CN118086149B - Streptomyces fusiformis QH1-20 strain with broad-spectrum antagonistic bacteria and application thereof - Google Patents
Streptomyces fusiformis QH1-20 strain with broad-spectrum antagonistic bacteria and application thereof Download PDFInfo
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- CN118086149B CN118086149B CN202410485214.6A CN202410485214A CN118086149B CN 118086149 B CN118086149 B CN 118086149B CN 202410485214 A CN202410485214 A CN 202410485214A CN 118086149 B CN118086149 B CN 118086149B
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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
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- 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
- A01N63/28—Streptomyces
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- 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
- C12R2001/465—Streptomyces
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
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- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
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- General Engineering & Computer Science (AREA)
- Pest Control & Pesticides (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- Dentistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Agronomy & Crop Science (AREA)
- Mycology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The application relates to the technical field of biological control of plant diseases, and particularly discloses a broad-spectrum antagonistic bacterium streptomyces fusiformis QH1-20 and application thereof; the streptomyces spindle QH1-20 is preserved in China general microbiological culture Collection center, address: the collection number of the institute of microbiology of China, national academy of sciences, no. 3, north Star West Lu, no. 1, of the Chaoyang district of Beijing is CGMCC No.28733; also discloses a microbial biocontrol agent containing streptomyces fusiformis QH 1-20; the streptomyces fusiformis QH1-20 or the microbial biocontrol agent is applied to inhibiting plant pathogenic bacteria. The broad-spectrum antagonistic bacteria streptomyces spindle QH1-20 disclosed by the application has a wide antibacterial spectrum, shows remarkable antagonistic activity on various plant diseases, provides a high-efficiency and stable disease prevention effect, is wide in application and has huge application potential.
Description
Technical Field
The application relates to the technical field of biological control of plant diseases, in particular to a broad-spectrum antagonistic bacterium streptomyces spindle QH1-20 and application thereof.
Background
With the gradual reduction of agricultural land area and the continuous increase of agricultural product demand in China, the improper production management method causes various problems such as continuous cropping obstacle of plants, worsening of soil conditions, improvement of resistance of pathogenic bacteria to pesticides and the like. These factors act together to cause diseases to frequently occur in agricultural product planting areas, so that the yield of crops is seriously affected, and the agricultural product is a major factor threatening agricultural production in China.
Currently, the control of plant diseases mainly depends on the use of chemical pesticides and the screening of disease-resistant varieties. The disease-resistant varieties have the problems of long breeding period, multiple disease varieties, quick updating and the like, and are faced with a plurality of continuously changing disease varieties, so that the application of the disease-resistant varieties is limited. The long-term and excessive use of chemical pesticides and fertilizers easily causes a series of environmental problems such as soil pollution, soil hardening, pesticide residues and the like, which not only endanger human health, but also threaten the safety of ecological environment. Therefore, the search for durable, low-toxicity and environmentally friendly control strategies has become a future development direction for plant disease control.
Biological control of plant diseases is a hotspot of current plant disease management due to its long-term efficacy and environmental friendliness. In the prior art, various biocontrol strains are reported, but the existing biocontrol strains have the problems of unstable disease prevention effect, narrow bacteriostasis spectrum and the like in the practical application process, and the problems severely limit the wide application of the existing biocontrol strains. In view of the richness of microbial resources in China, the development and screening of the biocontrol strain which is high in efficiency, stress resistance and broad-spectrum antibacterial activity can provide key technical support and resource basis for the prevention and control of plant diseases and the sustainable development of agriculture. Based on the above statement, the present application provides a broad-spectrum antagonistic bacterium Streptomyces fusiformis QH1-20 and its application.
Disclosure of Invention
The application provides a broad-spectrum antagonistic streptomyces fusiformis QH1-20 and application thereof, and aims to solve the problems that the existing biocontrol strain is unstable in disease prevention effect, narrow in bacteriostasis spectrum and the like in the practical application process.
In a first aspect, the application provides a broad-spectrum antagonistic bacterium Streptomyces fusiformis QH1-20, which adopts the following technical scheme:
A strain of broad-spectrum antagonistic bacteria streptomyces fusiformis QH1-20, said streptomyces fusiformis (Streptomyces netropsis) QH1-20 being deposited at the chinese microbiological bacterial deposit management committee common microbiological centre, 10 months 24, 2023, address: the collection number of the institute of microorganisms of China academy of sciences of China No. 3, north Star West Lu No. 1, the Korean region of Beijing is CGMCC No.28733.
In a second aspect, the application provides a microbial biocontrol microbial agent, which adopts the following technical scheme:
a microbial biocontrol agent comprising the broad-spectrum antagonistic bacterium Streptomyces fusiformis QH1-20 described in the first aspect.
Preferably, in the biocontrol agent, the streptomyces fusiformis QH1-20 exists in the form of cultured living bacteria, fermentation broth or bacterial suspension.
Preferably, the dosage form of the biocontrol microbial agent is wettable powder, water dispersing agent, water suspending agent or dispersible oil suspending agent.
In a third aspect, the application provides an application of a broad-spectrum antagonistic bacterium streptomyces spindle QH1-20 or a microbial biocontrol agent, which adopts the following technical scheme:
The use of a broad-spectrum antagonistic bacterium Streptomyces fusiformis QH1-20 as described in the first aspect or a microbial biocontrol agent as described in the second aspect described above for inhibiting plant pathogenic bacteria.
Preferably, the plant pathogenic bacteria include Botrytis cinerea (Botryosphaeria cinerea), rhizoctonia cerealis (Botryosphaeria berengerianade), brown rot of pear (Monilinia fructigena), black spot of pear (ALTERNARIA ALTERNATA), apple rot of apple (Cytospora mali QH 2), anthracnose of capsicum (Colletotrichu scovillei LJ 1), root rot of matrimony vine (Fusarium solani), phytophthora capsici (Phytophthora CAPSICI SP), root rot of carrot (Fusarium solani WQ 1), bakanae disease of rice (Fusarium moniliforme), etc.
Preferably, the application mode comprises seed soaking, seedling planting, root irrigation, plant spraying or fruit spraying of the agricultural products to be treated by adopting the microbial biocontrol microbial inoculum.
Preferably, the agricultural products to be treated comprise pears, apples, medlar, peppers, carrots, rice and the like.
In summary, the application has the following beneficial effects:
The broad-spectrum antagonistic bacteria streptomyces fusiformis QH1-20 disclosed by the application has remarkable antagonistic activity on various plant diseases, has a broad antibacterial spectrum, is efficient and stable in disease prevention effect, and is wide in application range and has a certain application potential.
Drawings
FIG. 1 is a diagram showing the growth state of strain QH1-20 in the modified Gaoshi first medium according to example 1 of the present application;
FIG. 2 is a graph showing the growth state of QH1-20 strain on different carbon sources in example 1 of the present application;
FIG. 3 is a physiological and biochemical profile of strain QH1-20 of example 1 of the present application;
FIG. 4 is a 16S rDNA sequence construction evolutionary tree of the QH1-20 strain of example 1 of the present application;
FIG. 5 is a graph showing the growth state of QH1-20 strain of example 2 according to the present application under different NaCl concentrations and pH conditions;
FIG. 6 is a graph showing the in-dish antagonistic activity of QH1-20 strain in example 3 according to the application against 10 pathogenic bacteria to be tested;
FIG. 7 is a graph showing the in vivo antagonistic activity against 6 diseases of QH1-20 strain in example 4 according to the application.
Detailed Description
The invention will be further elaborated in connection with the drawings and the specific embodiments described below, which are intended to illustrate the invention only and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
The pathogenic bacteria to be tested, the culture medium and the instrument and equipment related in the embodiment of the application are as follows:
1. test pathogenic bacteria
The botrytis cinerea (Botryosphaeria cinerea), the Rhizoctonia cerealis (Botryosphaeria berengerianade), the brown rot of pear (Monilinia fructigena) and the black spot of pear (ALTERNARIA ALTERNATA) are separated and identified by the institute of fruit trees of China academy of sciences (Sun et al 2017; sun Ping is equal, 2018);
Apple rot germ (Cytospora mali QH), pepper anthracnose germ (Colletotrichu scovillei LJ 1), medlar root rot germ (Fusarium solani) are isolated and identified by the laboratory (Ma Jiang, etc., 2020; sun Ping, etc., 2023);
phytophthora capsici (Phytophthora CAPSICI SP) is offered by the doctor of the agricultural university Zhou Jianbo, shanxi (Sun Ping et al, 2022);
Carrot root rot fungi (Fusarium solani WQ 1) are supplied by the institute of vegetable and flowers, inner Mongolia agricultural sciences (Han Fengying et al, 2020);
bakanae disease germ (Fusarium moniliforme) is supplied by the Shaanxi institute of microbiology Lu Meihuan doctor (Lu Meihuan et al 2020).
2. Culture medium
(1) Potato dextrose medium (PDA): 200g of potato extract, 20g of glucose, 20g of agar powder, 1000mL of distilled water and pH 7.2-7.4.
(2) Modified medium No. 1 gao: after the soluble starch 20g,NaCl 0.5g,KNO 3 1.0g,MgSO4·7H2O 0.5g,FeSO4 0.01.01 g, agar powder 20g, distilled water 1000mL and pH 7.2-7.4 and the culture medium are sterilized, 3.3mL of 3% potassium dichromate is added after the temperature is reduced to 50-55 ℃.
(3) Millet liquid medium: 10g of millet is boiled in distilled water for 20min, the filtered supernatant is fixed to 1000mL by distilled water, and 1g of glucose and 0.3g,NaCl 0.25g,CaCO 3 0.02.02 g of peptone are added.
(4) V8 agar medium (phytophthora capsici c spore-producing medium): v8 juice 200mL, caCO 3 g, agar 15g, distilled water 800mL.
(5) Carbon source utilization medium: firstly, preparing a basic culture medium according to 20g of (NH4)2SO4 2.64g,K2HPO4 5.65g,MgSO4·7H2O 1.0g,CuSO4·5H2O 0.0064g,FeSO4·7H2O 0.0011g,MnCl2·4H2O0.0079g,ZnSO4·7H2O 0.0015g, agar powder, and respectively adding 100g of lactose, galactose, xylose, maltose, starch, glucose or fructose serving as a carbon source, 1000mL of distilled water and pH of 7.2-7.4.
(6) V-P liquid medium:
V-P liquid medium: glucose 5g, K 2HPO4 g, peptone 5g, deionized water 1000mL, split charging (5 mL per tube), and sterilizing.
V-P reagent: and (3) solution A: 6.0g of alpha-naphthol, adding absolute ethyl alcohol for dissolution, and fixing the volume to 100ml, wherein solution B is: 40g of potassium hydroxide and distilled water are added for dissolution, and the volume is fixed to 100ml.
(7) Starch hydrolysis medium: 10g of peptone, 3g of beef extract powder, 30g of soluble starch, 15g of agar powder, 1000mL of deionized water and 7.6+/-0.1 of pH value.
(8) H 2 S Medium: beef extract 3g, yeast extract 3g, peptone 10g, feSO 4 0.2.2 g, naCl 5g, thiosulfate 0.3g, agar 12g, and distilled 1000mL of water.
(9) Gelatin liquefaction medium: 5g of peptone, 3g of beef extract powder, 120g of gelatin, 3 g of KNO (distilled water) and 1000mL of distilled water.
(10) Nitrate reduction medium
Nitrate-reducing liquid medium: potassium nitrate 0.2g, protein 5g dissolved in 1000mL distilled water, pH7.4, split charging test tube (5 mL per tube), 121 ℃ high pressure sterilization 15min.
Nitrate reducing agent: and (3) solution A: p-aminobenzenesulfonic acid 0.8g was dissolved in 2.5mol/L acetic acid solution 100mL, solution B: 0.5g of menadione was dissolved in 100mL of 2.5mol/L acetic acid solution.
3. Test instrument
An electric incubator (HPX-9162 MBE, shanghai Bo Xie Co., ltd.);
An ultra clean bench (SW-CJ-1 FD);
microscope (LEICA ICC50W, lycra instruments inc, germany);
electrophoresis apparatus (BG-Power 600K450W, beijing hundred Biotechnology Co., ltd.);
PCR instrument (624 BR47696, buret Bio-Rad Co., U.S.A.).
Example 1
Separation, screening and identification of streptomyces fusiformis QH1-20
1.1 Isolation and screening of strains
Collecting rhizosphere soil samples of the Qinghai matrimony vine orchard, removing surface soil of 5cm in the collecting process, collecting rhizosphere soil of 25cm, wrapping with kraft paper, and storing at 4 ℃.
Actinomycetes in the soil are separated by adopting a dilution plate coating method, and the specific separation and screening methods are as follows: 10g of the dried soil sample is ground and placed in 100mL of sterile water, the soil sample is diluted step by using the sterile water for 30min at 4 ℃ and 180r/min, 100 times of the diluted solution is sucked up, the diluted solution is uniformly coated on an improved Gao's first culture medium, and the soil sample is cultured for 2d at 28 ℃. Single bacterial colonies with different forms and colors are selected for streak purification, the purified bacterial strains are transferred into 20 percent glycerol and are preserved at the temperature of minus 20 ℃ for standby, and the separated and purified bacterial strains are named as QH1-20.
1.2 Identification of strains
1.2.1 Morphological observations and carbon Source utilization
The QH1-20 is coated on a modified Gaoshi first culture medium, and the growth state of the strain is observed and recorded after 2d culture at 28 ℃, and the result shows that the QH1-20 grows vigorously on the modified Gaoshi first culture medium, the aerial hyphae are in a velvet shape and yellow, and the mycelium in the medium is light yellow, and is shown in figure 1 in detail;
The method comprises the steps of coating QH1-20 on a carbon source utilization medium, observing and evaluating the utilization characteristics of xylose, lactose, starch, fructose, galactose, maltose and glucose, inoculating QH1-20 on different carbon source media by a streaking method, culturing for 2-4d at 28 ℃, observing and recording the characteristics of colony shape, transparency, color and the like, and the result shows that QH1-20 can grow vigorously on the medium with glucose, maltose and fructose as the sole carbon sources, and can also utilize starch, lactose, galactose and xylose, as shown in the figure 2.
1.2.2 Physiological Biochemical identification
(1) Gelatin liquefaction test
QH1-20 was inoculated into biochemical tubes containing gelatin liquefaction medium, the blank was not inoculated with strain, incubated at 28℃for 7d, and the biochemical tubes were placed in an environment at 4℃for 30min, if the contents did not solidify, positive reaction was observed, and if the contents did not solidify, negative reaction was observed.
(2) Nitrate reduction test
QH1-20 is inoculated in a nitrate reduction liquid culture medium, a blank culture medium is used as a reference, the culture is carried out for 7-14d at 28 ℃, after the culture is finished, 2-3 drops of nitrate reducing agent A liquid and 2-3 drops of nitrate reducing agent B liquid are respectively dripped, the color change is observed after a few seconds, and if the color changes to red, the positive reaction is obtained.
(3) Starch hydrolysis test
QH1-20 was inoculated onto a starch hydrolyzing medium and cultured at 28℃for 7d. The iodine drops are immersed on the surface of the culture medium, if the edge of the antagonistic bacteria point forms a transparent circle, the strain can produce amylase, and the larger the transparent circle is, the stronger the amylase producing capability of the strain is.
(4) H 2 S production test
QH1-20 was picked up and placed in a biochemical tube containing H 2 S medium and incubated at 28℃for 7d, and the surrounding colour was observed, if darkening indicated that H 2 S was produced.
(5) V-P test
Inoculating QH1-20 on the V-P liquid culture medium, dropwise adding V-P reagent (6 drops of A liquid and 2 drops of B liquid) after culturing, mixing uniformly, and continuing culturing for 0.5-4h, wherein the reddening within 4h is positive, and the color is not changed or the brown yellow is negative.
The physiological and biochemical identification results are shown in the following table 1 and fig. 3, and the results of all test groups in fig. 3 are: the left side is the blank control group and the right side is the QH1-20 group.
TABLE 1 physiological and biochemical characterization of Strain QH1-20
As can be seen from the results shown in table 1 and fig. 3: QH1-20 is capable of producing H 2 S, has nitrate reductase activity, but shows negative in starch hydrolysis, gelatin liquefaction and V-P assays.
1.2.3 Molecular sequence identification
QH1-20 is inoculated in millet liquid culture medium, shaking is carried out for 24 hours at 28 ℃ and 180R/min, 1ml of bacterial liquid is taken, centrifugation is carried out for 1min at 10000R/min, supernatant is discarded, ezup column type bacterial genome DNA extraction kit is used for extracting DNA, 7F (5'-AGAGTTTGATCCTGGCTCAG-3')/1492R (5'-GGTTACCTTGTTACGACTT-3') is utilized for amplifying 16S rDNA gene fragment, and the obtained PCR product is subjected to gel detection, and the sequence is recovered and sent to the sequencing of a worker. BLAST alignment is performed at NCBI after the detected sequences are spliced, and a Neighbor junction method is used for constructing an evolutionary tree, and the details are shown in FIG. 4.
The sequence alignment shows that QH1-20 and Streptomyces fusiformis (Streptomyces netropsis) are clustered together. Morphological characteristics, physiological and biochemical determination and molecular sequence identification result prove that QH1-20 is Streptomyces fusiformis (S.netropsis).
Through the separation, screening and identification, the application obtains the streptomyces fusiformis (Streptomyces netropsis) QH1-20 with stable and efficient disease prevention effect and broad-spectrum antagonistic bacteria, and the separated strain is preserved in the China general microbiological culture Collection center, address: the collection number of the institute of microorganisms of China academy of sciences of China No. 3, north Star West Lu No. 1, the Korean region of Beijing is CGMCC No.28733.
Example 2
Stability determination of Streptomyces fusiformis QH1-20
The growth of QH1-20 under different NaCl concentration and pH conditions was observed with the modified Gao's first medium as the basic medium. The concentration of the treated NaCl is 0.1%, 0.5%, 1.0%, 2.0%, 5.0%, 7.0%, 10.0% and 12.0% respectively; the pH was 5,6, 7, 8, 9, 10, 11, 12, respectively. Growth was observed and recorded after 5d and the results are shown in Table 2 below.
TABLE 2QH1-20 stability determination results
As can be seen from the results shown in table 2 and fig. 5: QH1-20 has higher salt-tolerant alkalinity. Salt tolerance measurement shows that QH1-20 can grow vigorously in a culture medium with 5% NaCl concentration, and a small amount of QH1-20 still grows in the culture medium with 7% NaCl, but the QH1-20 stops growing basically in 10% NaCl and above; the alkali resistance measurement shows that QH1-20 has high alkali resistance and can still grow normally in a culture medium with pH value of 12.
Example 3
In vitro bacteriostasis spectrum measurement of QH1-20
The antagonism effect of QH1-20 on Botrytis cinerea (Botryosphaeria cinerea), rhizoctonia cerealis (Botryosphaeria berengerianade), brown rot of pear (Monilinia fructigena), black spot of pear (ALTERNARIA ALTERNATA), anthracnose of capsicum (Colletotrichu scovillei LJ 1), phytophthora capsici (Phytophthora capsici), apple rot (Cytospora mali QH) and root rot of medlar (Fusarium solani), root rot of carrot (Fusarium solani WQ 1), bakanae disease of rice (Fusarium moniliforme) and 10 important fruit tree and crop plant pathogenic bacteria is measured by using a plate-in-dish counter method.
The specific test process is as follows: the QH1-20 biocontrol fungus cakes with the diameter of 6mm are picked by an inoculating needle and are inverted at the two sides of a PDA flat plate, after 2d of culture at 28 ℃,10 pathogen fungus cakes to be detected are respectively inoculated at the center of the flat plate; treatments with pathogenic bacteria inoculated only in the center of the plate were used as controls, and each treatment was repeated three times and incubated at 28 ℃. And when the control colony grows to be full of the flat plate, observing and recording the diameter of the inhibition zone, and calculating the inhibition rate.
Antibacterial ratio (%) = (1-antagonistic treatment plaque radius/control plaque radius) ×100%.
The results of the in vitro bacteriostasis spectrum measurements are shown in Table 3 below.
TABLE 3 detection results of the control of QH1-20 against 10 test pathogens
As can be seen from the results shown in table 3 and fig. 6: QH1-20 has remarkable inhibition effect on the growth of 10 tested pathogenic bacteria hyphae, and the inhibition zone reaches 3.4-11.01mm, so that the inhibition effect is obvious.
Example 4
Living body bacteriostasis spectrum determination of QH1-20
4.1QH1-20 preparation of fermentation broth
Selecting a small amount of QH1-20 thalli, inoculating the thalli into 200mL of millet culture medium, culturing for 2d at 28 ℃ and 180rpm to obtain antagonistic bacteria fermentation liquor, and diluting the antagonistic bacteria fermentation liquor to 1X 10 6 cfu/mL with sterile water for potting anti-effect treatment of phytophthora capsici.
4.2QH1-20 in vivo control treatment of Botrytis cinerea, rhizoctonia cerealis and brown rot of pear
Soaking healthy and fresh Huangguan pears in sodium hypochlorite with the mass concentration of 2% for 3min, washing with tap water and airing. Spraying antagonistic bacteria liquid on the wound of 5mm (diameter) x 3mm (depth) on the equatorial part of pear fruit by using a sterile puncher, respectively sticking 3 pathogenic bacteria cakes on the wound after 0.5h, and fixing by using a transparent adhesive tape to spray sterile water; yellow-crown pears inoculated with pathogenic bacterial cakes alone were used as controls, and each treatment was repeated 6 times. Sealing the treated fruits with a preservative film, placing the fruits in a 25 ℃ artificial climate box for 7d of humidification culture (controlling 16h illumination/8 h darkness), measuring the diameter of the disease spots, and calculating the bacteriostasis rate.
4.3QH1-20 in vivo control effect treatment of pepper anthracnose
Soaking healthy fresh chilli in sodium hypochlorite with the mass concentration of 2% for 3min, washing with tap water and airing. The pepper fruits are punctured by a sterile inoculation needle, antagonistic bacteria liquid is sprayed on the wound, and after 0.5h, pepper anthracnose bacteria cakes are stuck on the wound and fixed by transparent adhesive tapes, so that sterile water is sprayed; the peppers inoculated with the pathogenic bacterial cake alone were used as controls and each treatment was repeated 6 times. Placing in a climatic chamber at 25deg.C, culturing for 7d (controlling 16 hr light/8 hr darkness), measuring the diameter of the disease spot, and calculating antibacterial rate.
4.4QH1-20 potted plant anti-effect treatment of phytophthora capsici
Phytophthora capsici (PhytophthoracapsiciSP 90,90) is inoculated on a V8 agar culture medium, and after being cultured in the dark for 7 days in a 28 ℃ incubator, the zoosporangium is induced to generate by being cultured for 2 days in intermittent illumination (12 h illumination/12 h darkness). Placing 10mL of sterile water into a culture dish with sporangium, culturing in a 28 ℃ incubator under illumination for 30min, measuring spore concentration of the filtered zoospore liquid by using a hemocytometer, and finally diluting to 1X 10 5/mL to obtain phytophthora capsici spore suspension for later use.
Sterilizing the surface of the capsicum seed, then flushing with sterile water for 3 times, placing in a culture dish, and accelerating germination under constant temperature and dark light at 28 ℃. Seeds with bud length of 0.5cm are selected and inserted into seedling bowls, and the matrix is commercial nutrient soil and sterilized. And starting the test when the pepper seedlings grow to 3-4 leaves. And (3) suspending watering on the premise of ensuring healthy survival of the saplings 1d before inoculation of pathogenic bacteria or antagonistic bacteria. A root-filling inoculation method is adopted, a hole with the depth of 4cm is pricked within a diameter range of 3cm from the root base of the seedling, 4mL of antagonistic bacteria fermentation liquor (1X 10 6 cfu/mL) is injected by a liquid-transferring gun, sterile water is used as a control for inoculation, and after 5 days of culture at 25 ℃, phytophthora capsici fungus spore suspension (1X 10 5 cfu/mL) is inoculated. The experiment was set up with a total of 2 treatments, CK (only inoculated with sterile water) +phytophthora, antagonistic fermentation broth+phytophthora. 6 pepper seedlings are treated in total. Controlling the temperature to 25-28deg.C and humidity to above 75%, observing and recording the length of basal stem lesion after 7d inoculation, and calculating antibacterial rate.
4.5QH1-20 treatment of apple rot with antagonistic activity
Selecting two-year-old healthy apple branches, shearing the healthy apple branches into about 10cm, soaking the healthy apple branches in sodium hypochlorite with the mass concentration of 2% for 7 minutes, washing the healthy apple branches with sterile water for three times, and airing the healthy apple branches. Sealing both ends with paraffin, making 6mm wound on the middle part of apple branch with sterile puncher, spraying antagonistic bacteria liquid onto branch uniformly, inoculating apple rot pathogen after 30min, wrapping with sterile absorbent cotton (soaked with sterile water), and sealing with preservative film. Spraying sterile water after injury and inoculating branches of apple rot germ cakes as positive control, inoculating branches of apple rot germ cakes as negative control, carrying out 16h illumination/8 h dark moisturizing culture on all branches at 25 ℃, removing cotton after inoculation for 5d, measuring the diameter of the disease spots after 14d, and calculating the bacteriostasis rate.
Antibacterial ratio (%) = (1-treatment plaque diameter/control plaque diameter) ×100%.
Antagonistic activities of QH1-20 on gray mold, ring spot, brown rot of pear, anthracnose of capsicum, phytophthora capsici of capsicum and rot of apple were calculated by using antibacterial rate (control effect), and specific results are shown in Table 4 below.
TABLE 4 Living Activity control results of QH1-20 on 6 diseases
As can be seen from the results shown in table 4 and fig. 7: QH1-20 shows remarkable antibacterial activity on diseases to be detected. The living body inhibition activity of the composite material on gray mold, ring spot and brown rot of pears reaches 74% or above, and the composite material does not attack after pepper anthracnose, phytophthora capsici and apple rot are inoculated on pepper and apple branches treated by the QH1-20 fermentation liquor, so that the control effect of the QH1-20 on pepper anthracnose, phytophthora capsici and apple rot is as high as 100%.
The combination of the in vitro antibacterial activity and the in vivo antibacterial activity measurement results can be shown as follows: QH1-20 has remarkable antagonistic activity on various plant diseases, has a broad antibacterial spectrum, has high-efficiency and stable disease prevention effect, is widely applied and has a certain application potential.
The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.
Claims (6)
1. A broad-spectrum antagonistic bacterium streptomyces fusiformis QH1-20, characterized in that said streptomyces fusiformis (Streptomyces netropsis) QH1-20 was deposited at the chinese microbiological bacterial deposit management committee common microbiological center, address: the collection number of the institute of microorganisms of China academy of sciences of China No. 3, north Star West Lu No.1, the Korean area of Beijing is CGMCC No.28733.
2. A microbial biocontrol agent comprising the broad-spectrum antagonistic bacterium Streptomyces fusiformis QH1-20 according to claim 1.
3. The microbial biocontrol agent according to claim 2, wherein in said biocontrol agent, streptomyces fusiformis QH1-20 is present in the form of cultured living bacteria, fermentation broth or bacterial suspension.
4. The microbial biocontrol agent of claim 2, wherein the dosage form of said biocontrol agent is a wettable powder, a water-dispersible agent, an aqueous suspension agent or a dispersible oil suspension agent.
5. Use of a microbial biocontrol agent as defined in any one of claims 2-4 in the preparation of a product for inhibiting phytopathogens;
the plant pathogenic bacteria is Botryosphaeria cinerea、Botryosphaeria berengerianade、Monilinia fructigena、Alternaria alternata、Fusarium solani、Fusarium moniliforme or Phytophthora capsici.
6. The use of a microbial biocontrol agent according to claim 5 in the preparation of a product for inhibiting plant pathogenic bacteria, wherein the application mode comprises seed soaking of agricultural products to be treated, root irrigation after seedling planting, plant spraying or fruit spraying with the microbial biocontrol agent; the agricultural products to be treated are pears, apples, medlar, peppers, carrots or rice.
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