CN111944728B

CN111944728B - Pseudomonas chlororaphis and application thereof

Info

Publication number: CN111944728B
Application number: CN202010868222.0A
Authority: CN
Inventors: 王刚; 王淼; 王俊芳; 刘凤英; 张震
Original assignee: Henan University
Current assignee: Henan University
Priority date: 2020-08-26
Filing date: 2020-08-26
Publication date: 2022-02-22
Anticipated expiration: 2040-08-26
Also published as: CN111944728A

Abstract

The invention discloses Pseudomonas chlororaphis subsp. aurantiaca Zm-1 which is classified and named as Pseudomonas chlororaphis subsp. aurantiaca Zm-1, is preserved in China center for type culture Collection of Wuhan university in 2019, 12 and 25 months, and has the preservation number of CCTCC NO: M20191103. The pseudomonas chlororaphis Zm-1 has an antagonistic effect on pathogenic bacteria of peanut southern blight, the disease incidence rate of the peanut southern blight can be greatly reduced by spraying peanuts in a field environment, a good biocontrol effect is achieved, and the pseudomonas chlororaphis Zm-1 can be well colonized at the peanut rhizosphere, so that the peanuts can continuously resist the pathogenic bacteria of the peanut southern blight, the biocontrol effect is prolonged, and the yield is effectively improved.

Description

Pseudomonas chlororaphis and application thereof

Technical Field

The invention relates to pseudomonas aeruginosa and application thereof in biological prevention.

Background

The Sclerotium rolfsii Sacc, a pathogen of Sclerotium rolfsii, infects the stem base, the fruit stem, the fruit pod and the root of the peanut to cause fungal soil-borne disease. After the peanut is infected, the infected part is brown and soft rot, and white sericite mycelium grows on the surface. The contamination of peanut in seedling stage can cause withering and death of seedling stem leaves, and the yellowing and withering of leaves and plant death in adult stage. In recent years, the occurrence frequency of the southern blight of the peanuts is continuously enhanced, the serious plot disease rate reaches more than 60%, the yield and the quality of the peanuts are directly affected, and even the failure of harvest is caused. The peanut field is infected with diseases, the spread is fast, and once the peanut field is invaded, the disease is accumulated in successive years, and the disease is difficult to prevent and eradicate.

At present, an effective disease-resistant variety is not available for peanut southern blight diseases, the diseases are mainly prevented and controlled by a chemical prevention and control method, and due to the fact that a large amount of pesticides are used, not only is the peanut planting cost increased, but also the problems of pesticide residue, environmental pollution and the like exist. Biological control is a control method for plant diseases with wide prospect, and has become a hotspot for research and application at home and abroad at present.

The invention patent of invention publication No. CN108893418A 'an invention patent of Rhodopseudomonas palustris LY-6 microbial inoculum and preparation and application thereof in tomato southern blight control' discloses a Rhodopseudomonas palustris LY-6 microbial inoculum which can be used for biological prevention, wherein the liquid fermentation broth can inhibit the growth of southern blight to a certain extent, but the bacteriostatic rate is only 76.7%, which needs to be further improved, in addition, the supernatant obtained by centrifuging the fermentation broth is applied to crops, the Rhodopseudomonas palustris strain can not exist continuously at the root of peanut, and the continuity and stability of inhibiting southern blight are limited.

Disclosure of Invention

The invention aims to overcome the defect that the bacteriostatic effect of a strain for biologically preventing and treating peanut southern blight needs to be further improved in the prior art, and provides pseudomonas chlororaphis and application thereof.

In order to solve the technical problems, the invention provides the following technical scheme:

pseudomonas chlororaphis subsp. aurantiaca Zm-1 is classified and named as Pseudomonas chlororaphis, and has been preserved in China center for type culture Collection of Wuhan university in 2019, 12 months and 25 days, with the preservation number of CCTCC NO: M20191103.

The pseudomonas chlororaphis Zm-1 is obtained by separating from an unsealed field and an anemarrhena plant, is in a short rod shape, is small in individual, has two blunt circles at two ends, is gram negative, is negative in a catalase test, does not produce spores, is opaque, smooth, moist, orange and single-polar flagellum on an LB (Langerhans) flat plate, has motility, and produces possible biocontrol related factors such as catalase, phenazine and derivatives thereof. The pseudomonas chlororaphis, the mutant and the metabolite thereof can be used for biological control of the peanut southern blight according to a conventional method.

In a pot experiment, the pseudomonas chlororaphis Zm-1 strain is subjected to amplification culture in an LB culture medium to prepare a bacterial suspension with a certain concentration. Inoculating sclerotium rolfsii in the flowering period of the peanuts, placing 2g of wheat grain sclerotium rolfsii culture around the stem base of each plant, and carrying out aseptic moisture preservation for 60 h. And 3d, uniformly spraying the pseudomonas aeruginosa Zm-1 bacterial suspension by using a spray can. The number of diseased plants and the degree of disease were investigated at 7d after inoculation of southern blight, and the investigation was performed every 7 d. Inoculating sclerotium rolfsii 7d in field experiment, spraying with sprayer to obtain bacterial solution (bacterial suspension concentration 6 × 10)⁸cell.mL^-1) The base of the peanut stem is sprayed throughout. Spraying for the second time after 8d, and spraying clean water with the same amount as the control. Before spraying the biocontrol bacteria liquid, respectively investigating the number of diseased plants after spraying the biocontrol bacteria liquid for 20d for the first time and before harvesting for 2 d.

The Pseudomonas chlororaphis subsp. The average control effect of 6 repeated tests is 65.2 percent, and the average yield per mu of the tests is improved by 26 percent; in addition, pseudomonas chlororaphis Zm-1 can be well colonized at the peanut rhizosphere, so that the peanut can continuously resist pathogens of peanut southern blight, and the biocontrol effect of the peanut is prolonged. The invention provides a brand-new strain for biocontrol of the peanut southern blight, and the biocontrol effect of the strain is very competitive with the effect of the existing reported strain, so that the strain has good application and development prospects.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 is an electron microscope picture of Pseudomonas chlororaphis subsp. aurantiaca Zm-1 of the present invention;

FIG. 2 shows the results of measurement in the confrontation test;

FIG. 3 shows the results of PCR identification of orange colonies obtained from the rhizosphere of Arachis hypogaea.

Detailed Description

The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.

Examples

1. Isolation and characterization of Pseudomonas chlororaphis subsp

1.1 isolation of the Strain

Selecting healthy common anemarrhena, taking a root system of fresh common anemarrhena, washing the surface with tap water, removing particulate impurities on the surface of the root system, absorbing water by using filter paper, washing the surface of the root system with sterile water, diluting the washed suspension, coating a solid culture medium plate with a diluent, and culturing for 1-2 d at 28-30 ℃; after the colony grows out, the colony is purified and cultured at 4-6 ℃ for storage.

The culture medium is an improved LBS culture medium, and comprises the following specific components: tryptone (5 g), yeast extract (yeast extract)2.5g, sodium citrate (1 g), (NH)₄)₂SO₄ 2g，MgSO₄·7H₂O 0.2g，K₂HPO₄ 14g，KH₂PO₄6g, glucose 1g, agar powder 15g, distilled water 1000mL, pH 7.0.

1.2 measurement of morphological characteristics and physiological and biochemical characteristics of ZM-1 Strain

ZM-1 strain, round colony, convex surface, viscous, producing orange pigment, full margin. The thallus is rod-shaped, gram negative, polar flagellum, motile and free of spore. The cell size is 0.3 to 0.5X 1.1 to 1.2 μm.

TABLE 1 ZM-1 physio-biochemical characteristics

2. Experiment for confronting Pseudomonas chlororaphis subsp

2.1 Strain preparation

The activated culture of the Pseudomonas chlororaphis ZM-1 strain is transferred to LB liquid culture medium, the liquid loading of each bottle of culture medium is 30ml, shaking culture is carried out at 200rpm, the culture is carried out for 24h at 30 ℃, and the culture is placed in a refrigerator at 4 ℃ for standby. Inoculating sclerotium rolfsii to a PDA plate, culturing at 30 ℃ for 24h, and storing in a refrigerator at 4 ℃ for later use.

2.2 confrontation experiment

Beating the activated southern blight flat plate culture into a fungus cake, placing the fungus cake in the center of a PDA flat plate, dibbling 5 mu L of ZM-1 strain bacterial suspension at a symmetrical position 2cm away from the center of the flat plate, and taking the flat plate added with the same amount of sterile water as a reference; culturing at 30 ℃ for 24h, and counting the inhibition rate of the ZM-1 strain on sclerotium rolfsii.

The inhibition rate calculation was performed according to the following formula: biocontrol bacteria inhibition (%) (control colony diameter-treated colony diameter)/control colony diameter ] × 100%.

2.3 results of the confrontation test

As shown in figure 2, after the plate confrontation culture, the average value of a plurality of repeated tests is taken, and the bacteriostasis rate of the test strain ZM-1 to the sclerotinia sclerotiorum is calculated to be 73 percent.

3. Pot experiment

3.1 method for measuring biocontrol effect of pot experiment

Sterilizing sandy nutrient soil is filled in a plastic flowerpot with the diameter of 15cm, peanuts are sown, the sprouted peanut kernels are placed in a greenhouse in order with 2 peanut kernels per pot, and water is poured once a day.

Preparation of Pseudomonas chlororaphis ZM-1 bacterial suspension: the activated ZM-1 strain culture is transferred into LB liquid culture medium, and each bottle is cultivatedThe culture medium contained in the culture medium was 30mL, the culture was performed by shaking at 200rpm and at 30 ℃ for 24 hours, and the concentration of the culture medium was adjusted to 3X 10 by using a bacterial counting plate⁸cellmL^-1And putting the mixture in a refrigerator at 4 ℃ for later use.

Inoculation of southern blight: inoculating sclerotium rolfsii in the flowering period of the peanuts, placing 2g of wheat grain sclerotium rolfsii culture around the stem base of each plant, and carrying out aseptic moisture preservation for 60 h. And uniformly spraying the ZM-1 bacterial suspension by using a spray can after 3 days. The number of diseased plants and the degree of disease were investigated at 7d after inoculation of southern blight, and the investigation was performed every 7 d.

3.2 Classification Standard of southern blight of peanut

Level 0: no visible symptoms;

level 1, the area of the peanut leaf with symptoms of yellowing, wilting and the like does not exceed 25 percent of the total area;

2, the area of the peanut leaves with symptoms of yellowing, wilting and the like accounts for 25 to 50 percent of the total area;

grade 3, the area of the peanut leaves with symptoms of yellowing, wilting and the like accounts for 50 to 75 percent of the total area;

and 4, the area of the peanut leaves with symptoms of yellowing, wilting and the like exceeds 75 percent of the total area, and the plants die.

Disease index ═ Σ (each level of disease occurrence representative value × the number of disease plants in that level) × 100/(survey total number of plants × highest level of disease occurrence representative value)

The preventing and treating effect (%) is (contrast disease index-treatment disease index)/contrast disease index is multiplied by 100

Incidence (%) is the number of diseased plants/total investigated plants × 100%

3.3 biocontrol test result of pot experiment

After being treated by Pseudomonas chlororaphis subsp. aurantiaca Zm-1, the disease index and the morbidity of the southern blight disease of peanuts can be obviously reduced (table 2), and the average prevention effect is 82.5 percent in 6 repeated tests.

TABLE 2 biocontrol effect of potted plants

Note: the data in the table are the average of 6 replicates followed by the same letter indicating no significant difference (P ═ 0.05).

4. Field test

4.1 field biocontrol experimental method

Cell experimental design: randomly arranged, repeated 3 times, each zone having an area of 10m²Single-row double-grain uniform dibbling, peanut kernel seeding rate is 20-25g/m². And (4) medium-fertility cultivation, wherein topdressing is carried out when the peanuts are earthed and are subjected to needle-heading.

Inoculation of southern blight: after the peanut is sowed, southern blight bacteria are inoculated in the flowering phase, and 5g of wheat grain southern blight bacteria culture is placed around the stem base of the plant.

And (3) biocontrol bacterium suspension spraying design: inoculating sclerotium rolfsii 7d, spraying the bacterial liquid on the basal part of the peanut stem by a sprayer, wherein the concentration of the pseudomonas chlororaphis ZM-1 bacterial suspension is 6 multiplied by 10⁸cell.mL^-1. And (4) surveying and counting the number of diseased strains in each test cell before spraying the biocontrol bacteria liquid, spraying for the second time after 8 days, and spraying clean water with the same amount as the control. And (5) investigating the number of the diseased plants 20 days after the first spraying of the biocontrol bacteria liquid and 2 days before harvesting. The classification standard of the southern blight of the peanut is the same as the pot experiment.

4.2 field biocontrol experiment test results

TABLE 3 field control effect

TABLE 4 field test yield

Area of each experimental community is 10m²The land area per mu is about 667m²

The results show that: the spraying treatment of peanuts by Pseudomonas chlororaphis subsp. aurantiaca Zm-1 in a field environment can greatly reduce the incidence rate of southern blight of peanuts, achieve a better biocontrol effect and effectively improve the yield. The average control effect of 6 repeated tests is 65.2 percent, and the average yield per mu of the tests is improved by 26 percent.

5. Determination of colonization of Pseudomonas chlororaphis ZM-1 on peanut roots

Five-point sampling is carried out on the plot treated by the field control effect field (25 sampling points in 5 plots), 10 peanuts are selected at each sampling point, and soil samples are respectively taken before spraying the bacterial liquid and the day before harvesting. When taking a soil sample, punching and sampling near the rhizosphere of the peanut seedlings by using a sterile stainless steel puncher, wherein the hole depth is 3cm, three holes are punched in each seedling, and the soil sample is taken out and then is respectively filled into a sterile centrifuge tube. Taking a soil sample by a dichotomy, dissolving 10g of the soil sample into a triangular flask filled with 100mL of sterile water, shaking for 20min by a shaking table at room temperature at 200rpm, coating a PDA (personal digital assistant) plate by gradient dilution, culturing for 24h at 30 ℃, enabling pseudomonas chlororaphis ZM-1 to generate an orange pigment on a PDA culture medium to form an orange colony, counting the number of orange colonies on the plate, and calculating the colony forming unit of the unit soil sample. The experiment was repeated three times.

The soil sample treatment analysis shows that the ZM-1 strain has colonization capacity in peanut rhizosphere under field plot condition. The results showed that when the soil colonies of the ZM-1 strain were not sprayed, the strain on the plate without orange colonies grew. The ZM-1 strain was sprayed on soil samples and was tested to maintain the strain at 10 per gram of soil⁵Colony Forming Units (CFU). Orange colonies were specifically amplified using the PCR method, demonstrating the administered ZM-1 strain. Thus, the ZM-1 strain can be well colonized on the rhizosphere of the peanut. As shown in FIG. 3, the CK in the two lanes is two characteristic fragments amplified from ZM-1 strain by PCR identification of orange colonies, and lanes A-D and A/-D/are 4 colonies randomly selected by PCR identification.

The Pseudomonas chlororaphis subsp. aurantiaca Zm-1 is separated from a field rhizoma anemarrhenae plant, and the results of greenhouse potting and field experiments show that the strain has good control effect on the southern blight of peanuts and provides more choices for the biological control of the southern blight of the peanuts. The biocontrol effect shows that the modified strain has good application and development prospects.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The application of pseudomonas chlororaphis in preventing and treating peanut southern blight is characterized in that the application method comprises the following steps:

s1, preparation of a suspension of Pseudomonas chlororaphis ZM-1 bacteria: transferring the activated ZM-1 strain culture into a liquid culture medium for culture to obtain a fermentation liquid, and adjusting the thallus concentration of the fermentation liquid to 1-6 x 10⁸cellmL^-1Putting the mixture in a refrigerator at 4 ℃ for later use;

s2, spraying the bacterial suspension on the base of the peanut stem by a sprayer,

the Pseudomonas chlororaphis subsp. aurantiaca Zm-1 has been preserved in the China center for type culture collection of Wuhan university in 2019, 12 months and 25 days, and the preservation number is CCTCC NO: M20191103; the cell size is 0.3-0.5 multiplied by 1.1-1.2 μm, the thallus is rod-shaped, gram negative, polar flagellum, motile and free of spore; the colony was round, convex on the surface, sticky, producing an orange pigment, all round.

2. The use of claim 1, wherein the strain is cultured in an improved LBS medium, and the specific components are as follows: tryptone 5g, yeast extract 2.5g, sodium citrate 1g, (NH)₄)₂SO₄ 2g，MgSO₄·7H₂O 0.2g，K₂HPO₄ 14g，KH₂PO₄6g, glucose 1g, agar powder 15g, distilled water 1000mL, pH 7.0.