CN113832049A - Coptis southern blight biocontrol bacterium pseudomonas fluorescens HT1 and application thereof - Google Patents

Coptis southern blight biocontrol bacterium pseudomonas fluorescens HT1 and application thereof Download PDF

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CN113832049A
CN113832049A CN202110929652.3A CN202110929652A CN113832049A CN 113832049 A CN113832049 A CN 113832049A CN 202110929652 A CN202110929652 A CN 202110929652A CN 113832049 A CN113832049 A CN 113832049A
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coptis
pseudomonas fluorescens
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southern blight
pseudomonas
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唐涛
游景茂
王帆帆
郭杰
郭晓亮
段媛媛
毛婷
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INSTITUTE OF CHINESE HERBAL MEDICINES HUBEI ACADEMY OF AGRICULTURAL SCIENCES
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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
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Abstract

The invention relates to the technical field of microorganisms, and discloses a fluorescent pseudomonas (P.fluoroscecens) HT1 for biocontrol bacteria of southern blight of coptis, wherein the fluorescent pseudomonas (P.fluoroscecens) HT1 is preserved in China center for type culture collection (CCTCC M2021901) at 7-19 months in 2021; applications are also disclosed. The invention screens and obtains pseudomonas fluorescens (P.fluoroscens) HT1 from the rhizosphere soil of the coptis for the first time, can be used for preventing and treating the coptis southern blight, provides a green channel for preventing and treating the coptis southern blight, can reduce the application amount of chemical pesticides in the planting process of the coptis, has the advantages of long prevention time, no toxicity, no harm and the like, and can obtain greater environmental benefits and economic benefits for agricultural production.

Description

Coptis southern blight biocontrol bacterium pseudomonas fluorescens HT1 and application thereof
Technical Field
The invention relates to the technical field of microorganisms, in particular to a fluorescent pseudomonas HT1 for biocontrol bacteria of southern blight of coptis and application thereof.
Background
The coptis chinensis is a perennial herb of the coptis genus of the Ranunculaceae family, has wide antibacterial, antiviral and antioxidant effects by using roots and stems, is commonly used for reducing blood sugar and resisting tumors of diabetes, and the latest research also shows that active substances in the coptis chinensis detoxification soup can treat viral pneumonia through multiple targets and multiple channels. The autonomous state of the Enshi soil family is one of main producing areas of coptis chinensis, and with continuous enlargement of planting scale and continuous cultivation for many years, the diseases of the rhizome of Chinese goldthread such as Lichuan and stone column are serious, large-area yield reduction is caused, and even the rhizome of Chinese goldthread is a destructive soil-borne disease caused by Sclerotium rolfsii, and the disease is serious in 5-9 months every year under the high-temperature and high-humidity condition that the air temperature is higher than 28 ℃. The prevention and treatment of the southern blight of the coptis are mainly based on chemical prevention and treatment at present, but no medicament for preventing and treating the southern blight of the coptis is registered at present, and the safety risk of the medicament registered for preventing and treating the southern blight of other crops is used for reference.
Bacteria are the highest content of organisms in the rhizosphere soil, while Volatile Organic Compounds (VOCs) secreted by bacteria are an important signal that plants and bacteria do not interact directly. Many studies have shown that various bacteria in soil can improve plant growth, enhance plant immunity, and act as biocontrol agents against pathogen infection. In recent years, volatile substance-antagonistic bacteria capable of producing strong antibacterial activity have been increasingly used for the control of plant diseases. Therefore, screening antagonistic microorganisms to replace chemical agents for preventing and treating the southern blight of coptis is necessary.
Disclosure of Invention
Based on the problems, the invention provides a fluorescent pseudomonas HT1 and application thereof, and the fluorescent pseudomonas is screened from coptis rhizosphere soil for the first time aiming at the coptis southern disease, and the strain inhibits the hypha growth of southern disease pathogenic bacteria and the occurrence of the southern disease on coptis through volatile metabolites.
In order to solve the technical problems, the invention provides Pseudomonas fluorescens (P.fluorescens) HT1, wherein the Pseudomonas fluorescens (P.fluorescens) HT1 is preserved in China center for type culture collection (CCTCCM 2021901) at 7-19 months in 2021.
In order to solve the technical problems, the invention also provides application of the fluorescent pseudomonas HT1 in preparation of medicines or biocontrol microbial agents for preventing and treating the southern blight of coptis.
Further, the medicine contains volatile metabolites of pseudomonas fluorescens HT1, the volatile metabolites can inhibit the growth of sclerotia of the sclerotia of the sclerotia of the same of sclerotia of the strain of the same of sclerotia of the sclerotia of the sclerotia of the strain of the sclerotia of the strain of the sclerotia of the strain of.
Furthermore, the biocontrol microbial inoculum contains pseudomonas fluorescens HT1, and the pseudomonas fluorescens HT1 can inhibit growth of mycelial of southern blight of coptis.
Further, the biocontrol microbial inoculum is fermentation liquor of pseudomonas fluorescens HT1, and the preparation method of the fermentation liquor comprises the following steps: inoculating fluorescent Pseudomonas HT1 in NA culture medium, performing shake culture at 28 deg.C and 150r/min for 24 hr, measuring effective viable bacteria concentration by plate colony counting method, and adjusting to 107cfu/mL to obtain the biocontrol microbial inoculum HT1 microbial inoculum.
Compared with the prior art, the invention has the beneficial effects that: the invention screens and obtains fluorescent pseudomonas (P.fluoroscens) HT1 from the rhizosphere soil of coptis for the first time, the fluorescent pseudomonas HT1 has the inhibition rate of 62% on the growth of the sclerotium of coptis, the inhibition rate of 92% on the pathogenicity of the sclerotium of coptis, and the control effect on the sclerotium of coptis in a pot test reaches 69.02%, so the invention can be used for controlling the sclerotium of coptis, provides a green channel for controlling the sclerotium of coptis, can reduce the application amount of chemical pesticides in the planting process, has the advantages of long control time, no toxicity, no harm and the like, and can obtain great environmental and economic benefits for agricultural production.
Drawings
FIG. 1 shows a plate of HT1 screened by plate-opposing method according to an embodiment of the present invention;
FIG. 2 is a biological identification chart of HT1 according to an embodiment of the present invention;
FIG. 3 is a graph showing the results of the inhibition of hypha growth of southern blight of Coptis by HT1 in example of the present invention;
FIG. 4 is a graph showing the results of the inhibition of hypha growth of southern coptis bacterium by volatile metabolites (VOCs) of HT1 in an example of the present invention;
FIG. 5 is a graph showing the results of the inhibition of the pathogenicity of southern coptis bacteria by volatile metabolites (VOCs) of HT1 in an example of the present invention;
FIG. 6 is a graph showing the results of the inhibition of sclerotia germination of southern blight of Coptis by volatile metabolites (VOCs) of HT1 in example of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.
Example (b):
in this example, rhizosphere soil in 5-10cm depths of different coptis root planting plots in the arundo donax, lichuan, lakebei province planting foundation land was collected, and the plots were taken back to the laboratory after being subpackaged and labeled, and bacteria were separated after being naturally air-dried. The separation method comprises the following steps: separating by plate dilution method, weighing 5g of air-dried rhizosphere soil sample, adding 45 g of air-dried rhizosphere soil sampleShaking culturing in sterile water at 150r/min for 1 hr, filtering with sterile gauze to obtain suspension, and diluting by 10-fold serial dilution method to obtain 10-5-10-3And (3) taking 50 mu L of diluent, respectively, uniformly coating the diluent on an NA plate, culturing the NA plate in a constant-temperature incubator at 28 ℃ for 48 hours, selecting single colonies with different colors, glossiness, sizes and types, streaking and purifying strains, uniformly numbering, transferring the strains into an NA slant culture medium, storing the strains at 4 ℃ for a short time, storing the strains by using sterilized glycerol with the final concentration of 25%, and storing the strains at-80 ℃ for later use.
Screening bacteria having antagonistic action on pathogenic bacteria of southern blight of Coptis by using a plate confronting method, which comprises the following steps: selecting and culturing 2d Coptis sclerotiorum cake (5mm) in the center of PDA plate, inoculating different bacteria obtained by screening at symmetrical positions on two sides of the plate, culturing in a constant temperature incubator at 28 ℃ for 48h, observing and measuring the antibacterial efficiency, repeating the experiment for 3 times, and screening out the strain with the highest antibacterial efficiency, namely Pseudomonas fluorescens (P.fluorescens) HT1 shown in figure 1.
In this example, the obtained pseudomonas fluorescens HT1 was biologically identified, and the identification result shown in FIG. 2 shows that the pseudomonas fluorescens is gram-negative bacteria, rod-shaped, has the cell size of 2.0-3.2 μm × width of 0.5-1.0 μm, has flagella, no spores, round colony shape, smooth surface and is opaque. The gene is identified as P.fluoroscens according to the phylogenetic analysis and biological characteristics of 16S rRNA, and is preserved in China Center for Type Culture Collection (CCTCC) at 19 months 7 and 19 months 2021, the preservation address is No. 299 of the eight paths in the Wuchang district, Wuhan City, Hubei province, the preservation number is CCTCC M2021901, and the classification is named as P.fluoroscens HT 1.
In the embodiment, a plate confronting culture method is adopted, a sclerotium rolfsii (from Lichuan Jian Zhuxi) cake with consistent age and 5mm diameter is inoculated at the center of a PDA culture medium plate, a strain Pseudomonas fluorescens HT1 colony is inoculated at a position 2.5 cm away from two opposite sides of a hypha block, the sclerotium rolfsii of an unanswered strain HT1 is used as a reference, the process is repeated for 3 times, the sclerotium rolfsii is placed in a constant temperature incubator at 28 ℃ for culture for 3 days, and then the size of a bacteriostatic zone is measured, which is shown in an attached figure 3, and the left figure is the reference, and the result shows that the strain HT1 has a good antagonistic effect on the growth of sclerotium rolfsii.
In this example, a two-dish opposing culture method was used, and NA and PDA solid media were poured on both sides of the two dishes to prepare plates. A5 mm diameter mycelium block of southern blight of Coptis on one side of PDA plate was plated, and P.fluorescens HT1 was plated on one side of NA plate, and two plates plated with southern blight of Coptis alone without plating P.fluorescens HT1 were used as control. After the strains are subjected to the opposite culture in a constant-temperature incubator at 28 ℃ for 3 days, the sizes of the sclerotium rolfsii colonies are observed and measured, and the results are shown in figure 4, wherein the left picture is a control group, and the results show that volatile metabolites (VOCs) of the strain P.fluoroscens HT1 have a remarkable inhibition effect on the growth of sclerotium rolfsii strains.
In this example, NA solid media were poured into two halves of dishes to prepare a flat plate and a piece of sterile absorbent paper was laid, one side of the NA flat plate was coated with p.fluoroscens HT1, the sterile absorbent paper was wetted with sterile water and placed into healthy coptis leaves, hypha blocks (5mm) of southern coptis were inoculated on the coptis leaves by stabbing, and the two halves of dishes without p.fluoroscens HT1 were used as a control group. After the leaves are oppositely cultured in a constant-temperature incubator at 28 ℃ for 3 days, the sizes of southern blight spots on the leaves of the coptis are observed and measured, and the result is shown in figure 5, wherein the left picture is a control group, and the result shows that the volatile metabolites (VOCs) of the strain P. fluoroscens HT1 have obvious inhibition effect on the pathogenicity of southern blight bacteria of the coptis.
In this example, two dishes are prepared by inverting NA and PDA solid media to form plates on both sides of the two dishes, respectively, and inoculating sclerotium of NA of coating p.fluoroescens HT1 is used as a control group. After the rhizoctonia solani is cultured for 3 days in a constant temperature incubator at 28 ℃, the size of the rhizoctonia solani colonies is observed and measured, the result is shown in figure 6, and the result shows that volatile metabolites (VOCs) of the strain P.fluoroscens HT1 have obvious inhibition effect on the rhizoctonia solani colonies.
Inoculating strain HT1 in NA culture medium, performing shake culture at 28 deg.C and 150r/min for 24 hr, determining effective viable bacteria concentration by plate colony counting method, and adjusting to 107cfu/mL, obtaining HT1 microbial inoculum, and storing at 4 ℃ for later use.
Then, healthy soil is taken and potted, healthy 3-year-old coptis seedlings are transplanted, 5-7 sclerotia of southern blight coptis are inoculated to the rhizosphere of potted coptis after 1-month culture, and after 3-day culture in a constant-temperature incubator at 28 ℃, HT1 microbial inoculum is sprayed, a blank NA spraying culture medium is used as a control, the experiment is repeated for 3 times, and the disease condition of coptis is checked after 7 days, which is shown in Table 1, wherein the disease condition grading standard of southern blight of coptis is as follows:
level 0: the coptis chinensis is healthy, disease-free and normal in growth;
level 1: small scabs at the base of the coptis stem;
and 3, level: the root of the coptis root is rotten and the disease spots spread to part of the leaves
And 5, stage: the whole coptis is rotten and withered.
Calculating disease indexes and related control effects:
disease index ∑ (number of disease-grade plants × number of disease-grade)/(total number of plants × number of highest disease-grade) × 100
Control effect (%) (disease index of control group-disease index of treatment group)/disease index of control group x 100.
As shown in Table 1, the pot culture control effect of the HT1 microbial inoculum on the southern blight of coptis chinensis is 69.02%.
TABLE 1 prevention and treatment effects of HT1 microbial inoculum on southern blight of Coptis in potting experiment
Figure BDA0003210868860000041
In conclusion, the inhibition rate of Pseudomonas fluorescens HT1 on the growth of the sclerotium of southern coptis is as high as 62%, the inhibition rate of volatile metabolites on the growth of sclerotium of southern coptis is as high as 96%, the inhibition rate on the germination of sclerotium of southern coptis is as high as 92%, the inhibition rate on the pathogenicity of southern coptis is 92%, and the prevention and treatment effect on southern coptis in a pot experiment is 69.02%. By the popularization and use of Pseudomonas fluorescens HT1 in this example, the use of pesticide on Coptidis is expected to be reduced by 20%.
The above is an embodiment of the present invention. The embodiments and specific parameters in the embodiments are only for the purpose of clearly illustrating the verification process of the invention and are not intended to limit the scope of the invention, which is defined by the claims, and all equivalent structural changes made by using the contents of the specification and the drawings of the present invention should be covered by the scope of the present invention.

Claims (5)

1. The fluorescent pseudomonas fluorescens HT1 is characterized in that the fluorescent pseudomonas (P. fluorosceens) HT1 is preserved in China center for type culture collection (CCTCCM 2021901) at 7 months and 19 days of 2021.
2. The use of the biocontrol bacterium for southern blight of coptis as claimed in claim 1, namely pseudomonas fluorescens HT1 in the preparation of medicines or biocontrol bacterium agents for preventing and treating southern blight of coptis.
3. The use according to claim 2, wherein the medicament comprises a volatile metabolite of pseudomonas fluorescens HT1, wherein the volatile metabolite inhibits hyphal growth and sclerotia germination of southern coptis, and wherein the volatile metabolite inhibits virulence of southern coptis.
4. The use of claim 2, wherein the biocontrol bacterial agent contains pseudomonas fluorescens HT1, and the pseudomonas fluorescens HT1 can inhibit the growth of mycelial of southern blight of Coptis.
5. The application of claim 2, wherein the biocontrol microbial inoculum is a fermentation broth of pseudomonas fluorescens HT1, and the preparation method of the fermentation broth is as follows: inoculating fluorescent Pseudomonas HT1 in NA culture medium, performing shake culture at 28 deg.C and 150r/min for 24 hr, measuring effective viable bacteria concentration by plate colony counting method, and adjusting to 107cfu/mL to obtain the biocontrol microbial inoculum HT1 microbial inoculum.
CN202110929652.3A 2021-08-13 2021-08-13 Coptis southern blight biocontrol bacterium pseudomonas fluorescens HT1 and application thereof Pending CN113832049A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116162564A (en) * 2022-08-18 2023-05-26 西南科技大学 Achromobacter JY-2-3R strain for biocontrol of aconitum carmichaeli and application thereof

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CN106613557A (en) * 2017-01-20 2017-05-10 青岛蔚蓝生物集团有限公司 Microbial control method for plant southern blight
CN111621447A (en) * 2020-07-02 2020-09-04 河南省科学院生物研究所有限责任公司 Application of bacillus amyloliquefaciens WS3-1 in prevention and treatment of peanut southern blight

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CN106613557A (en) * 2017-01-20 2017-05-10 青岛蔚蓝生物集团有限公司 Microbial control method for plant southern blight
CN111621447A (en) * 2020-07-02 2020-09-04 河南省科学院生物研究所有限责任公司 Application of bacillus amyloliquefaciens WS3-1 in prevention and treatment of peanut southern blight

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116162564A (en) * 2022-08-18 2023-05-26 西南科技大学 Achromobacter JY-2-3R strain for biocontrol of aconitum carmichaeli and application thereof
CN116162564B (en) * 2022-08-18 2023-12-01 西南科技大学 Achromobacter JY-2-3R strain for biocontrol of aconitum carmichaeli and application thereof

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Application publication date: 20211224