3 Co-cultivation of Complex microbial inoculum
And (3) streaking and activating HNU24 and HNU6-13 strains stored in a glycerin tube at the temperature of-20 ℃ on an LB solid culture medium flat plate, and culturing for 2 days in an incubator at the temperature of 28 ℃. Respectively picking single colonies in 3mL liquid LB test tubes, performing shake culture at 30 ℃ and 200rpm overnight, respectively taking 2mL culture bacteria liquid to enter 100mL LB liquid culture medium, performing culture at 30 ℃ and 200rpm, and taking the bacteria liquid to perform fluorescence confocal microscope observation after 7 days.
Example 4 rhizosphere co-colonization with Complex microbial Agents
The rhizosphere colonization of the composite microbial inoculum is carried out on tomatoes. Tomato seeds are subjected to surface disinfection by adopting a 2% sodium hypochlorite solution for 10min, soaked in 75% alcohol for 30s, washed by sterile distilled water for 5 times, placed on an MS plate, subjected to germination acceleration for 3 days at 28 ℃, and then transferred to a constant temperature box at 28 ℃ for culture for 7 days to take roots. 16h light, 8h dark. Selecting seedlings with root systems of 5cm, and adding MS liquid culture medium.
And (3) streaking and activating HNU24 and HNU6-13 strains stored in a glycerin pipe at the temperature of-20 ℃ on an LB solid culture medium flat plate, and culturing for 2 days in an incubator at the temperature of 28 ℃. Respectively picking single colony in 3mL liquid LB test tube, shake culturing at 30 deg.C and 200rpm overnight, centrifuging at 4 deg.C, collecting thallus, washing thallus with sterile water for 3 times, adjusting OD 600 And (3) when the temperature is 1.0, soaking the tomato seedlings in the bacterial suspension for 20min, then transplanting the tomato seedlings into a liquid MS culture medium, culturing for 7 days, taking out the tomato seedlings, washing the tomato seedlings with sterile water for 3 times to remove non-adhesive bacteria, soaking roots in a 0.1% crystal violet solution for 10min, washing the roots with flowing sterile water for 1min after taking out, shearing off the roots at the base parts of the tomato seedlings, placing the roots in a 2mL 75% ethanol solution for full oscillation, taking out roots, and measuring the absorbance value of the ethanol solution at the wavelength of 570 nm.
The results show (figure 5), the amount of biofilm formed by single bacteria of HNU24 and HNU6-13 in the rhizosphere of tomato seedlings is obviously lower than that of the complex microbial inoculum, and the capability of single bacteria in the form of biofilm colonization in the rhizosphere is weaker than that of double bacteria in complex.
Example 5 biocontrol experiment of plant probiotic complexing agent on bacterial wilt
Experimental setup experiments the following 5 experimental groups were set up: a: treating with clear water; group B: inoculating forced pathogenic Serratia solani EP1; group C: inoculating HNU24 and then inoculating EP1; group D: inoculating HNU6-13 and then inoculating EP1; group E: inoculating HNU6-13 (the inoculation amount of HNU6-13 is 1/10 of that of HNU 24) after inoculating HNU24, and simultaneously inoculating EP1.
Respectively activating HNU24 and HNU6-13 on an LB culture medium, selecting a single colony, and culturing the single colony in 5mL of liquid LB culture medium until the single colony is OD 600 To 1.0 as seed solution, inoculating 1mL seed solution into 500mL LB liquid culture medium, culturing at 28 deg.C and 200rpm for 24h, centrifuging at 6000rpm and 25 deg.C, collecting thallus, adding sterile water, and resuspending thallus to OD 600 To 2.0. Selecting tomato seedlings which grow well and grow uniformly, grouping according to experimental settings, repeating the tomato seedlings in each group by 3 times, and repeating the tomato seedlings by 10 times for treatment. Counting is started after the solanacearum EP1 is inoculated, the disease condition of tomato plants is observed every day, and disease indexes of the disease-affected tomatoes are graded according to the disease standard of bacterial wilt to judge the disease degree.
The results show (figure 6 and figure 7) that the incidence of the bacterial wilt in tomatoes can be reduced to a certain extent by using single bacillus beleisi HNU24 and bacillus frenkensis HNU6-13, while the incidence of the bacterial wilt can be reduced to a greater extent by using the double-bacterium compound biological agent, and the reduction range is more than 50%.
Example 6 antagonistic experiment of plant probiotic compound on banana wilt bacteria
Respectively activating HNU24 and HNU6-13 on LB culture medium, respectively selecting single colony to culture in 5mL liquid LB culture to OD 600 To 1.0. The banana fusarium oxysporum FOC4 is cultured on a PDA plate for 3 days respectively, then biocontrol bacteria are inoculated at the position 1cm away from the edge of fungal hyphae, and the antagonistic effect is observed after 48 hours, so that the antagonistic effect on the banana fusarium oxysporum under the mixed condition of HNU24 and HNU6-13 (HNU 24: HNU6-13= 5) is better than that of the single culture of HNU24, and HNU6-13 does not show the antagonistic activity on the banana fusarium oxysporum (figure 8).
Claims (7)
1. The Frankia furnacis HNU6-13 is characterized in that the strain preservation information is as follows: the name of the depository: guangdong province microbial strain preservation center; the address of the depository: the institute of microbiology, guangzhou, junior, midlu 100, lou 5, guangdong province academy of sciences; the preservation date is as follows: 30 months at 2022; the preservation number is as follows: GDMCC No.62498; and (3) classification and naming: franconicobacter pumps.
2. The use of the bacterium freudenreichii HNU6-13 of claim 1 for the control of tomato plant bacterial wilt and/or banana wilt. Enhancing the application of plant rhizosphere colonization.
3. Use of the freckles HNU6-13 of claim 1 for synergistically enhancing bacillus belgii HNU24 for the control of tomato plant bacterial wilt and/or banana wilt. Enhancing the application of plant rhizosphere colonization.
4. A complex microbial inoculum, characterized in that the effective components of the complex microbial inoculum comprise Bacillus belgii HNU24 and the Francisella HNU6-13 of claim 1. The bacterial quantity ratio is preferably HNU24: HNU6-13= 2. The name of the depository of the bacillus belgii HNU24: guangdong province microbial strain preservation center; the address of the depository: "Zhou 59 building 5" of the institute for microbiology, guangzhou, ministry of sciences, guangdong province, junior 100, guangzhou; the preservation date is as follows: 10 and 15 days in 2020; the preservation number is: GDMCC No.61231; and (3) classification and naming: bacillus sp.
5. The complex microbial inoculum according to claim 4, which is characterized in that the preparation method comprises the following steps: re-suspending the cultured and obtained matrix-removed thallus HNU24 and HNU6-13 to bacterial liquid OD respectively with sterile water 600 And (3) mixing uniformly according to the proportion of HNU24: HNU6-13=2 from 1 to 10, and obtaining the compound microbial inoculum.
6. The use of the complex microbial inoculum of any one of claims 4 to 5 in the control of tomato plant bacterial wilt and/or banana wilt.
7. Use of a complex bacterial agent as claimed in any one of claims 4 to 5 for enhancing rhizosphere colonization of plants.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211218878.3A CN115369064B (en) | 2022-09-30 | 2022-09-30 | Development and application of multifunctional composite microbial inoculum for enhancing rhizosphere colonization and control effect of biocontrol bacillus bailii |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211218878.3A CN115369064B (en) | 2022-09-30 | 2022-09-30 | Development and application of multifunctional composite microbial inoculum for enhancing rhizosphere colonization and control effect of biocontrol bacillus bailii |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115369064A true CN115369064A (en) | 2022-11-22 |
| CN115369064B CN115369064B (en) | 2023-07-07 |
Family
ID=84072917
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211218878.3A Active CN115369064B (en) | 2022-09-30 | 2022-09-30 | Development and application of multifunctional composite microbial inoculum for enhancing rhizosphere colonization and control effect of biocontrol bacillus bailii |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115369064B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114181865A (en) * | 2021-12-21 | 2022-03-15 | 海南师范大学 | Bacillus with efficient antagonistic action on serratia solanacearum and application thereof |
| CN117187077A (en) * | 2023-07-24 | 2023-12-08 | 广东省科学院南繁种业研究所 | Composite flora and application thereof, and control method of bacterial wilt of plants |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106635922A (en) * | 2017-01-22 | 2017-05-10 | 浙江农林大学 | Salt-tolerant biocontrol bacterium B268 for bacterial wilt of horsetail beefwood and application of salt-tolerant biocontrol bacterium B268 |
| CN108463549A (en) * | 2015-12-14 | 2018-08-28 | 巴斯夫欧洲公司 | The fermentation of enhancing |
| CN110777090A (en) * | 2019-10-17 | 2020-02-11 | 中国科学院上海高等研究院 | Kitchen waste treatment strain and application thereof |
| CN111040974A (en) * | 2019-12-31 | 2020-04-21 | 华南农业大学 | Banana endophytic Bacillus belgii and application thereof |
| CN112351688A (en) * | 2018-06-22 | 2021-02-09 | 巴斯夫欧洲公司 | Composition for animals and use thereof |
| CN112522144A (en) * | 2020-12-09 | 2021-03-19 | 华南农业大学 | Bumper-fragrant endogenous Bacillus belgii and application thereof |
| CN114181865A (en) * | 2021-12-21 | 2022-03-15 | 海南师范大学 | Bacillus with efficient antagonistic action on serratia solanacearum and application thereof |
| WO2022157213A1 (en) * | 2021-01-20 | 2022-07-28 | Inbiose N.V. | Production of oligosaccharides comprising ln3 as core structure in host cells |
-
2022
- 2022-09-30 CN CN202211218878.3A patent/CN115369064B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108463549A (en) * | 2015-12-14 | 2018-08-28 | 巴斯夫欧洲公司 | The fermentation of enhancing |
| CN106635922A (en) * | 2017-01-22 | 2017-05-10 | 浙江农林大学 | Salt-tolerant biocontrol bacterium B268 for bacterial wilt of horsetail beefwood and application of salt-tolerant biocontrol bacterium B268 |
| CN112351688A (en) * | 2018-06-22 | 2021-02-09 | 巴斯夫欧洲公司 | Composition for animals and use thereof |
| CN110777090A (en) * | 2019-10-17 | 2020-02-11 | 中国科学院上海高等研究院 | Kitchen waste treatment strain and application thereof |
| CN111040974A (en) * | 2019-12-31 | 2020-04-21 | 华南农业大学 | Banana endophytic Bacillus belgii and application thereof |
| CN112522144A (en) * | 2020-12-09 | 2021-03-19 | 华南农业大学 | Bumper-fragrant endogenous Bacillus belgii and application thereof |
| WO2022157213A1 (en) * | 2021-01-20 | 2022-07-28 | Inbiose N.V. | Production of oligosaccharides comprising ln3 as core structure in host cells |
| CN114181865A (en) * | 2021-12-21 | 2022-03-15 | 海南师范大学 | Bacillus with efficient antagonistic action on serratia solanacearum and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| GUNASEKARAN YAZHINI等: "Alleviation of Sodic Stress in Rice by Exploring the Exopolysaccharide-Producing Sodic-Tolerant Bacteria", 《AGRICULTURE》, vol. 12, no. 9 * |
| 曹宇等: "贝莱斯芽孢杆菌HNU24高效拮抗茄雷尔氏菌和促进植物生长活性的研究", 《海南师范大学学报(自然科学版)》, vol. 35, no. 1 * |
| 郝旭等: "不同条件对阪崎克罗诺肠杆菌生物被膜形成的影响", 《现代食品科技》, no. 10 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114181865A (en) * | 2021-12-21 | 2022-03-15 | 海南师范大学 | Bacillus with efficient antagonistic action on serratia solanacearum and application thereof |
| CN117187077A (en) * | 2023-07-24 | 2023-12-08 | 广东省科学院南繁种业研究所 | Composite flora and application thereof, and control method of bacterial wilt of plants |
| CN117187077B (en) * | 2023-07-24 | 2024-03-08 | 广东省科学院南繁种业研究所 | Composite flora and application thereof, and control method of bacterial wilt of plants |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115369064B (en) | 2023-07-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN115369064B (en) | Development and application of multifunctional composite microbial inoculum for enhancing rhizosphere colonization and control effect of biocontrol bacillus bailii | |
| Mansoori et al. | Evaluation of Pseudomonas and Bacillus bacterial antagonists for biological control of cotton Verticillium wilt disease | |
| CN111172080A (en) | Bacillus belgii and application thereof | |
| CN111394261B (en) | Saline-alkali-tolerant trichoderma asperellum and application thereof | |
| CN102154157B (en) | Bio-control strain 4AT8 capable of preventing and curing various bacterial diseases | |
| CN113322209B (en) | Plant growth-promoting composite microbial inoculum produced based on ecological relationship among strains and application thereof | |
| CN114196585B (en) | Burkholderia for preventing and treating tomato bacterial wilt and application thereof | |
| CN113528395B (en) | Myxococcus xanthus prey on tomato bacterial wilt and application thereof in biological prevention and control of tomato bacterial wilt | |
| CN102174431A (en) | Bio-control strain for preventing and controlling bacterial fruit blotch of watermelon | |
| CN107674850B (en) | Pseudomonadaceae fragrans Sneb811 for killing root-knot nematodes, metabolite and application | |
| CN107467075B (en) | Application of bacillus pumilus as rice growth promoter | |
| CN112899203A (en) | Composite flora for preventing and controlling fusarium oxysporum outbreak of corn seeds and application thereof | |
| Abdelzaher | Biological control of root rot of cauliflower (caused by Pythium ultimumvar. ultimum) using selected antagonistic rhizospheric strains of Bacillus subtilis | |
| CN102428964B (en) | Bio-control combined bacterium agent CB28 admixture for preventing cucumber downy mildew | |
| CN112442451B (en) | Endophytic mycorrhizal fungi for promoting growth of blueberries and application thereof | |
| CN113373069B (en) | Straw saprophytic fungus and application thereof | |
| CN112458021B (en) | Biocontrol pseudomonas fluorescens and culture method and application thereof | |
| CN110643538B (en) | Pseudomonas fluorescens XY2F4 and application thereof in preventing and treating crop verticillium wilt | |
| CN111893053B (en) | Pseudomonas fluorescens 22G5 and application of active ingredient 7-hydroxy tropolone thereof in preventing and treating crop verticillium wilt | |
| CN114480171A (en) | Rhizobium japonicum and application thereof | |
| CN114032182A (en) | Fungus with functions of antagonizing garlic root rot pathogenic bacteria and promoting growth | |
| CN1141383C (en) | Waxy bacillus and its prepn | |
| CN116555091B (en) | Salt-tolerant myxobacteria and application thereof | |
| CN111454848A (en) | Actinomucor elegans GD48, fermentation product, microbial inoculum and application thereof | |
| Phondekar et al. | Isolation and identification of native strains of Pseudomonas fluorescens and test their in vitro bio-efficacy against bacterial wilt of potato caused by Ralstonia solanacearum |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |