WO2021073142A1 - Strain of fluorescent pseudomonas 22g5 and application thereof in preventative treatment of verticillium wilt in crops - Google Patents

Abstract

A strain of Pseudomonas (Pseudomonas donghuensis) 22G5 and an application thereof in the preventative treatment of verticillium wilt in crops. The Pseudomonas 22G5 has been stored in the General Microbiology Center of the China Microbiological Culture Collection and Management Committee on 08 July 2019, and the storage code is CGMCC No.18084. The Pseudomonas strain 22G5 itself is not pathogenic to plants, has a significant inhibitory effect on cotton verticillium wilt caused by Verticillium dahliae (Verticillium dahliae), can significantly reduce the occurrence of cotton verticillium wilt, and has a good biological preventative treatment effect. Genomic studies have shown that Pseudomonas donghuensis Pseudomonas donghuensis 22G5 has a gene cluster that synthesizes a type of siderophore 7-hydroxytrophinolone; the compound can be used as an iron ion chelating agent to inhibit the iron element necessary for the growth of Verticillium dahliae, thereby inhibiting the growth of pathogenic bacteria. The strain can be used to develop biological pesticides against verticillium wilt disease in crops, and provide new ideas and new methods for using biology for the preventative treatment of verticillium wilt.

Description

A Strain of Pseudomonas fluorescens 22G5 and Its Application in Controlling Verticillium Wilt of Crops Technical field

The invention belongs to the technical field of plant disease biocontrol. More specifically, it relates to a strain of Pseudomonas donghuensis 22G5 and its application in preventing and controlling Verticillium wilt of crops.

Background technique

Cotton is an important economic crop in my country, providing renewable raw materials for the textile industry. Verticillium dahliae (Verticillium dahliae Kleb.) caused by Verticillium dahliae (Verticillium dahliae Kleb.) is a devastating disease. The spread of the disease not only significantly reduces the quality of cotton fiber, but also seriously affects the economic output of cotton. The direct economic loss caused by Verticillium dahliae Verticillium wilt caused by Verticillium dahliae exceeds billions of dollars every year. In 1982, cotton verticillium wilt had spread to nearly 130,000 hectares of agricultural soil in my country. By 1993, cotton verticillium wilt had become a major obstacle to high and stable cotton yields in my country, with the diseased area reaching 2.666 million hectares. At present, about half of my country's cotton planting areas are affected by Verticillium wilt, and the economic loss amounts to 1.5 to 2 billion yuan per year.

Verticillium wilt is a soil-borne seed-borne vascular disease, which spreads mainly through the bacteria-carrying cottonseed, cottonseed cake, cottonseed hulls, diseased plant residues, soil, fertilizer, running water, and farmland management tools. Pathogens invade from the roots, and then systematically infect cotton plants. Therefore, disease can be manifested in various growth stages from the seedling stage to the adult stage. Due to the unclear key mechanism of the pathogenic Verticillium dahliae and the lack of high-resistance cotton germplasm resources, the prevention and control of cotton verticillium wilt has not yet made a breakthrough. The current direction of prevention and control is mainly to reduce the use of synthetic fungicides, as far as possible to use Verticillium wilt resistant/disease-resistant varieties as a basis, to improve soil ecological conditions and to induce cotton plant disease resistance combined with comprehensive control measures. The comprehensive prevention and control in practice production is mainly divided into three aspects. (1) Use agricultural control methods such as crop rotation, deep soil plowing, and timely cultivation to increase soil permeability, strengthen the control of pathogen infection, and improve the resistance of cotton plants; (2) use melanamine, chemical pesticides and other pesticides Pesticide prevention and control; (3) Microbial inocula prevention and treatment using biocontrol bacteria to inhibit Verticillium wilt Among the three control methods, pesticide control is the most widely used because of its convenience and efficiency, but it also causes serious pollution to the environment. In recent years, research on how to improve the tolerance of crops to plant diseases, increase yield, reduce irrigation costs, and reduce the use of chemical fertilizers and pesticides has become the core of the development of ecological agriculture. Biological control is the use of beneficial microorganisms to fight against pathogenic microorganisms, inhibit the growth of pathogenic bacteria, and improve the immunity of plants themselves, thereby playing a way of preventing and controlling plant diseases. However, the current strains and products for biological control of cotton verticillium wilt are very limited, and only a few products containing active microbial ingredients such as active bacillus, Trichoderma or Pseudomonas fluorescens can be used to control verticillium wilt. In addition, the host of Verticillium dahliae is very wide, such as cotton, soybean, tobacco, Arabidopsis, potato, tomato, citrus, cherry, strawberry, apple, barley, oats, blueberry, fruit crops and greenhouse vegetables, which seriously harm agriculture produce.

In short, looking for effective biological control strains and control methods is the development direction and effective way to prevent and control the verticillium wilt of crops.

Summary of the invention

The technical problem to be solved by the present invention is to overcome the shortcomings and deficiencies of the existing verticillium wilt disease prevention and control technology, provide a kind of biocontrol bacteria that has a better inhibitory effect on the verticillium wilt disease of various crops, and is to use microorganisms instead of chemical synthesis Fungicides provide new development resources and can be used as biological pesticides for development and utilization.

The purpose of the present invention is to provide a strain of Pseudomonas 22G5.

Another object of the present invention is to provide the application of the Pseudomonas 22G5 in preventing and controlling Verticillium wilt of crops.

The above objectives of the present invention are achieved through the following technical solutions:

According to the present invention, a strain of Pseudomonas donghuensis 22G5 was obtained through screening and identification, and was deposited in the General Microbiology Center of the China Microbial Culture Collection and Management Committee on July 8, 2019, with the preservation number CGMCC No. 18084.

The study found that the strain Pseudomonas 22G5 formed an obvious inhibition zone on the plate containing Verticillium dahliae strains V07df2, V08df1, V15QY1 and V991 (strains provided by the Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences). The pot experiment did not affect the growth of cotton, and could effectively inhibit the infection of Verticillium dahliae strain V991 on cotton plants. It shows that the strain 22G5 of the present invention can be used to develop biological pesticides against verticillium wilt of crops. In addition, the genome information of the strain 22G5 shows that this strain has extremely high homology and similarity with the Pseudomonas model strain Pseudomonas donghuensis HYS(T), and has the specific siderophore compound 7- of the genus Pseudomonas. The tropolone synthesis gene cluster.

The discovery of this strain is beneficial to alleviate the problem of chemical abuse, and also provides resources for the use of biological control methods to prevent and control the verticillium wilt disease of plants.

Due to the wide host range of Verticillium dahliae, the application of the Pseudomonas 22G5 in preventing and controlling verticillium wilt of crops should also fall within the protection scope of the present invention.

Preferably, the crop verticillium wilt disease includes but is not limited to cotton verticillium wilt disease caused by Verticillium dahliae Kleb or Verticillium alboatrum.

Preferably, the Verticillium wilt disease includes cotton, soybean, tobacco, Arabidopsis, potato, tomato, citrus, cherry, strawberry, apple, barley, oats, blueberry and the like.

In addition, the biocontrol agent for the verticillium wilt disease of crops containing the Pseudomonas 22G5 should also fall within the protection scope of the present invention.

Preferably, the concentration of Pseudomonas 22G5 in the biocontrol preparation is OD ₆₀₀ =2.0.

As an alternative embodiment, the present invention also provides a method for preventing and controlling verticillium wilt of crops by inoculating the above-mentioned biocontrol agent to plant materials.

Preferably, the inoculation may adopt a soil mixing method.

Preferably, the inoculation may adopt the root dip method.

The invention has the following beneficial effects:

The present invention provides a Pseudomonas 22G5 strain, which has a broad-spectrum and significant inhibitory effect on various Verticillium dahliae strains, and can reduce the occurrence of Verticillium wilt in cotton.

The research of the present invention shows that Pseudomonas strain 22G5 can form an inhibition zone on a plate containing V07df2, V08df1, V15QY1 and V991 bacterial liquid; the inoculation test found that 22G5 can well inhibit the infection of cotton plants by pathogens and reduce cotton Incidence of Verticillium Wilt. Moreover, the strain 22G5 itself has no pathogenicity and can be used to develop biological pesticides against bacterial diseases of crops, providing new ideas and new ideas for the use of biological control of verticillium wilt.

Description of the drawings

Figure 1 shows the experimental steps for large-scale screening of Verticillium wilt antagonistic strains.

Figure 2 shows the evolutionary relationship between Pseudomonas strain 22G5 and other strains of the same genus that have been reported. It is a cluster map based on 10 housekeeping genes.

Figure 3 shows the prediction of the secondary metabolite synthesis gene cluster contained in Pseudomonas strain 22G5 that may be related to the growth inhibitory activity of Verticillium dahliae.

Figure 4 shows the inhibitory activity of Pseudomonas strain 22G5 on the growth of Verticillium dahliae.

Detailed ways

Hereinafter, the present invention will be further described with reference to the attached tables of the specification and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the following examples are all commercially available, among which Verticillium dahliae strains V07df2, V08df1, V15QY1 and V991 are provided by the Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, but are not limited thereto.

Example 1 Isolation and Screening of Pseudomonas 22G5

The experimental procedures for large-scale screening of Verticillium wilt antagonistic strains are shown in Figure 1a:

1. Sample collection

Samples were collected from Anwei, Zhejiang, Jiangsu, Xinjiang, Jilin, Heilongjiang, Hunan, Shanghai and other places. Weeds, corn, cotton, Soil-bearing root system samples of soybeans and other plants. The soil-bearing root samples were separated by the dilution-coated plate method. Pick soil, roots and other samples respectively and place them in a 50ml collection tube, add 45ml of water and mix and shake, generally 4 to 5 times of shaking, draw 800μl of the mixed solution, and use ddH ₂ O to perform a gradient of ^{10 1} -10 ⁵ on a 96-well plate dilution. Choose 10μl solution of suitable concentration to spread on LB medium, generally choose 10 ³ or 10 ⁴ dilution concentration for plate coating. The plate is incubated in the dark in a constant temperature incubator at 28 ℃ for 24 to 48 hours, and then kept in a refrigerator at 4 ℃. Cultivate in 7-15 days. To collect microbial strains, add 720μl of culture solution to a 96-well plate. There are many single colonies of different shapes, sizes, and colors on the petri dish. Use toothpicks to pick the bacteria randomly and cultivate them at 28°C on a shaker and 220rpm overnight. stand-by.

2. Strain screening

Strain screening was carried out by the plate inhibition zone test. The cotton Verticillium wilt V15QY1 was activated on the LB medium, and a single colony was picked and placed in a 10 ml centrifuge tube containing 3 ml of culture solution, and cultured with shaking at 28° C. and 220 rpm for 24 hours. Three ml aliquots were centrifuged in two 1.5 ml tubes, and centrifuged at 4000 rpm for 5 min. Aspirate the supernatant, add 500μl of water, mix evenly and transfer to the same 10ml centrifuge tube, add 2ml of water to make a 3ml solution. Adjust and calculate the OD value to ensure OD ₆₀₀ = 0.2. Prepare 0.8% (mass fraction) of Top Agar, prepare 6 ml of Top Agar for each medium, and keep the water bath at a constant temperature of 55°C. Mix the pathogen and Agar on the plate, and let it stand for 1 hour to dry. Take 5μl from the 96-well plate and spot it on the plate, spot 16 samples in each petri dish, dry it, and then incubate it upside down in a 28°C incubator, and seal the membrane. Place the petri dish in an incubator at 28°C. Take out the petri dish from the incubator after 24 hours and observe whether there is a clearly visible circle of inhibition around the pre-selected strains. A strain with good antagonistic activity against Verticillium dahliae was screened, and the number of the strain library in our laboratory was 22G5 (Figure 1b).

Example 2 Identification and genome sequencing of Pseudomonas fluorescens 22G5

1. Morphological identification

Strain 22G5 is a gram-negative bacteria, non-spore forming, single or multiple flagella provide movement. After culturing on LB medium for 24 hours, larger colonies can be formed, the colonies do not produce pigment, the surface is convex, smooth, thicker, and easy to provoke.

2. Molecular identification

In order to clarify the bacterial species classification of the 22G5 strain obtained in Example 1, we analyzed the 16S ribose DNA (rDNA) sequence of the 22G5 strain for molecular biological identification of bacteria. The results showed that the 16S rDNA sequence of 22G5 was similar to pseudomonas. The model strain Pseudomonas donghuensis HYS(T) has the highest homology of 99%, indicating that the strain belongs to the species Pseudomonas donghuensis. Pseudomonas is a type of Gram-negative bacteria containing multiple species. Due to their diversity at the morphological structure level, genome level, and metabolic level, they are widely present in a variety of soils, water bodies, and hosts. At present, the most studied Pseudomonas include the opportunistic pathogen Pseudomonas aeruginosa in animals, the plant opportunistic Pseudomonas syringae, and the fluorescent pseudomonas with plant growth promoting effect. Pseudomonas fluorescens. In order to reveal the evolutionary relationship between the 22G5 strain and these Pseudomonas strains of different genus, the MEGA software Neighbor-joining method was used to construct based on 10 housekeeping genes (acsA, aroE, dnaE, guaA, gyrB, mutL, ppsA, pyrC). , recA and rpoB) evolutionary tree, analyzed the evolutionary relationship between 22G5 and Pseudomonas donghuensi model strains of different species. The results showed that 22G5 was evolutionarily related to Pseudomonas donghuensi model strains Pseudomonas donghuensis HYS (T) closely clustered together (Figure 1a), and has the closest evolutionary relationship with other Pseudomonas putida species. In summary, the 22G5 strain was identified as Pseudomonas donghuensis 22G5. The strain was deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee on July 8, 2019, with the preservation number CGMCC No. 18084, and the preservation address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Zip Code Is 100101.

3. Genomic characteristics

The 22G5 genome size is 6,546,541 bases; the GC content is 60.7%; it has 7862 protein coding regions (CDS), and the average length of the protein coding sequence is 725bp (Table 1). The prediction results of secondary metabolite compounds show that 22G5 only has the pyoverdine synthetic gene cluster (synthesized by nonribosomal peptides, NRPS genes) commonly found in pseudomonas. In addition, two NRPS-like gene clusters are also predicted in 22G5, which have certain homology compared with the known synthetic gene clusters of Mangotoxin and Chejuenolide in other known strains, but the specific structure of their products And the function is not clear (Figure 3b). Very importantly, we found that 22G5 also has a synthetic gene cluster of Pseudomonas donghuensis HYS(T) siderophore 7-hydroxytrophinol (Figure 3b). Iron is an indispensable element for all living organisms, and iron nutrition is an important limiting factor for the growth of microorganisms and plants in the agricultural environment. In a low-iron environment, certain microorganisms can produce a special organic compound that can bind with iron, called siderophores (also known as siderophores), which specifically chelate iron ions, and use a specific transport system to transfer iron Transfer to the body to meet the growth needs of the microorganisms themselves, thereby reducing the iron concentration in the environment, so that the pathogenic microorganisms cannot grow and reproduce due to lack of iron, thereby achieving the purpose of controlling plant diseases. 7-hydroxytropolone (7-hydroxytropolone) is a new type of siderophore, currently only reported in Pseudomonas donghuensis that it can be synthesized naturally by Pseudomonas spp. The gene cluster for synthesizing it has 12 genes, including Synthetic genes and regulatory genes (Figure 3a).

Table 1 Genomic characteristics of Pseudomonas strain 22G5

To	22G5
Gene size (bp)	6,546,541
Number of contigs	3185
GC content (%)	60.7
Number of CDS	7862
CDS average size (bp)	725
rRNA quantity	4
tRNA number	75

Example 3 Determination of antibacterial activity of Pseudomonas 22G5

In order to better study the biocontrol potential of the strain 22G5 of the present invention, we have studied the antibacterial spectrum of the strain. The specific operations are as follows:

Inhibition zone experiment

In order to explore whether 22G5 has broad-spectrum resistance to multiple strongly pathogenic strains of Verticillium dahliae, the same method as the strain screening in Example 1 was used, and the pathogenic bacteria V15QY1 was replaced with the same amount of the same culture conditions and methods. V07df2, V08df1 and V991 are multiple highly pathogenic strains, and the rest of the steps remain unchanged. The experimental results found that the strain 22G5 also has a strong antibacterial effect on Verticillium dahliae V07df2, V08df1 and V991 (Figure 3).

Example 4 Determination of the Control Effect of Pseudomonas 22G5 on Verticillium Wilt of Cotton Plants

The Verticillium dahliae strain V991 and the biocontrol strain 22G5 were activated on the LB medium, and single colonies were picked up in 50ml LB medium, 28°C, 220rpm shaker and cultured for 24h. Take 50 mL _{of 22G5 bacterial solution with OD 600} ＝2 and mix the same amount of V991 bacterial solution to form the treatment group; add 50 mL of LB bacterial solution to V991 bacterial solution and mix as a control. Use ddH ₂ O to dilute the mixed solution of different treatments 1:10, and use the 10-fold diluted mixed solution to irrigate individual cotton plants. Observe the growth and incidence of cotton seedlings every day and make records.

In the biological control test of cotton plant TM-1 with a sample of 12, after 58 days of treatment, compared with the treatment group with deciduous verticillium wilt V991 and the antagonistic strain 22G5 (V991+22G5), there is no antagonistic 22G5 protection control The infection of cotton plants in the group was more serious (Figure 4a). Observations were made on both 23 and 58 days of treatment, and the number of plants in each group was counted. The disease index of the treatment group watered with the biocontrol bacteria 22G5 was lower than that of the control group (Table 2). Similarly, in the repeated test of plant biocontrol with a sample of 14, compared with the control group (V991), the disease index of the plants in the treatment group (V991+22G5) also showed a downward trend, again verifying the antagonistic effect of 22G5 (Figure 4b, Table 2).

Table 2 Statistics of disease index of 22G5 plant biocontrol experiment

Disease index (DI) is also called disease index and infection index, which refers to the average disease number calculated from a certain number of plants or plant organs according to the disease level of each diseased individual plant. According to the diseased area of the leaves of the infected plants, the number of diseased leaves of each cotton plant is divided into 5 levels: 0 level: no disease on the leaves of the plant; level 1: less than or equal to 25% of the number of diseased leaves on the plant; level 2: the number of diseased leaves on the plant The number of leaves is greater than 25% and less than or equal to 50%; level 3: the number of leaves on the plant is greater than 50% and less than or equal to 75%; level 4: the number of leaves on the plant is 75% to 100%. After statistics, the disease index is calculated according to the formula. A value of 0 means that the population is free of disease, and a value of 100 means that the population has the most serious disease.

(dc: disease grade of plants; nc: number of diseased plants corresponding to each grade; nt: total number of plants)

Experimental results show that the strain 22G5 can significantly reduce the incidence of Verticillium wilt on cotton plants (Figure 4). This is also the first time that a strain of Pseudomonas donghuensis has been found to inhibit Verticillium wilt.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, etc. made without departing from the spirit and principle of the present invention Simplified, all should be equivalent replacement methods, and they are all included in the protection scope of the present invention.

Claims (8)

1. A strain of Pseudomonas (Pseudomonas donghuensis) 22G5, whose classification is named Pseudomonas donghuensis, was deposited in the General Microbiology Center of the China Microbial Culture Collection and Management Committee on July 8, 2019, and the preservation number is CGMCC No.18084.
2. An application of the Pseudomonas 22G5 of claim 1 in the prevention and treatment of verticillium wilt of crops.
3. The application according to claim 2, characterized in that Pseudomonas 22G5 is used to prevent and treat verticillium wilt of crops through the production of 7-hydroxy hinokitiol.
4. The application according to claim 2, characterized in that the verticillium wilt of crops comprises a fungal disease caused by Verticilium dahliae or Verticillium alboatrum.
5. The application according to claim 2, wherein the crops include cotton, soybeans, tobacco, Arabidopsis, potatoes, tomatoes, citrus, cherries, strawberries, apples, barley, oats, blueberries.
6. The application according to claim 2, characterized in that the solution containing Pseudomonas 22G5 is inoculated to crops for the prevention and control of verticillium wilt.
7. The application according to claim 6, wherein the concentration of Pseudomonas 22G5 in the solution containing Pseudomonas 22G5 is OD ₆₀₀ =2.0.
8. The application according to claim 6, wherein the method of inoculation includes soil mixing method and root dip method.

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