CN104017744A - Preparation method and application of pseudomonas chlororaphis for resisting disease and promoting growth - Google Patents

Abstract

The invention discloses a preparation method and application of a disease-resistant Pseudomonas chlororaphis agent; the bacterial strain is Psedunomonas chlororaphis HT66CCTCC NO: M2013467. The main metabolites of this strain were phenazine-1-carboxylic acid and phenazine-1-carboxamide. Inoculate the strain into the microbial culture medium, and obtain high-concentration bacteria and metabolites after fermentation, which can be mixed with powdered activated carbon, bentonite, bentonite, kaolin, diatomaceous earth, zeolite and calcium carbonate to prepare microorganisms Bacteria agent, the number of viable bacteria in the finished product is 1-8.5×10 ⁹ cfu/mL. It is confirmed by plate experiments that the bacterial agent has a good control effect on rice sheath blight, cotton fusarium wilt, wheat take-all and watermelon wilt pathogens, and also has a good promotion effect on the growth of some plants.

Description

Preparation method and application of Pseudomonas chloropinus agent for disease resistance and growth promotion

technical field

The invention belongs to the field of biological pesticides and biological fertilizers, and relates to a preparation method and application of a disease-preventing and growth-promoting microbial preparation; in particular, it relates to a preparation method and application of a disease-resistant and growth-promoting Pseudomonas chloropina agent. the

Background technique

The research results of the Food and Agriculture Organization of the United Nations show that the natural loss rate of crop diseases and insect pests is more than 37%, seriously affecting food security and environmental ecological security. According to the survey, there are 1,600 kinds of disease organisms in common crops in my country, causing losses of nearly 100 billion RMB every year, seriously hindering the development of my country's agriculture. At present, due to the extensive use of various chemical pesticides in agricultural production, many types of diseases and insect pests have developed severe resistance to these drugs, resulting in a continuous decline in control effects. According to reports, the types of pests and diseases that have broken out in my country have more than doubled in the past ten years. At the same time, some pesticides remain in soil, plants and water bodies to varying degrees, posing a potential threat to human health. When many agricultural and sideline products in my country are exported to foreign countries, they are often banned by foreign "green barriers" because of excessive pesticide residues. The annual loss is as high as tens of billions of dollars, which has aroused widespread concern from the whole society. the

Among the disseminated diseases of crops, there are mainly fungal diseases, bacterial diseases, viral diseases and nematode diseases. Among them, fungal diseases are the diseases with the largest number of known plant diseases, accounting for about 80%-90% of the disease types; and fungal diseases can appear in various parts of plants, and the types of symptoms are also the most. the

Rice sheath blight, also known as moire, is commonly known as flower foot stalk, rotten foot blast, eyebrow spot. It is caused by infection by Rhizoctonia solani, and it mostly occurs under high temperature and high humidity conditions. It is common in rice-growing countries in Asia, America, and Africa. It is distributed in various rice areas in my country, but it is the most serious damage in southern rice areas, causing a significant decrease in seed setting rate and thousand-grain weight, and even plant death. It is one of the main diseases in current rice production. Due to the wide occurrence area and high epidemic frequency, the losses caused by it often exceed that of rice blast. the

Fusarium wilt of watermelon is also known as vine cutting disease and wilting disease. Its pathogen is Fusarium oxysporum f.sp.niveum, which belongs to the fungus of half-knowledge fungus and belongs to the order Polypodium, and Tumoraceae survives in the soil. The time can be as long as 10 years. Watermelon can be diseased from the seedling stage to the adult plant stage, but the peak period of the disease is the flowering and fruit setting stage. In recent years, the occurrence of Fusarium wilt of watermelon in our country has been serious, resulting in a large reduction in watermelon production, generally 30% to 40% in diseased fields, and more than 80% or even no harvest in severely diseased fields, causing serious economic losses to watermelon production. the

Pythium ultimum Trow was originally isolated from rotting cress seedlings in the UK, and it has been reported to be widely distributed. It not only inhabits the soil, but also widely infects soybeans, kidney beans, peas, sweet potatoes, pine seedlings, coffee, Apple, citrus, peach, cotton, chrysanthemum, dahlia, pumpkin, watermelon, sugar cane, alfalfa, tomato and more than 150 kinds of economic plants cause many diseases such as seedling dieback, damping off, root rot, foot rot and wilting. the

Plant growth-promoting rhizobacteria (PGPR) are a group of rhizobacteria that colonize the plant rhizosphere and are closely related to plant roots. The results of a large number of greenhouse and field experiments show that many PGPR strains have obvious effects of promoting plant growth, preventing and controlling soil-borne diseases, improving plant resistance to abiotic stress, and promoting soil bioremediation under greenhouse and field conditions. PGPR is one of the effective ways to eradicate crop continuous cropping obstacle (replantation disease). the

Pseudomonas is a class of Gram-negative bacteria widely distributed in nature, and it is also a common microbial population in the plant rhizosphere. Many Pseudomonas can produce metabolites such as siderophore, antibiotics, extracellular hydrolase and hydrocyanic acid to protect plants from pathogenic microorganisms and induce plant resistance. At the same time, it can also secrete indole compounds to promote plant growth. As plant rhizosphere growth-promoting bacteria, Pseudomonas has been extensively studied by scientists all over the world. There are many kinds of antibiotics produced by Pseudomonas, including prorolnitrin (Prn), phenazine-1-carboxylic acid (phenazine-1-carboxylic acid, PCA), 2,4-diacetylgarcinol (2 ,4-diaacetylphloroglucinol, DAPG), luteolin (pyoluteorin, Plt), pyocyanin (pyocyanin, PYO), lipopolypeptide, etc. Phenazine compounds can be used as electron carriers to consume reducing substances such as NAD(P)H in cells, and at the same time generate reactive oxygen species (reactive oxygen species, ROS), causing target cells to poison and die. Therefore, phenazine compounds have broad-spectrum antifungal activity and are not easy to develop drug resistance. It can be seen that while vigorously developing high-efficiency and low-toxic chemical pesticides, screening and application of Pseudomonas that can secrete phenazine compounds for the prevention and treatment of crop fungal diseases is an economical and beneficial to sustainable agricultural production. effective way of development. the

Contents of the invention

The purpose of the present invention is to overcome the serious pollution in the production process of chemical pesticides used in the prevention and treatment of plant fungal diseases in the above-mentioned prior art, the high residues in soil, water and crops, and the increasingly serious drug resistance, etc., to provide a disease-resistant The preparation method and application of the growth-promoting Pseudomonas chloropina agent. The invention provides a microbial strain that can simultaneously prevent and treat various plant pathogenic fungi and promote plant growth——Pseudomonas chlororaphis HT66 (Psedunomonas chlororaphis), and use the bacterial strain to prepare microbial inoculants, and use the bacterial strain to control Plant diseases such as rice sheath blight, watermelon wilt, and beet chrysanthemum wilt. the

In order to achieve the above purpose, the inventor isolated and purified a green pigment-producing Pseudomonas HT66 for the first time, carried out PCR cloning and sequencing of its 16S rDNA, and analyzed its evolutionary relationship. The similarity is 99%. After whole-cell fatty acid analysis and comparison with the Sherlock database of MIDI Company, the similarity SI between the bacteria and Pseudomonas chloropinus in environmental samples was 0.343. the

When the Pseudomonas chloropinus HT66 grows on a solid agar medium, the colonies are round, with neat and regular edges, and the surface is moist and smooth; within 1 to 2 days of its growth, the colonies are khaki, and on the third day After ~4 days, more dark green substances will appear on the surface of the colony. the

After the Pseudomonas chloropinus HT66 was cultured in liquid for 2 days, the bacterial liquid was relatively thick and showed an obvious earthy yellow color; after standing overnight, a large amount of green precipitates appeared in the bottom layer of the fermentation liquid. After separation, purification and identification, the precipitate was a mixture of phenazine-1-carboxylic acid and phenazine-1-amide. the

The purpose of the present invention is achieved by the following technical solutions:

In the first aspect, the present invention relates to a kind of Pseudomonas chlororaphis HT66 (Psedunomonas chlororaphis HT66) CCTCC NO: M2013467. the

Preferably, the partial sequence of the 16S rDNA of the Pseudomonas chloropinus is shown in SEQ ID NO.1. the

Further preferably, the partial sequence of the 16S rDNA of Pseudomonas chloropinus is obtained by sequencing after PCR amplification with bacterial universal primers 27F and 1429R, wherein the sequences of 27F and 1429R are respectively as shown in SEQ ID NO.2 And shown in SEQ ID NO.3. the

Preferably, the main metabolites of Pseudomonas chloropinus are phenazine-1-carboxylic acid (phenazine-1-carboxylic acid, CAS No. 2538-68-3, referred to as PCA) and phenazine-1-carboxylate Phenazine-1-carboxamide (CAS No. 550-89-0, referred to as PCN). the

Preferably, the Pseudomonas chloropinus can suppress Rhizoctonia solani (Rhizoctonia solani), Fusarium oxysporum f.sp.niveum (Fusarium oxysporum f.sp. ) and Septoria steviae Ishiba Yokoyama et Tani. the

In a second aspect, the present invention relates to a disease-resistant Pseudomonas chlororaphis agent for promoting growth, using microbial culture medium as a raw material, after Pseudomonas chlororaphis HT66 (Psedunomonas chlorophis HT66) CCTCC NO: M2013467 is expanded and cultivated by cells Prepared. the

Preferably, the carrier of the bacterial agent is one or more of powdered activated carbon, bentonite, bentonite, kaolin, diatomaceous earth, zeolite, and calcium carbonate. the

In the third aspect, the present invention relates to the use of the above-mentioned anti-disease and growth-promoting Pseudomonas aeruginosa agent in the preparation of biological pesticides. the

Preferably, the biopesticide is used to control rice sheath blight, watermelon wilt, stevia leaf blight or soybean, kidney bean, pea, sweet potato, pine seedling, coffee, apple, citrus, peach, cotton, chrysanthemum, dahlia, pumpkin, Biopesticide for root rot in watermelon, sugarcane, alfalfa, tomato crops. the

In the fourth aspect, the present invention relates to the use of the above-mentioned disease-resistant and growth-promoting Pseudomonas chloropsis agent in the preparation of microbial fertilizers. the

Compared with the prior art, the present invention has the following beneficial effects:

1. Pseudomonas bacteria used for biological control include Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri and Pseudomonas chloropinus; except Pseudomonas aeruginosa Except for Pseudomonas aeruginosa and P. There is no report of pathogenicity so far, so it is environmentally friendly, safe and reliable. the

2, Pseudomonas chloropinus used in the present invention, its wild strain liquid culture time phenazine-1-carboxamide concentration is up to 420mg/L, is the wild strain with the highest phenazine-1-carboxamide output in the world at present, Therefore, its biological defense function is powerful. the

3. The development cost of the bacterial agent of the present invention is low: Pseudomonas chloropinus has low nutritional requirements and good stability, and the carrier and auxiliary agents selected for processing the bacterial agent are cheap, and the production process is simple and easy. the

4. The bacterial agent of the present invention can effectively prevent and treat fungal diseases such as rice sheath blight, wheat take-all, watermelon wilt, and cotton wilt. the

Description of drawings

Other characteristics, objects and advantages of the present invention will become more apparent by reading the detailed description of non-limiting embodiments with reference to the following drawings:

Figure 1 is a schematic diagram of the solid agar plate culture characteristics of Pseudomonas chloropinus HT66;

Figure 2 is a schematic diagram of the phylogenetic tree analysis of Pseudomonas chloropinus HT66;

The liquid chromatogram of Fig. 3 Pseudomonas aeruginosa HT66 metabolite;

Figure 4 is the antibacterial experiment pictures of Pseudomonas chloropinus HT66. Among them, the upper left picture is the pathogen of rice sheath blight, the upper right picture is Stevia leaf spot blight, the lower left picture is Pythium ultima, and the lower right picture is Fusarium wilt of watermelon . the

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those skilled in the art can make some adjustments and improvements without departing from the concept of the present invention. These all belong to the protection scope of the present invention. the

Pseudomonas chlororaphis HT66 (Psedunomonas chlorophis HT66) of the present invention, this bacterial strain has been preserved in the China Type Culture Collection Center (CCTCC for short), and the address of the preservation unit is: China. Wuhan. Wuhan University, zip code: 430072, preserved The date is: October 12, 2013, and the deposit number is: CCTCC NO: M2013467. the

Example 1

1. Acquisition and identification of Pseudomonas chloropinus HT66

First, select a vigorously growing rice plant without any visible lesions in a dry paddy field, remove the stem part of the rice and the 5 cm thick surface soil, and dig out a rhizosphere soil sample with a sterile medicine spoon; weigh 1 g Soil samples were placed in a 250mL Erlenmeyer flask filled with 50mL KMB medium, and ampicillin (final concentration was 50 μg/mL), chloramphenicol (final concentration was 15 μg/mL) and cycloheximide (final concentration The concentration is 100μg/mL) to reduce the contamination of fungi in the soil. Then place the flask on a shaking table with a rotating speed of 200rpm at 28°C for 2 hours; take a sample, dilute it 1000 times, and spread it on a solid KMB plate containing the same antibiotic, and use the asynchronous culture method to test the resistance of rice sheath blight to the single colony obtained. In the experiment of pathogenic bacteria, the strain with the highest antibacterial activity was Pseudomonas aeruginosa HT66, as shown in Figure 1. the

Fig. 1 is a schematic diagram of the solid agar plate culture characteristics of Pseudomonas chloropinus HT66. Strain HT66 grew on KMB solid medium for 12 hours to form milky white colonies with a diameter of about 1.5mm, round shape, raised surface, smooth, viscous, easy to pick up, and neat edges; with the extension of culture time, The colony gradually turns yellow; observed under a microscope, the bacteria are rod-shaped, 0.3μm-0.7μm×1.0μm-1.2μm, Gram staining is negative, and there are no spores. After 24 hours, green crystals gradually appeared on the surface of the colony, which was a mixture of phenazine-1-carboxylic acid and phenazine-1-carboxamide. the

The strain HT66 was cultured in KMB medium to the end of logarithmic growth, and its genomic DNA was extracted with a rapid bacterial genomic DNA extraction kit (product of Shanghai Jierui Bioengineering Co., Ltd.). Using the genome sample as a template, the bacterial 16s rDNA universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1429R (5'-TACGGHTACCTTGTTACGACTT-3') were used for PCR experiments, and the 16s rDNA sequence obtained by sequencing after amplification, As shown in SEQ ID NO.1. Blast analysis was performed on the sequence in the NCBI nucleic acid database, and the results showed that most of the microorganisms highly homologous to HT66 belonged to the genus Pseudomonas. Select the first 15 species, download their 16S rDNA sequences, and use MEGA5 software to construct evolution Tree. Fig. 2 is a schematic diagram of the phylogenetic tree analysis of strain HT66. It can be seen from Fig. 2 that the confidence level of the homology between the strain HT66 and Pseudomonas chloroacinatus GP72 is 93, so it is preliminarily proved that the bacterium is a strain of Pseudomonas chlorospinatus. the

2. Whole-cell fatty acid analysis of Pseudomonas chloropinus HT66

Using the four-section line method, inoculate the fully activated HT66 strain on the KMB plate, and use the inoculation loop to collect about 40 mg of the bacteria in the third area after 24 hours. The whole-cell fatty acid extraction samples of HT66 were obtained by saponification, methylation, n-hexane extraction and alkaline washing, and then the fatty acid composition was analyzed using MIDI's automatic microbial identification system. Then, using covariance matrix analysis, principal component analysis and pattern recognition analysis techniques, the analysis results of gas chromatography were compared with MIDI's clinical microbiological database and environmental microbiological database. The results are shown in Table 1. the

Table 1. Sherlock MIS microbial automatic identification system report

database similarity index strain name

RTSBA6 0.343 Pseudomonas-chlororaphis/aureofaciens/aura RCLIN6 0.228 Pseudomonas-putida-biotype A

As shown in Table 1, the similarity index between the strain HT66 and Pseudomonas aeruginosa in the environmental microbial database is 0.343, which is higher than the reliability standard of 0.25, thus further proving that the bacteria is a strain of Pseudomonas aeruginosa . the

Three, the preparation method of Pseudomonas chloropinus HT66 inoculum

The preparation method of Pseudomonas aeruginosa HT66 bacterial agent comprises the steps:

1. Activation of Pseudomonas chloropinus strains: Prepare solid LB or KMB culture medium respectively; take out the frozen Pseudomonas chloropinus HT66, inoculate it on the solid medium and activate it at 26-32°C for 48 hours, then Transfer to a new solid medium, continue to pass 2 to 3 times;

2. Seed preparation of Pseudomonas chloropinus: Prepare liquid LB or KMB medium; pick a ring of activated bacteria, inoculate into 50mL liquid medium (packed in a 250mL Erlenmeyer flask), and place at 26-32°C The constant temperature shaker was shaken for 10-20 hours, and the shaker speed was 150-5000 rpm. Then, the seed liquid is further transferred to 1000-5000 mL of liquid medium, and cultivated under the same conditions to obtain a large number of vigorous and robust seeds; wherein, the composition of the LB medium is: The base contains 10 grams of tryptone, 5 grams of yeast extract, 10 grams of sodium chloride, and pH 7.5; the solid medium is 1.2% agar powder added to the corresponding liquid medium. the

Wherein, the King's B medium (KMB): every liter of culture medium contains 20 grams of tryptone, 0.392 grams of K2HPO, 15 milliliters of glycerol, 0.732 grams of MgSO4, and pH7.2; Add 1.2-1.5% agar powder to the liquid medium. the

3. Fermentation and cultivation of Pseudomonas chloropinus: Prepare a large volume of liquid LB or KMB medium, put it into a fermenter and sterilize it at 121°C for 20 minutes; when the temperature of the fermenter is cooled to 26-32°C, add % into the seed solution of Pseudomonas chloropinus; then cultured with aeration and agitation at 26-32°C, the rotation speed of the stirring paddle in the fermenter was 150-300 rpm, and the aeration ratio was 1:1 (VVM). The time is 30-40 hours. the

There are no special requirements for the composition of the fermentation medium. The carbon source is starch, glucose, sucrose or glycerin, and the nitrogen source can be peptone, soybean meal, meat extract, corn steep liquor, etc., and an appropriate amount of inorganic salts such as phosphate and magnesium sulfate are added. The pH value of the fermentation broth was kept at 7.0-8.0. the

4. Add carrier: Weigh an appropriate amount of powdered activated carbon, bentonite, bentonite, kaolin, diatomaceous earth, zeolite or calcium carbonate, etc., and add it to the fermentation broth of Pseudomonas chloropinus in a mass ratio of 5-50%. And mix well before use. the

4. Analysis of metabolites of Pseudomonas chloropinus HT66

After fully activating HT66 on the plate, inoculate it in a 250mL Erlenmeyer flask containing 50mL KMB medium, 180rpm, culture at 28°C for 16h, then transfer to a 2L Erlenmeyer flask containing 500mL KMB medium, 180rpm, 28°C Cultivate for 48h. 1 mL was sampled regularly, the pH was adjusted to 2.0 with 6 mol/L hydrochloric acid, and the fermentation broth was repeatedly extracted 3 times with an equal volume of ethyl acetate. The organic solvents were combined, and the sample was fully evaporated to dryness using a rotary evaporator and a vacuum oven, and the sample was redissolved in 5 mL of methanol (chromatographic grade). Adopt C18 reverse high performance liquid chromatography column (46 * 250mm, 5 μ m, WondaSil, Japan) to analyze, mobile phase is composed of: 0-2min, 8% acetonitrile-92% 25mM ammonium acetate; 2-20min, acetonitrile concentration from 8% When it rises to 60%, the ammonium acetate concentration drops from 92% to 40%; 20-21 minutes, 8% acetonitrile-92% 25mM ammonium acetate, the ultraviolet detection wavelength is 254nm, and the column temperature is set at 30°C. The liquid chromatogram of the metabolites of Pseudomonas chloropinus HT66 is shown in Figure 3. the

As can be seen from Figure 3, phenazine-1-carboxylic acid (phenazine-1-carboxylic acid, CAS number is 2538-68-3, referred to as PCA) and phenazine-1-carboxamide (Phenazine-1-carboxamide, CAS number is 550-89-0, referred to as PCN) retention times were 9.532 minutes and 17.217 minutes. the

Example 2

The present embodiment prepares the Pseudomonas chloropinus agent on the basis of Example 1 and carries out biological assay to it; specifically as follows:

1. Preparation of bacteria

Prepare solid KMB solid and liquid medium, inoculate the Pseudomonas chlorosinum HT66 frozen at -80°C on the solid medium, activate it at 28°C for 48 hours; then transfer to a new solid medium and continue passage 2 times; then pick a ring of activated bacteria, inoculate into 50mL liquid culture medium (packed in 250mL Erlenmeyer flask), place on a constant temperature shaker at 28°C for 16 hours, and shake at a speed of 180 rpm. Then, the seed solution was further transferred to 1000 mL of liquid medium, and cultivated under the same conditions; finally, a larger volume of liquid KMB medium was prepared, put into a fermenter and sterilized at 121°C for 20 minutes; When the temperature is cooled to 28°C, add the seed solution of Pseudomonas chloropinus at a ratio of 1%; then culture with aeration and stirring at 28°C, the rotation speed of the stirring blade in the fermenter is 250 rpm, and the aeration ratio is 1:1 (VVM), the fermentation time was 36 hours, and sampling and determination of bacterium liquid OD value and phenazine-1-amide output at regular intervals. Finally, the number of bacteria in the final unit volume of fermentation broth was determined by gradient dilution method. Under this condition, the bacterial cell concentration of Pseudomonas chloropinus HT66 was 7.9×10 ⁹ cfu/mL. According to the standard curve, the yield of phenazine-1-carboxamide reached 424.87 mg/L.

2. Determination of indole acetic acid

The determination method of the plant growth-promoting factor indole-3-acetic acid (IAA) is mainly detected after improvement of the method of Bric JM (Bric JM, 1991) et al. The general steps are as follows: fully activate the HT66 strain on the KMB medium, inoculate a single colony of the strain HT66 with a toothpick in the center of the LB medium containing 5mM L-tryptophan, 0.06% SDS and 1% glycerol. Use Whatman No. 1 filter paper to cover the surface of the colony, and culture at a constant temperature in a 28°C incubator for 3 days. Salkowski's reagent (0.5mM FeCl ₃ , 35% HClO ₄ ) was added dropwise to the center of the filter paper for treatment, and a red circle appeared immediately at and near the HT66 colony, indicating that HT66 could produce the plant growth-promoting factor indole-3-acetic acid.

3. Antifungal experiments of bacterial strains

In the center of the potato dextrose medium plate, the hyphae blocks of rice sheath blight, Pythium ultima, stevia spot blight and watermelon wilt pathogen with a diameter of 8 mm were inserted into the center of the potato dextrose medium plate. A piece of sterilized filter paper with a diameter of 8 mm is symmetrically placed in each petri dish, and the center of the filter paper is 25 mm away from the central mycelium block. Take the HT66 bacterial agent sample prepared above, dilute it 100 times, and put 10 μL of bacterial dilution on the filter paper. Three parallel plates were carried out for the antibacterial experiment of each pathogenic fungus, and the plates were placed in a constant temperature incubator at 28°C for 5 days to measure the radius of the inhibition zone. the

Fig. 4 is a picture of the antibacterial experiment of Pseudomonas aeruginosa HT66. Among them, the upper left picture is the pathogen of rice sheath blight, the upper right picture is Stevia leaf spot blight, the lower left picture is Pythium ultima, and the lower right picture is Fusarium wilt of watermelon. It can be seen from Figure 4 that Pseudomonas chloropinus HT66 has obvious inhibitory effects on the pathogens of rice sheath blight, Pythium ultima, stevia spot blight and watermelon wilt. the

Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art may make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. the

Claims (10)

1. A Pseudomonas chlororaphis HT66 (Psedunomonas chlororaphis HT66) CCTCC NO: M2013467. 2. Pseudomonas chloropinus as claimed in claim 1, is characterized in that, the partial sequence of the 16S rDNA of described Pseudomonas chloropinus is as shown in SEQ ID NO.1. 3. Pseudomonas chloropinus as claimed in claim 2 is characterized in that, the partial sequence of the described 16S rDNA of Pseudomonas chloropinus carries out sequencing after PCR amplification with universal primer 27F and 1429R of bacteria Obtained, wherein the sequences of 27F and 1429R are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively. 4. Pseudomonas chloropinus as claimed in claim 1, is characterized in that, the main metabolite of described Pseudomonas chloropinus is phenazine-1-carboxylic acid and phenazine-1-formamide; The CAS number of the phenazine-1-carboxylic acid is 2538-68-3, and the CAS number of the phenazine-1-carboxamide is 550-89-0. 5. Pseudomonas chloropinus as claimed in claim 1, is characterized in that, described Pseudomonas chloropinus can suppress rhizoctonia solani (Rhizoctonia solani), Fusarium oxysporum in the phytopathogenic fungi (Fusariumoxysporumf.sp.niveum), Pythium ultimum Trow, and Septoriasteviae Ishiba Yokoyama et Tani. 6. A disease-resistant Pseudomonas chlororaphis agent for promoting growth, characterized in that, using a microbial culture medium as a raw material, Pseudomonas chlororaphis HT66 (Psedunomonas chlorophis HT66) CCTCC NO: M2013467 is prepared after cell expansion culture And get. 7. the anti-disease and growth-promoting Pseudomonas chlorophyll agent as claimed in claim 6, is characterized in that, the carrier of described bacterial agent is powdered activated carbon, bentonite, bentonite, kaolin, diatomaceous earth, zeolite, carbonic acid One or more of calcium. 8. A use of the disease-resistant and growth-promoting Pseudomonas chlorophyll agent as claimed in claim 6 in the preparation of biological pesticides. 9. the purposes of the anti-disease and growth-promoting Pseudomonas chloropina agent as claimed in claim 8 in the preparation of biopesticide, it is characterized in that, described biopesticide is control rice sheath blight, watermelon wilt, stevia rebaudiana Biopesticide for spot blight or root rot of soybean, kidney bean, pea, sweet potato, pine seedling, coffee, apple, citrus, peach, cotton, chrysanthemum, dahlia, pumpkin, watermelon, sugar cane, alfalfa, tomato crops. 10. the purposes of a kind of Pseudomonas aeruginosa agent for disease resistance and growth promotion as claimed in claim 7 in the preparation of microbial fertilizer.