CN110643538B

CN110643538B - Pseudomonas fluorescens XY2F4 and application thereof in preventing and treating crop verticillium wilt

Info

Publication number: CN110643538B
Application number: CN201910978568.3A
Authority: CN
Inventors: 关雪莹; 陶晓园; 张海琳
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2019-10-15
Filing date: 2019-10-15
Publication date: 2021-04-27
Anticipated expiration: 2039-10-15
Also published as: CN110643538A; WO2021073143A1

Abstract

The invention discloses Pseudomonas fluorescens (Pseudomonas proteins) XY2F4 and application thereof in preventing and treating crop verticillium wilt. The pseudomonas fluorescens XY2F4 is preserved in China general microbiological culture Collection center in 2019, 06 and 24 months, and the preservation number is CGMCC No. 18017. The pseudomonas fluorescens strain XY2F4 has no pathogenicity to plants, has obvious inhibiting effect on cotton Verticillium wilt caused by Verticillium dahliae, can obviously reduce the cotton Verticillium wilt, and has good biological control effect. Genome research shows that the Pseudomonas fluorescens strain Pseudomonas protegens XY2F4 has a series of synthetic gene clusters of biological control related compounds, can be used for developing biological pesticides for crop verticillium wilt diseases, and provides a new thought and a new method for biologically controlling crop verticillium wilt diseases.

Description

Pseudomonas fluorescens XY2F4 and application thereof in preventing and treating crop verticillium wilt

Technical Field

The invention belongs to the technical field of plant disease biocontrol. More particularly, relates to Pseudomonas fluorescens proteins XY2F4 and application thereof in preventing and treating crop verticillium wilt.

Background

Cotton is an important economic crop in China and provides a renewable raw material for the textile industry. Verticillium wilt of cotton caused by Verticillium dahliae Kleb is a destructive disease, and the spreading of the disease not only obviously reduces the quality of cotton fibers, but also seriously affects the economic yield of cotton. Worldwide, each year, verticillium wilt caused by verticillium dahliae causes direct economic losses in excess of billions of dollars. In 1982, the cotton verticillium wilt has spread to nearly 13 million hectares in agricultural soil area in China. By 1993, the cotton verticillium wilt becomes a main obstacle for high and stable yield of cotton in China, and the disease area reaches 266.6 million hectares. At present, about half of the area of a cotton planting area in China is affected by verticillium wilt diseases, and the economic loss reaches 15-20 billion yuan each year.

The verticillium wilt belongs to soil-borne seed-borne vascular bundle diseases and is mainly propagated and spread through cotton seeds with bacteria, cotton seed cakes, cotton seed hulls, diseased plant residues, soil, fertilizers, running water, farmland management tools and the like. The germs invade from the roots and further systemically infect and harm cotton plants, so that diseases can be shown in each growth stage from a seedling stage to a plant-growing stage. As the pathogenic bacteria verticillium dahliae disease treatment key mechanism is not clear and the cotton high-resistance germplasm resources are lacked, the prevention and treatment of the cotton verticillium wilt have not made a breakthrough progress so far. The current prevention and control direction mainly is a comprehensive control measure combining reduction of the use of artificially synthesized bactericides, utilization of verticillium wilt resistant/disease-tolerant varieties as much as possible, improvement of soil ecological conditions and induction of disease resistance of cotton plants. The comprehensive control in practical production is mainly divided into three aspects. (1) The soil permeability is increased through agricultural control methods such as crop rotation, soil deep ploughing, timely intertillage and the like, the control on germ infection is enhanced, and the resistance of cotton plants is improved; (2) pesticides such as mepiquat chloride and chemical pesticides are used for pesticide control; (3) the microbial agent has the function of inhibiting verticillium wilt by biocontrol bacteria. Of the three control modes, the pesticide is most widely applied at present due to the convenience and high efficiency of control, but causes serious pollution to the environment. In recent years, research on how to improve the tolerance of crops to plant diseases, improve the yield, reduce the irrigation cost and reduce the use amount of fertilizers and pesticides becomes the core of the development of ecological agriculture. Biological control is a way of controlling plant diseases by using beneficial microorganisms to resist pathogenic bacteria microorganisms, inhibiting the growth of pathogenic bacteria and improving the immunity of plants. However, the strains and products for biological control of cotton verticillium wilt are very limited at present, and only a few products containing active microbial components such as active bacillus, trichoderma or fluorescent pseudomonas can be used for controlling verticillium wilt. In addition, hosts of verticillium wilt pathogens are very wide, and crops such as cotton, soybean, tobacco, arabidopsis thaliana, potato, tomato, citrus, cherry, strawberry, apple, barley, oat, blueberry, fruit tree crops, greenhouse vegetables and the like seriously damage agricultural production.

In a word, the search for effective biological control strains and control methods is a development direction and an effective way for controlling crop verticillium wilt diseases.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects and shortcomings of the existing verticillium wilt disease control technology, provides a biocontrol bacterium with better inhibiting effect on verticillium wilt diseases of various crops, provides a new development resource for replacing a chemically synthesized bactericide with microorganisms, and can be developed and utilized as a biological pesticide.

The invention aims to provide pseudomonas fluorescens XY2F 4.

The invention also aims to provide application of the pseudomonas fluorescens XY2F4 in preventing and treating crop verticillium wilt diseases.

The above purpose of the invention is realized by the following technical scheme:

the invention obtains a fluorescent Pseudomonas (Pseudomonas protegens) XY2F4 by screening and identifying, and the fluorescent Pseudomonas is preserved in China general microbiological culture Collection center (CGMCC) 24.06.2019 with the preservation number of CGMCC No. 18017.

The strain pseudomonas fluorescens XY2F4 forms an obvious antibacterial ring on a flat plate containing verticillium dahliae strains V15QY1, V07DF2, V08DF2, V08T2 and V16DF4 (provided by Jiangsu province academy of agricultural science and plant protection institute), does not influence the growth of cotton in a greenhouse pot experiment, and can effectively inhibit the infection of verticillium dahliae V15QY1 on the cotton plant. The strain XY2F4 of the invention can be used for developing biological pesticides aiming at crop verticillium wilt. In addition, the genome information of the strain XY2F4 shows that the strain has extremely high homologous similarity with the well-known biocontrol strains, namely, the Pseudomonas fluorescens strains CHA0 and Pf-5, has a gene cluster synthesized by a series of biocontrol related compounds of the Pseudomonas fluorescens strains CHA0 and Pf-5 together, and has evolved a certain number of novel compound metabolic pathways and fascicular (Flp pilus) fasciculate fiber protein coding gene clusters (related to the adhesion and colonization of bacteria in the roots of plants), thereby indicating that the strain has better capability of exchanging substances/signals with host plants and colonization in the roots of plants.

The discovery of the strain is beneficial to relieving the abuse problem of chemical agents, and provides resources for preventing and treating the plant verticillium by using a biological control method.

Because the host range of the verticillium dahliae is wide, the application of the pseudomonas fluorescens XY2F4 in the aspect of preventing and treating the verticillium wilt of crops is also within the protection range of the invention.

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

Preferably, the verticillium wilt disease comprises cotton, soybean, tobacco, arabidopsis, potato, tomato, citrus, cherry, strawberry, apple, barley, oat, blueberry, fruit crops and greenhouse vegetables.

In addition, a biocontrol agent for crop verticillium wilt diseases containing the pseudomonas fluorescens XY2F4 also belongs to the protection scope of the invention.

Preferably, the concentration of Pseudomonas fluorescens XY2F4 in the biocontrol formulation is OD₆₀₀＝1.0～2。

As an alternative embodiment, the invention also provides a method for controlling the verticillium wilt disease of crops, and the biocontrol agent is inoculated to plant materials.

Preferably, the inoculation can be carried out by a soil mixing method.

Preferably, the inoculation can be by root dipping.

The invention has the following beneficial effects:

the invention provides pseudomonas fluorescens XY2F4, which has broad-spectrum and obvious inhibiting effect on various verticillium dahliae strains and can reduce the verticillium wilt of cotton.

The research of the invention shows that the pseudomonas fluorescens strain XY2F4 can form a bacteriostatic circle on a flat plate containing V15QY1, V07DF2, V08DF2, V08T2 and V16DF4 bacterial liquid; an inoculation test shows that XY2F4 can well inhibit infection of pathogenic bacteria to cotton plants and reduce the incidence of cotton verticillium wilt. In addition, the strain XY2F4 has no pathogenicity, can be used for developing biological pesticides for crop bacterial diseases, and provides a new idea for biologically preventing and treating crop verticillium wilt.

Drawings

FIG. 1 shows the relationship of P.fluorescens strain XY2F4 in evolution with other strains of the same species that have been reported. FIG. 1a is a cluster map based on 10 housekeeping genes; FIG. 1b shows the analysis of XY2F4 for the consensus and unique genes of the two most similar P.fluorescens strains CHA0 and Pf 5; FIG. 1c is annotation information for 960 genes specific to XY2F 4;

FIG. 2 shows the secondary metabolite synthesis gene cluster and structure contained in P.fluorescens strain XY2F4 and possibly related to the growth inhibitory activity of verticillium dahliae.

FIG. 3 shows the inhibitory activity of P.fluorescens strain XY2F4 on the growth of Verticillium dahliae.

FIG. 4 shows that P.fluorescens strain XY2F4 significantly reduced the cotton verticillium wilt disease.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available. Among them, strains V15QY1, V07DF2, V08DF2, V08T2, V16DF4 and V991 were provided by plant protection research institute of agricultural academy of sciences of Jiangsu province, but not limited thereto.

Example 1 isolation and screening of Pseudomonas XY2F4

1. Sample collection and strain culture: collecting soil-carrying plant root system samples in soil, water and near plant roots in various places such as Yangtze river basin, Xinjiang cotton production area, Hainan cotton south breeding base, northeast agricultural production area and the like, bagging and storing the samples, and carrying the samples back to a laboratory for strain separation. Diluting and coating flat plate method for soil-bearing root system sampleIsolation of the strain was performed. Respectively selecting samples such as soil, root system and the like, placing the samples into a 50ml collecting pipe, adding 50ml of water, mixing and oscillating for 4-5 times, generally, sucking 800 mu l of mixed solution, and using ddH₂O on 96-well plates 10¹-10⁵Gradient dilution of (3). The solution is spread on LB medium at a suitable concentration of 10. mu.l, typically 10³Or 10⁴Coating the plate with the diluted concentration, carrying out dark inversion culture on the plate in a constant-temperature incubator at 28 ℃ for 24-48 h, and continuously culturing in a refrigerator at 4 ℃ for several days. Selecting single colonies with different shapes, sizes and colors for purification and culture. 720. mu.l of the culture medium was added to a 96-well plate, picked with toothpicks and cultured with shaking overnight at 28 ℃ and 220rpm for future use.

2. Screening of verticillium wilt antagonistic strains: screening of verticillium wilt resistant strains is carried out by adopting a plate zone test. Activating cotton verticillium wilt V15QY1 on LB culture medium, picking out the monoclone of cotton verticillium wilt, placing the monoclone in a 10ml centrifuge tube containing 3ml culture solution, shaking and culturing for 24h at 28 ℃ and 220rpm by a shaking table. The conidia are collected by centrifugation at 4000rpm for 5 min. The supernatant was aspirated and resuspended in 3ml of water. Adjusting and calculating OD value to ensure OD₆₀₀0.2. 0.8% (mass fraction) of agar was prepared, and 6ml of agar was prepared for each medium, and the water bath was kept at a constant temperature of 55 ℃. Mixing the germs and agar, spreading on a plate, standing for 1h, and air drying. Pipetting 5. mu.l of the solution into a 96-well plate, spotting 16 samples on each petri dish, air-drying, performing inverted culture in a constant temperature box at 28 ℃, and sealing the membrane. And (3) placing the culture dish in an incubator at 28 ℃, taking out the culture dish from the incubator after 24 hours, and observing whether a clearly visible inhibition zone exists around the preselected strain. A strain with good inhibitory activity against the growth of Verticillium wilt disease was selected, and the strain is numbered XY2F4 in the laboratory strain pool (FIG. 3 a).

Example 2 identification and genome sequencing of Pseudomonas fluorescens XY2F4

1. Morphological identification

Strain XY2F4 provides movement for gram-negative bacteria, non-sporulation, single or multiple flagella. After 24 hours of culture on an LB culture medium, a large colony can be formed, the colony can produce pigment, the surface is convex, smooth and viscous, and the colony is easy to pick up.

2. Molecular identification

To clarify the classification of the species of strain XY2F4 obtained in example 1, we analyzed the 16S ribose DNA (rDNA) sequence of strain XY2F4 for the molecular biological identification of bacteria. The results showed that the homology of the 16SrDNA sequence of XY2F4 with Pseudomonas fluorescens strain CHA0 and Pseudomonas proteins CHA0 and Pseudomonas saponiphila DSM9751 strain reached up to 99%, indicating that this strain belongs to the general group of Pseudomonas. Pseudomonads are a group of gram-negative bacteria containing multiple species, which are widely present in a variety of soils, water bodies, hosts due to their diversity at the morphological, genomic and metabolic levels. Currently, the most studied pseudomonads include the opportunistic pathogens Pseudomonas aeruginosa, the plant opportunistic pathogens Pseudomonas syringae, and Pseudomonas fluorescens, which have plant growth promoting effects, in animals. In order to reveal the relationship of XY2F4 strain evolutionarily with strains of different genera of these Pseudomonas, an evolutionary tree based on 10 housekeeping genes (acsA, aroE, dnaE, guaA, gyrB, mutL, ppsA, pyrC, recA, rpoB) was constructed using the Neighbor-joining method using MEGA 5.0 software, and the evolutionary relationship of XY2F4 with model strains of different genera of Pseudomonas was analyzed, which revealed that XY2F4 was evolutionarily closely clustered with model strains CHA0 and Pf-5 of p. In conclusion, the XY2F4 strain was identified as Pseudomonas fluorescens (Pseudomonas proteins) XY2F 4. The strain is preserved in China general microbiological culture Collection center (CGMCC) in 2019 at 24.06.78 with the preservation number of CGMCC No.18017, the preservation address of No. 3 Siro 1 of Beijing, Chaoyang, and the postal code of 100101.

3. Genomic characterization

The size of the XY2F4 gene is 6,811,381 bases; GC content 63.6%; there were 6153 protein coding regions (CDS) and the average length of the protein coding sequence was 969bp (Table 1). The genome information was uploaded to the NCBI database at 2017, 12/06, with the accession number PIZE 00000000. Overall, the XY2F4 genomic profile was very similar to that reported for the biocontrol strain Pseudomonas proteins CHA0, Pf-5 (FIG. 1 a). Analysis of consensus genes and unique genes revealed that XY2F4 shares 5033 genes with strain CHA0, Pf-5 and 960 unique genes (FIG. 1 b). These specific genes mainly include genes related to the metabolism of membrane-transported amino acids and their derivatives, and carbohydrate metabolism (FIG. 1 c). In particular, XY2F4 has unique Type II and Type VI secretion systems, tryptophan metabolism, butanediamine and polyamine metabolism, maltose and maltodextrin metabolism gene clusters, and fasciculated fibrin encoding a Type of fimbriae (Flp fimbriae) which are involved in adhesion and colonization of bacteria in roots of plants, as compared with biocontrol strains Pseudomonas proteins CHA0, Pf-5 (table 2), suggesting that this strain has evolved some new substance metabolic pathways and protein secretion systems that interact with hosts and has better ability to colonize roots of plants, as compared with the standard strain.

TABLE 1 Pseudomonas fluorescens strain XY2F4 genomic characteristics

	XY2F4	CHA0	Pf-5
				Genome size (bp)	6,811,381	6,867,980	7,074,893
Number of contigs	50	1	1
				GC content (%)	63.6	63.4	63.3
Number of encoding genes	6153	6219	6342
				Average encoded Gene Length (bp)	969	971	977
Number of ribosomal RNA-encoding Gene clusters	8	11	16
				Number of transport RNA-encoding Gene clusters	60	64	70

TABLE 2 Gene clusters specific to Pseudomonas fluorescens strain XY2F4

We focused on biocontrol-associated genes and gene clusters in the XY2F4 genome, and the results showed that, similar to the standard strains CHA0, Pf-5, XY2F4 also has gene clusters for the synthesis of biocontrol-associated secondary metabolic compounds, including the gene clusters for the synthesis of iron vectors pyochelin and pyoverine, bacteriocin, 2, 4 diacetylphloroglucinol (2, 4-DAPG), rhizoxin and its derivatives, etc. (FIG. 2).

Example 3 determination of the bacteriostatic Activity of Pseudomonas XY2F4

In order to better study the biocontrol potential of the strain XY2F4 of the invention, the bacterial inhibition spectrum of the strain is studied. The specific operation is as follows:

XY2F4 has the ability to inhibit the growth of other pathogenic fungi. The same method as that for screening the strain in example 1 was used, and the pathogenic bacteria V15QY1 were changed to the same amounts of V07DF2, V08DF2, V08T2 and V16DF4 under the same culture conditions and method, and the rest of the procedures were unchanged. Experimental results show that the strain XY2F4 has strong bacteriostatic action on verticillium wilt diseases V07DF2, V08DF2, V08T2 and V16DF4 (figure 3 a).

In addition, XY2F4 also inhibited the growth of Verticillium dahliae. The verticillium dahliae to be tested is a common verticillium wilt strongly pathogenic strain V991. Respectively picking out the pathogenic bacteria V991 and the biocontrol bacteria XY2F4 to be monocloned in a 10ml centrifuge tube containing 3ml of culture solution, and carrying out shake culture at 28 ℃ and 220rpm for 24 h. Adjusting and calculating OD value by using sterile water to ensure OD₆₀₀0.2. Gradient dilution of XY2F4 with sterile water (dilution 10)⁶、10⁵、10⁴、10³、10²10-fold and undiluted), 200. mu.l of each of the 7 XY2F4 cultures at different concentrations were aspirated and 200. mu.l of OD was added₆₀₀Conidium solution of verticillium dahliae V991 of 0.2 was co-cultured for 1 hour. Then, 200. mu.l of the co-culture mixture was aspirated for plate coating, membrane sealing, and inverted culture at 28 ℃ for 24 hours. Counting single clone, and observing the single spore number of the verticillium dahliae of different groups. In the experiment, the number of V991 single spore growth is obviously reduced along with the increase of the concentration of XY2F4, and when the dilution factor of XY2F4 is 10 times, the V991 single spore growth can be completely inhibited (FIG. 3 b). The results show that XY2F4 has a remarkable inhibiting effect on verticillium wilt bacteria in a plate experiment.

Example 4 determination of the Effect of Pseudomonas XY2F4 on the prevention of Cotton plant verticillium wilt

Activating verticillium dahliae strain V15QY1 on PDA solid plate, activating strain XY2F4 on LB solid plate, respectively picking up single colony in 200ml culture bottle containing 50ml culture solution, shake culturing for 24h at 28 deg.C and 220rpm, adjusting to OD with clear water₆₀₀Is 1-2. Adjusted to OD in this example₆₀₀2. Mixing 50ml of XY2F4 bacterial liquid with 50ml of V15QY1 bacterial liquid to form a treatment group; 50mL of LB was added to 50mL of V15QY1 bacterial suspension, and the mixture was used as a control. By ddH₂And O, diluting the two groups of mixed solutions at a ratio of 1:10, inoculating the cotton single plants with the mixed solution diluted by 10 times, repeating the steps for 30 in each group, and observing the growth and morbidity of cotton seedlings every day. FIG. 4 shows the results of the measurement of verticillium wilt in cotton plants by the soil mixing method, and it can be seen that the plants shown by the arrows in the figure have all the leaves yellow and withered and have no viable green leaves. The disease index is a comprehensive index considering the morbidity and the severity, and the experimental result shows that the disease index of the cotton plants in the treatment group containing XY2F4 is reduced compared with the control group only treated by verticillium wilt bacterium V15QY1 (figure 4 a). In the repeated experiment with the sample size of 30, the consistent result is obtained, namely, the cotton leaf yellowing and wilting fall of the treatment group is reduced (disease index of the control group is 80% vs. disease index of the treatment group is 55.8%), and the XY2F4 is verified to have stronger resistance to cotton verticillium wilt again (fig. 4 b).

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. Pseudomonas fluorescens XY2F4, which is classified and named as Pseudomonas proteins and is preserved in China general microbiological culture Collection center (CGMCC) 24.06.2019 with the preservation number of CGMCC No. 18017.

2. The application of pseudomonas fluorescens XY2F4 in preventing and treating Verticillium wilt of cotton, which is characterized in that the Verticillium wilt is a fungal disease caused by Verticillium dahliae Kleb.

3. The use according to claim 2, characterized in that pseudomonas fluorescens XY2F4 is inoculated into crops for verticillium wilt control.

4. The use according to claim 3, wherein the inoculation method is a soil-mixing method or a root dipping method.

5. Use according to claim 3 or 4, wherein the Pseudomonas fluorescens XY2F4 is inoculated at OD₆₀₀＝1.0～2.0。