CN112522133A - Pseudomonas aeruginosa EZ-35, metabolite thereof and application thereof - Google Patents
Pseudomonas aeruginosa EZ-35, metabolite thereof and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
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- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
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Abstract
The invention discloses pseudomonas aeruginosa EZ-35, a metabolite thereof and application thereof, and aims to solve the technical problem of poor prevention and control effect on rice sheath blight in the prior art. The Pseudomonas aeruginosa (Pseudomonas aeruginosa) CGMCC NO.20621 can effectively inhibit rhizoctonia solani and can be used for preventing and treating plant diseases caused by the rhizoctonia solani, thereby achieving the purpose of reducing pesticide application, ensuring sustainable development of agriculture and having wide application prospect; the pseudomonas aeruginosa can metabolize and secrete a large amount of volatile substance 3-methylbutyric acid, and the 3-methylbutyric acid has good function of inhibiting rhizoctonia solani.
Description
Technical Field
The invention relates to the technical field of biological control, in particular to pseudomonas aeruginosaPseudomonas aeruginosa) EZ-35, metabolites thereof and uses thereof.
Background
The rice sheath blight disease is a disease caused by rhizoctonia solani infection and occurring on rice, is one of three main diseases affecting rice production, can occur in the whole growth period of rice, but has the greatest influence on the rice yield due to the disease from the booting stage to the grain filling stage. After the plants are ill, the mild can cause early withering of leaf sheaths and leaves, grain filling is influenced, a large amount of blighted grains are formed, the severe can cause normal heading of rice, even the whole plant is rotted and withered, the rice yield is seriously influenced, the rice setting rate is reduced, the thousand seed weight is reduced, and the blighted grains are increased. The yield is reduced by 15-20% generally, and the yield of a seriously ill field can reach 60-70%. However, no rice varieties with good resistance to sheath blight have been found. Thus, effective chemical control or biological control is required for such diseases.
At present, the prevention and control of rice sheath blight disease are mainly controlled by fungicidal chemical agents, such as jinggangmycin, carfentrazone-ethyl and the like which are widely used in China for many years; however, the continuous use of large amounts of a single fungicide increases the risk of resistance of the pathogen and also presents undesirable environmental problems.
There are also many alternative agricultural control measures in the prior art, such as fertilization, soil improvement and agricultural management, but such measures are not only time consuming and labor intensive, but also fail to achieve optimal control. It is well known that preparations derived from microorganisms are considered to be ideal substitute agents for future chemical pesticides due to their characteristics of high efficiency, low toxicity, low residue, no pollution, and low possibility of drug resistance.
It has been shown that volatile organic compounds (mVOCs) can regulate various biotic and abiotic stresses faced by plants, with the potential to be alternatives to harmful pesticides and fungicides. Such as the endophytic bacteria Pseudomonas stutzeri E25 and stenotrophomonas maltophilia CR71 may be released as dimethyl disulfideVOC with base (DMDS) as the main substance for inhibiting the growth of Botrytis cinerea ()Rojas-SolísEtc., 2018); VOC produced by Bacillus subtilis GB03 can directly inhibit the mycelial growth of Botrytis cinerea and interfere with the attachment of pathogenic fungi to hydrophobic leaves (R) ((R))Sharifi et al.，2016）。
However, due to the complex and diverse nature of microorganisms and the enormous amount and variety of mVOCs produced by their metabolism, it is not clear whether there are more effective strains of microorganisms and their metabolic mVOCs that can specifically inhibit the growth of Rhizoctonia solani.
However, there is a need to research and screen out a new microbial strain and a preparation thereof capable of specifically inhibiting rhizoctonia solani, which can be used for preventing and treating rice sheath blight diseases, so as to reduce the use of chemical preparations, thereby promoting the sustainable development of the rice industry.
Disclosure of Invention
The invention aims to provide pseudomonas aeruginosa EZ-35 capable of specifically inhibiting the growth of rhizoctonia solani, and provides an application way according to the characteristics of a secreted product of the pseudomonas aeruginosa EZ-35 so as to realize the biological control of rice sheath blight or extract an antibacterial active substance with good effect.
Research shows that during the growth of rice, the root system can release various signal molecules to assemble a unique root system microbial community. These microorganisms play an important role in the growth and development of rice. By releasing various metabolites, such as plant hormones and mvocs, they can not only promote plant growth, but also help plants resist various diseases. To date, there have been few studies on inhibition of the growth of rhizoctonia solani by rice-related bacteria, and there has been no report on whether rice rhizosphere bacteria can inhibit the growth of rhizoctonia solani by releasing VOCs.
Based on the above, the inventor conducts a series of rhizosphere microorganism separation and screening researches on healthy rice rhizosphere soil, and obtains a pseudomonas aeruginosa EZ-35 capable of inhibiting the growth of rhizoctonia solani by screening, wherein the strain is preserved in China general microbiological culture Collection center (CGMCC, address: No. 3 Xilu 1. on the North Kyoho in the Beijing City of Tokyo province) in 9 months and 9 days in 2020 and the preservation number is CGMCC number 20621.
The pseudomonas aeruginosa CGMCC number 20621, the metabolite thereof or the liquid fermentation product thereof can be used for preparing a biocontrol agent for the rice sheath blight; researches show that the strain can secrete a large amount of bacteriostatic active substance 3-methylbutyric acid (A)3- methyl-Butanoic acid）。
The biocontrol agent for the rice sheath blight is prepared by the following method:
marking lines of pseudomonas aeruginosa on a solid culture medium, culturing for 70-72 h in a dark place at 30-32 ℃, selecting a single colony, inoculating into a liquid culture medium, culturing for 70-72 h in a dark place at 30-32 ℃ at 150-180 r/min, and preparing the biocontrol agent according to conventional steps.
Based on the properties of pseudomonas aeruginosa CGMCC number 20621, the pseudomonas aeruginosa can be applied to the prevention and control of rice sheath blight.
Pseudomonas aeruginosa CGMCC number 20621 can be used for preparing bacteriostatic active substance 3-methylbutyric acid3- methyl-Butanoic acid）。
Compared with the prior art, the invention has the main beneficial technical effects that:
1. the pseudomonas aeruginosa CGMCC number 20621 can specifically inhibit rhizoctonia solani and can be used for preventing and treating plant diseases caused by the rhizoctonia solani, so that the aim of reducing pesticide application is fulfilled, sustainable development of agricultural health is ensured, and the pseudomonas aeruginosa CGMCC number 20621 has wide application prospect.
2. The pseudomonas aeruginosa CGMCC number 20621 can metabolize and secrete a large amount of volatile substance 3-methylbutyric acid3-methyl-Butanoic acid) (ii) a The invention also discovers 3-methylbutyric acid (A) for the first time3-methyl- Butanoic acid) Has good function of inhibiting rhizoctonia solani.
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FIG. 1 is a photograph showing the effect of Pseudomonas aeruginosa in inhibiting Rhizoctonia solani, the experimental group is the P-buckling fumigation Rhizoctonia solani of Pseudomonas aeruginosa, and the control group is the P-buckling fumigation Rhizoctonia solani without inoculating culture medium.
FIG. 2 is a chromatogram of Pseudomonas aeruginosa producing volatile organic compounds.
FIG. 3 is a photograph comparing the effect of 3-methylbutyric acid on inhibiting Rhizoctonia solani; wherein A is a photograph for comparing the effect of 3-methylbutyric acid on inhibiting rhizoctonia solani, wherein 50 uL of pure 3-methylbutyric acid and 150 uL of sterilized water (with the concentration of 25%) are dripped into a test group, and 200 uL of sterilized water is dripped into a control group; and B is a photograph for comparing the effect of 3-methylbutyric acid on inhibiting rhizopus microsporus, wherein 200 uL of pure 3-methylbutyric acid is dropwise added into the test group, and 200 uL of sterilized water is dropwise added into the control group.
FIG. 4 is a photograph for comparing evaluation of the effect of 3-methylbutyric acid in inhibiting Rhizoctonia solani from infecting rice leaves in vitro.
FIG. 5 is a comparison graph of the chromatographic peak areas of 3-methylbutyric acid released by different strains cultured for the same time; wherein 35 (EZ-35) is the pseudomonas aeruginosa, 34 is the enterobacter shenghuensis, 62 is the bacillus aryabhattai, and the two strains are obtained by collecting and screening the strains in a healthy rice pot culture of a greenhouse of Chinese academy of agricultural sciences by Wann.
Detailed Description
The following examples are intended to illustrate the present invention in detail and should not be construed as limiting the scope of the present invention in any way.
The instruments and devices referred to in the following examples are conventional instruments and devices unless otherwise specified; the related reagents or products are all conventional reagents or products on the market if not specifically indicated; the test methods involved are conventional methods unless otherwise specified.
Example 1: screening and identification of biocontrol strains
(1) Screening
Healthy rice rhizosphere soil samples were collected in a pot experiment by Wann in a greenhouse of Chinese academy of agricultural sciences in 2018 and 12 months (covering soil about 5 cm from the root surface of rice was removed, and loose soil bodies combined with rice root systems were randomly collected in an S-shape).
The control group was sterile water and the test group was a soil sample suspension diluted in a gradient.
The specific operating method of the test group is as follows:
the method comprises the steps of coating suspensions of gradually diluted soil samples on a beef extract peptone flat plate by using a gradient dilution coating method, selecting single bacteria to fall into a liquid beef extract peptone culture medium to obtain bacteria liquid, uniformly coating the bacteria liquid on the beef extract peptone culture medium to culture for 24 hours, buckling the inoculated flat plate on a PDA flat plate which is inoculated with 10 uL of rhizoctonia solani bacteria liquid (the rhizoctonia solani is purchased from China agricultural microbial strain preservation management center and has a strain number of 36246) in the center to culture, culturing at 32 ℃ in the dark, counting the bacteriostasis rate, and screening strains with the bacteriostasis effect.
The related culture medium comprises the following components in percentage by weight:
beef extract peptone medium: 3 g of beef extract, 10 g of peptone, 5 g of sodium chloride, 18 g of agar and 1L of deionized water, wherein the pH value is 7.2-7.4;
liquid beef extract peptone medium: 3 g of beef extract, 10 g of peptone, 5 g of sodium chloride and 1L of deionized water, wherein the pH value is 7.2-7.4;
PDA culture medium: 200 g of potatoes, 20 g of glucose and 1L of tap water, and the pH value is natural.
Bacteriostatic rate = (rhizopus microsporus colony size of blank medium cross-wise-rhizopus microsporus colony size of the bacteria cross-wise)/rhizopus microsporus colony size of blank medium cross-wise × 100%;
a strain EZ-35 is obtained by screening, and tests show that the bacteriostasis rate of rhizoctonia solani reaches 39.22 percent, as shown in figure 1.
(2) Identification
a. And (4) staining the screened strain according to a gram staining method, and displaying that the strain is a negative bacterium.
b. The screened strains are cultured on an LB solid culture medium, and physiological and biochemical characteristics such as colony characteristics, color and the like are observed according to a common bacteria system identification manual, and the results are shown as follows:
the fungus is yellow, large and flat on the plate, and has metallic luster and ginger taste.
c. PCR amplification is carried out by adopting bacterial universal primers GM3F and GE4F to amplify a 1200 bp product, sequencing is carried out, sequencing results are compared by BLAST and are compared with the sequences in GenBankThe results of homology comparison with those of Pseudomonas aeruginosaPseudomonas aeruginosa) There is 99% homology.
The strain is pseudomonas aeruginosa, is preserved in China general microbiological culture Collection center (CGMCC) in 9 months and 9 days in 2020, and the preservation number is CGMCC 20621.
Example 2: identification of pseudomonas aeruginosa bacteriostatic metabolite
The volatile organic compound is solid or liquid with carbon as basic element, can be rapidly volatilized at 20 ℃ and in the environment of 0.01 kPa, and enters a gas phase state. The results of the previous long-term research of the inventor show that the microorganism can produce a large amount of volatile organic compounds with rich varieties and multiple functions, and the substances mainly play the following roles: as signal species between and within communities; cell-to-cell signaling substances; possible carbon release channels; growth promoting and growth inhibiting factors. If acting as an inhibitor, the interaction is inhibited by phytopathogenic microorganisms, then the microorganisms which release volatile organic compounds have the potential to be used for the biological control of plant diseases.
(1) Collection of volatile organic compounds
Adding 0.1 g of agar into a 20 mL headspace bottle for culturing beef extract peptone, sealing the bottle mouth with kraft paper, sterilizing at 121 ℃ for 30 min, inclining the bottle at an angle of 30 degrees after sterilization, cooling and solidifying, adding 100uL of prepared bacterial liquid into the bottle, shaking the bottle body to uniformly distribute the bacterial liquid on the inclined plane of a culture medium, covering the bottle body with the kraft paper, culturing for 48 hours at a constant temperature of 30 ℃ under a dark condition, removing the kraft paper, sealing with a hollow spiral cover with a polytetrafluoroethylene spacer, setting the headspace bottle only containing the culture medium and not containing the bacteria as a blank control, and repeating four times of treatment.
After 5 days of culture, 50/30 μm DVB/CAR/PDMS extraction fibers (from commercial products)SupelcoCompany) for extraction, the extraction head being fitted on an SPME Manual extraction handle (Supelco，BellefontePA, USA) before use, aging is carried out according to the manufacturer's instructionsThe process is carried out, and then the extraction is carried out for 12 h under the constant temperature condition of 30 ℃.
(2) Analysis of volatile organic Compounds
7890A-5975C gas chromatography-mass spectrometer was used immediately after the extraction was completed (Agilent TechnologiesUSA) was performed.
Chromatographic conditions are as follows: the sample inlet temperature is 250 ℃, and the sample introduction time is 2.7 min; in a non-flow splitting mode, the carrier gas is high-purity helium with the purity of 99.999 percent, and the flow rate of the column is 1 mL/min;
column box temperature program: the initial temperature is 50 ℃, the temperature is kept for 2 min, the temperature is increased to 180 ℃ at the speed of 8 ℃/min, the temperature is increased to 240 ℃ at the speed of 10 ℃/min, and the temperature is kept for 6 min.
Mass spectrum conditions: the ionization mode is EI, 70 eV; the ion source temperature is 230 ℃, the quadrupole rod temperature is 150 ℃, and the transmission line temperature is 250 ℃; the scanning mode is full scanning, and the scanning range is 35-450 amu.
The measured mVOCs mass spectrum is compared and identified in an NIST/EPA/NIH database, and the strain can produce various volatile organic compounds, wherein the release amount of 3-methylbutyric acid is relatively large, as shown in figure 2.
The experimental study of this example tested 9 P.aeruginosa strains of VOCs, 3 of which were capable of producing 3-methylbutyric acid, and one of which released much more P.aeruginosa strain than the other two strains, designated P.aeruginosa EZ-35, as shown in FIG. 5.
(3) Identification of bacteriostatic effect of bacteriostatic active substance
A sterilized 2 ml centrifuge tube cover is added into a PDA culture medium, 50 uL of pure 3-methylbutyric acid and 150 uL of sterilized water are added into the cover to serve as a test group (with the concentration of 25%), 200 uL of sterilized water is added into the cover of a control group, 10 uL of rhizoctonia solani strain solution is added into the center of the culture medium, the culture is carried out at the temperature of 32 ℃ under the dark condition, the growth condition of the rhizoctonia solani is observed, and the test result is shown in figure 3.
(4) Evaluation of Effect of bacteriostatic active substance on inhibiting Rhizoctonia solani from infecting in-vitro rice leaves
Rhizoctonia solani was inoculated on PDA medium and pre-cultured for 48 hours. Cutting rice leaves into 5 cm-long segments, and placing on PDA culture medium; 200 uL of 3-methylbutyric acid (at concentrations of 0, 12.5%, 25%, and 50%, respectively) was added to a 200 uL container in a petri dish. Each treatment was repeated 5 times, and observed every 24 hours, and the results are shown in fig. 4.
The result shows that pseudomonas aeruginosa CGMCC number 20621 can effectively inhibit the growth of rhizoctonia solani and can generate a plurality of volatile organic compounds, wherein the first research of the invention finds that 3-methylbutyric acid is lethal to rhizoctonia solani, and the 3-methylbutyric acid can obviously inhibit the infection of the rhizoctonia solani on rice leaves.
The invention is explained in detail above with reference to the drawings and the embodiments; however, it will be understood by those skilled in the art that various changes in the specific components and parameters of the above embodiments may be made without departing from the spirit of the invention, or equivalents thereof may be substituted for elements thereof, thereby forming a plurality of specific embodiments, which are common variations of the invention and will not be described in detail herein.
Claims (7)
1. Pseudomonas aeruginosaPseudomonas aeruqinosa) EZ-35 with the preservation number of CGMCC number 20621.
2. A biocontrol agent comprises at least one of Pseudomonas aeruginosa CGMCC number 20621 containing copper, its metabolite, and its liquid fermentation product.
3. A method for preparing the biocontrol agent of claim 2, comprising the steps of:
marking pseudomonas aeruginosa CGMCC number 20621 on a solid culture medium, culturing for 70-72 h at 30-32 ℃ in a dark place, selecting a single colony, inoculating the single colony into a liquid culture medium, culturing for 70-72 h at 30-32 ℃ at 150-180 r/min in a dark place to obtain a liquid fermentation product, and preparing the biocontrol agent according to conventional steps.
4. The biocontrol agent of claim 3, wherein the metabolite comprises 3-methylbutyric acid.
5. The application of the pseudomonas aeruginosa CGMCC number 20621 in the prevention and treatment of rice sheath blight disease in claim 1.
6. The use of pseudomonas aeruginosa CGMCC number 20621 or/and metabolite thereof as claimed in claim 1 in the preparation of 3-methylbutyric acid.
7. The application of 3-methylbutyric acid secretion of pseudomonas aeruginosa CGMCC number 20621 disclosed in claim 1 in preventing and treating rice sheath blight disease.
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| CN114317319A (en) * | 2021-11-10 | 2022-04-12 | 福建省农业科学院植物保护研究所 | Pseudomonas aeruginosa and metabolite and application thereof |
| CN115039794A (en) * | 2022-06-30 | 2022-09-13 | 中国烟草总公司辽宁省公司 | Biocontrol microbial inoculum for preventing and treating tobacco target leaf spot disease as well as preparation method and application thereof |
| CN116649374A (en) * | 2023-07-21 | 2023-08-29 | 青岛农业大学 | Pseudomonas aeruginosa QSE-TM7 and application thereof in preparation of biological agent for preventing and treating tomato gray mold |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116649374A (en) * | 2023-07-21 | 2023-08-29 | 青岛农业大学 | Pseudomonas aeruginosa QSE-TM7 and application thereof in preparation of biological agent for preventing and treating tomato gray mold |
| CN116649374B (en) * | 2023-07-21 | 2024-05-28 | 青岛农业大学 | Pseudomonas aeruginosa QSE-TM7 and application thereof in preparation of biological agent for preventing and treating tomato gray mold |
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