TW201726910A - Pseudomonas mendocina strain isolated from mud and use thereof - Google Patents

Abstract

The present invention relates to a pseudomonas mendocina strain and a use thereof. The pseudomonas mendocina strain named as A6 is deposited at Food Industry Research and Development Institute, Taiwan (R.O.C.) with a patent deposit number for microorganisms, BCRC910628. The strain has enzyme activities of protease, [beta]-glucanase and chitinase, as well as various antipathogen abilities for biologic control, and improvement of plant growth, increase of crop and fruit production.

Description

Pseudomonas sinensis strain isolated from mud and its use

The present invention relates to a strain of Pseudomonas mendocus isolated from mud, in particular to a Mendoza pseudomonocyte named as A6 (the Institute of Food Industry Development, Patent Microbial Deposit No. BCRC 910628). The strain can produce a variety of enzymes and a variety of anti-pathogenic capabilities, and can be used for biological control purposes to promote plant growth and increase crop fruit yield.

Crop disease control has always been the key to the success of crop cultivation. With the continuous improvement of scientific and technological standards and the problems people encounter in the production process, most of the traditional farmers continue to use a large number of chemical pesticides in terms of economy and speed. . However, the use of pesticides has too much impact on the ecological environment; such as: 1. pesticide poisoning and residual poison; 2. improper use of harmful plants or resistance; 3. harm to non-target organisms; 4. environmental pollution.

In recent years, based on the awareness of environmental protection, chemical pesticides have caused serious damage to the environment, making bio-pesticide a safe substitute for future agricultural drugs. The primary goal of organic agriculture is to keep the ecological community of soil life, plants, animals and humans in harmony and maintain a harmonious state of production and life. Therefore, whether the development and application of biological pesticides can become a substitute for chemical pesticides, Agricultural development plays a key role.

The advantages of biological pesticides are as follows: 1. The mechanism of action is complex and it is not easy to produce drug resistance; 2. The pests and diseases that can not be inhibited by chemical pesticides are controlled; 3. There is no residue problem; 4. It is easy to decompose and will not cause environmental accumulation.

In addition, biological pesticides mainly control the biological activity of plant diseases through various microorganisms, and have been known as a means for controlling plant diseases and developing insecticides, but most of the insecticides on the market have been applied. The agent is still a synthetic compound. These chemical fungicides are toxic to wildlife and other non-standard species, so many chemical fungicides are classified as carcinogens by the US Environmental Protection Agency (EPA).

Pseudomonas spp. is a rod-shaped, non-spore-forming Gram-negative bacterium with one or more polar flagella. It is an aerobic bacterium and has strong adaptability to the environment. It can utilize nearly 100 species. Organic matter, distributed in soil and water. In addition, Pseudomonas spp. has a variety of physiological properties, can use a wide range of carbon sources to remove environmental organic pollutants, including petroleum and petrochemical hydrocarbons, chlorine-containing organic solvents, polychlorinated pesticides and pesticides. Furthermore, some species of Pseudomonas spp. can be used in cereal seeds or soil to prevent the growth of crop pathogens, for example: Pseudomonas sp. can prevent carnation wilt, tomato bacterial spots, courgettes and tomato Disease (Ralstonia solanacearum). Most Pseudomonas spp. can secrete siderophores, which restrict pathogenic bacteria from absorbing free ferrous ions in the soil, thereby achieving competitive effects and playing an important role in biological control. It has also been found that strains of Pseudomonas spp. can promote plant growth, produce allelochemicals and induce systemic acquired resistance (SAR) and induced systemic resistance (ISR), and via The roots are probiotics in the root zone, which makes the root tissues and leaves of the plants resistant to pathogenic bacteria.

The root ring probiotic is defined as: some bacteria can invade the roots of plants and promote plant growth after treating seeds or propagules. The root-borne bacteria that promote plant growth include many types of soil bacteria, which promote plant growth indirectly or directly by one or more different mechanisms. The main functions are: 1. Produce plant hormones and promote plant growth; 2. Non- Symbiotic nitrogen fixation; 3. Resistance to plant pathogen growth; 4. Dissolving organic phosphorus, minerals or other nutrients in the soil for plant utilization. Plant growth-promoting rhizobacteria (PGPR) promotes plant growth and promotes viability. It also secretes extracellular hydrolase, protease or volatile organic compounds. Related to, for example, Bacillus spp. secretion contains hydrolyzing enzymes, proteases, which can decompose proteins into amino acids for plant absorption. Bacillus subtilis and Pseudomonas cepacia secrete phytic acid, which transforms the soil into free form for plant uptake.

The present invention is based on the Pseudomonas spp. strain, and its anti-biomass and enzyme activity yields bacteriostatic ability and promotes plant growth. Therefore, the present invention screens Pseudomonas meadocina strain from mud and applies it to agricultural biological control and plant promotion. The purpose of growth.

The main object of the present invention is to provide a strain which is a novel strain of Pseudomonas Mendoca with the code A6, which produces a specific and beneficial enzyme such as a protease, --glucanase, chitinase, and the strain can be used for plant protection and to promote plant growth.

The above main object of the present invention is achieved by the following techniques:

A strain of Pseudomonas sinensis isolated from mud, deposited in the Food Industry Development Research Institute of the consortium, registered as BCRC 910628, and named A6, with a unique gene sequence.

A strain for use according to the first aspect of the patent application, for producing at least one enzyme in the following group consisting of: a protease, a beta-glucan decomposing enzyme (β- Glucanase), chitinase.

A strain for use according to the first aspect of the invention is for inhibiting the activity of a phytopathogenic bacterium, wherein the phytopathogenic bacterium comprises at least one of the following: a tomato bacterial spot (Xanthomonas campestris pv. vesicatoria), tomato green Ralstonia solanacearum.

A use of the strain according to the first aspect of the invention is for promoting plant growth, wherein the genus of the plant comprises at least one of the following: Cucurbitaceae, Solanaceae, and Musa ( Musaceae), Orchidaceae, Caricaceae.

The strain as described above is used for promoting plant growth use, wherein the Cucurbitaceae includes Cucumis melo var. saccharinus and Cucumis anguria.

The strain as described above is used for promoting plant growth use, wherein the Solanaceae includes eggplant (Solanum melongena) and tomato (Solanum lycopersicum).

The strain as described above is used for promoting plant growth, wherein the Musaceae is a banana (Musa sapientum L.).

The strain as described above is used for promoting plant growth use, wherein the Orchidaceae is Phalaenopsis aphrodite Reichb.f.

The strain as described above is used for promoting plant growth use, wherein the Caricaceae is Carica papaya.

The advantages of the invention are:

The present invention tests the activity of protease, β-glucanase and chitinase in different culture days, and finds that the protease, β-glucanase and chitinase have the maximum enzyme activity in one day. In terms of bacteriostatic ability, the pathogenic bacteria against tomato bacterial wilt (Ralstonia solanacearum) can be reduced to a radius of 0.8 cm. In terms of virulence testing, nine common crops of Cucurbitaceae, Solanaceae, Brassicaceae and Poaceae were inoculated and found to be uninfected. At the same time, in the promotion of plant growth, Cucurbitaceae, Solanaceae, Musaceae, Orchidaceae, Caricaceae were inoculated separately and found to promote these plants. Growing. Therefore, Pseudomonas meadocina A6 is a species of Plant Growth-Promoting Rhizobacteria (PGPR), which promotes plant growth, enhances crop yield, and has a variety of anti-pathogenic effects.

For a more complete and clear disclosure of the technical content, the purpose of the invention and the effects thereof achieved by the present invention, it is explained in detail below, and please refer to the drawings and drawings:

The invention relates to a strain of Pseudomonas mendoc, which is deposited in a mud separation method, which is deposited in the Food Industry Development Research Institute of the consortium, and has the registration number: BCRC 910628, which is named as A6 has a unique gene sequence (parameter list). .

This strain of Pseudomonas mendoc, codenamed A6, produces specific and beneficial enzymes such as protease, beta-glucanase, chitinolytic enzymes (β-glucanase) Chitinase).

The following is a detailed description of the colony screening of the present invention, as well as its growth condition test and strain property test results.

<Pseudomonas sp. bacterial molecular identification analysis>

(1) Isolation and purification of bacterial strains:

A single strain isolated and purified from the mud was drawn on a NA medium (Nutrient Agar) in a four-zone method, placed in a 30 ° C incubator, cultured for 16 to 18 hours, and a single colony of bacteria was picked. A total of three single colonies were isolated and purified on NA medium.

(2) Extraction of bacterial genetic DNA (Genomic DNA):

Genomic DNA was extracted using a DNA extraction purification reagent set and stored at -20 °C for testing.

(3) Augmentation, sequencing and sequence alignment of bacterial 16S rRNA gene sequences:

Bacterial 16S rRNA gene with universal primer

16S-F: 5 ́-AGAGTTTGATCATGGCTCAG-3 ́ and

16S-R: 5 ́-GGTTACCTTGTTACGACTT-3 ́

Increased by PCR. The PCR reaction volume was 20 μl and included: 13.8 μl ddH ₂ O, 2 μl 10X PCR buffer, 1 μl 2.5 mM dNTP, 0.2 μl Taq DNA polymerase, 0.5 μl forward primer, 0.5 μl reverse primer, 2 μl A6 genomic DNA. The PCR reaction solution was uniformly mixed and placed in a PCR automatic circulation controller (Perkin Elmer, USA) to set the temperature and time of each stage: the denature temperature was 94 ° C for 30 seconds, and the annealing temperature was 54. The °C time was 30 seconds, the extension temperature was 72 ° C, and the time was 1 minute, for a total of 30 cycles. The PCR product was placed on a gel for colloidal electrophoresis. 1 μl of 6-fold concentration of the dye was applied and 5 μl of the PCR product was mixed evenly. 1.5% agarose gel and 0.5 running buffer [TAE (Tris) -acetate-EDTA)buffer system], electrophoresed at 50 volts for 5 minutes, then conditioned to 100 volts for 20 minutes, stained with ethidium bromide, observed under UV light and labeled with DNA Compare and estimate the molecular size. The DNA product is subjected to nucleotide sequence sequencing and nucleotide sequence alignment for fungal identification.

The isolated strain was amplified by the 16S rRNA gene using a universal primer, and it was found that all strains isolated from the mud could be amplified to 16S rRNA gene fragments, both of which were approximately 1.5 kb in size (see the first panel). After sequencing, it was found that the isolated strain belonged to Pseudomonas sp.

(4) 16S-23S internal transcribed spacer (ITS) gene sequence, sequencing and sequence alignment:

The A6 strain of the present invention was selected and compared with the 16S rRNA gene sequence of the same genus and the 23S rRNA gene sequence for multiplex sequence alignment program (CLUSTALW) to align each other with the conserved region, and the highest similarity fragment was selected for the most suitable temperature and GC ratio. Conditions are used for primer design (see Table 1). The A6 genomic DNA was amplified by PCR to obtain the ITS fragment, and then the fragmentation and sequencing were performed. Finally, the sequence results were obtained, and the obtained nucleotide sequences were aligned to confirm that the fragment sequence of this strain was Pseudomonas mendocs. Species.

A6 uses a specific primer to increase the ITS sequence. Since the ITS sequence is repetitive and several tRNA codon sequences may be inserted between the repeat sequences, the clearest product region is selected for product recovery. A6 has a distinct fragment at about 1.5 kb on the agarose gel (see Figure 2). The above fragments are recovered and sequenced. The sequence after sequencing was 95% identical to Pseudomonas mendocina ymp (CP000680.1) and 94% identical to P. putida KT2440 (AE015451.1).

Table 1. Pseudomonas spp. 16S-23S ITS region synthesis primer sequence

(5) Rec A gene sequence increase and sequence alignment:

With a specificity primer:

Forward: 5'-CBTGYGCNTTCGTCGAYGC-3';

Reverse: 5'-ATCTGGAAYTCRGCYTGRC-3';

The sequence of the specific functional gene rec A gene was amplified and subjected to electrophoresis. Electrophoresis results showed that the scorpion fragments were observed at 520 bp and 650 bp. The 520 bp fragment was cut and sequenced. The sequencing results showed that the rec A gene was 97% identical to Pseudomonas mendocina (see the third figure). .

<Impact of nitrogen source on growth>

(1) Pre-cultivation

First, a single colony was taken from the culture medium by inoculation, and inoculated into a 15 ml sterile round bottom tube containing 3 ml of LB culture solution, and cultured in a shaking incubator at 28 ° C and 150 rpm for 24 hours.

(2) Main training

Using a basic synthetic medium (ingredients: 0.5% NaCl, 0.01% CaCl ₂ ‧2H ₂ O, 0.02% MgSO ₄ ‧7H ₂ O, 0.1% KH ₂ PO ₄ , 0.1% K ₂ HPO ₄ , 0.5% glucose), respectively NH ₄ Cl, NH ₄ NO ₃ ,, KNO ₃ , Urea and NaNO _{3 were} used as nitrogen sources (concentration of 1 g/L) to prepare culture medium, which was prepared by autoclaving and then cultured for 24 hours before sterile operation. The tube was pipetted with 2 ml of the bacterial fermentation broth, and inoculated into a 500 ml triangular conical flask containing 200 ml of basic synthetic medium (containing different nitrogen sources), and then cultured in a shaking incubator at 28 ° C and 150 rpm. During the culture period, the OD value and pH value of the bacterial fermentation broth were measured and serially diluted in the aseptic processing table. Then, 100 μl of the bacterial fermentation broth was applied to the LB solid medium, and the number of colonies was calculated 48 hours later. Total bacterial count (CFU/mL) = number of dish colonies × dilution factor × 10.

With the growth of the test bacteria, the pH of the NH ₄ Cl treatment was maintained between 5.5 and 5.9, while the NaNO ₃ and NH ₄ NO ₃ were maintained at 6.5-7.4, and the remaining treatment groups were between 6.0 and 6.5. (Please refer to the fourth picture).

Among the six nitrogen sources tested, NH ₄ Cl, NH ₄ NO ₃ , NaNO ₃ and other three nitrogen sources, the total number of colonies (CFU) reached 10 ⁹ CFU /mL on the second day, of which NH ₄ Cl On the fourth day, the total number of colonies remained at 10 ⁹ CFU /mL, and the total number of colonies (CFU) in the other two groups decreased from the third day. The KNO ₃ and Urea treatment groups had the best effect, and the total number of colonies on the second day had reached 10 ¹⁰ CFU/mL, but the total number of colonies in the Urea treatment group had decreased to 10 ⁷ CFU/mL since the fourth day (see Figure 5). .

<The effect of carbon source on growth>

Using a basic synthetic medium (components of 0.5% NaCl, 0.01% CaCl ₂ ‧2H ₂ O, 0.02% MgSO ₄ ‧7H ₂ O, 0.1% KH ₂ PO ₄ , 0.1% K ₂ HPO ₄ , 0.1% KNO ₃ ), respectively The culture solution was prepared by replacing the original glucose with glucose, lactose, mannitol, sorbitol, sucrose, starch, arabinose, etc. as the test carbon source (concentration: 5 g/L), and after autoclaving, the test was carried out according to the above method.

With the growth of the test bacteria, the pH value of Glucose treatment decreased gradually with the length of sound during the test; the arabinose treatment group decreased to pH 6.0 on the second day, then gradually increased and maintained at pH 6.3; lactose treatment The group was maintained at pH 6.4; the remaining treatment groups were maintained at pH 6.6 (see Figure 6).

Among the 7 carbon sources tested, the starch treatment group had the best effect. The total number of colonies on the second day had reached 10 ¹⁰ CFU/mL, and then gradually decreased. On the 7th day, it decreased to 10 ⁸ CFU/mL, still better than other. The treatment group was the highest; the lactose treatment group had the worst effect, and the total number of colonies was maintained at 10 ⁵ CFU/mL for 7 days (see Figure 7).

<Effects of different media on the growth of Pseudomonas meadocina A6>

For the medium suitable for the growth of Pseudomonas meadocina A6, the following mediums were selected: carbon-nitrogen source medium, LB, NB, MISP, TM, and the effects of different media on the growth of Pseudomonas meadocina A6 were tested.

(1) Pre-cultivation

First, a single colony was taken from the culture medium by inoculation, and inoculated into a 15 ml sterile round bottom tube containing 3 ml of LB culture solution, and cultured in a shaking incubator at 28 ° C and 150 rpm for 24 hours.

(2) Main training

The bacteria solution was cultured for 24 hours before the culture, and 500 μl of the bacterial fermentation broth was pipetted from the aseptic console. The cells were inoculated into a 125 ml triangular conical flask containing 49.5 ml of the above culture solution, and then placed in a shaking culture at 28 ° C and 150 rpm. The box culture was carried out for 3 days. During the culture, 1 ml of the bacterial fermentation broth was serially diluted at 24 hours, and 100 μl of the bacterial fermentation broth was applied to the LB solid medium for the plate, and the number of colonies was calculated 48 hours later. Total bacterial count (CFU/mL) = number of dish colonies × dilution factor × 10.

After 24 hours of culture, the total number of colonies of TM medium reached 1.32 × 10 ¹⁰ CFU/mL, but after 48 hours, it dropped to 1.2 × 10 ⁸ CFU/mL; while A6 was cultured for 24 hours with MISP, the total colonies were The number is 7.56 × 10 ⁹ CFU/mL. After 48 hours of culture, the total number of colonies is as high as 9.3 × 10 ¹⁰ CFU/mL, and at 72 hours, there is still 3.35 × 10 ⁸ CFU/mL; the other three media, except for carbon and nitrogen sources The total number of colonies in the 48 hours of culture was 4.45 × 10 ⁹ CFU/mL, and the total number of colonies was × 10 ⁸ CFU/mL. A6 was cultured for 3 days using the above five culture solutions, and the total number of colonies in MISP medium was the highest, so MISP medium was more suitable for A6 growth (refer to Fig. 8).

<Pseudomonas meadocina A6 physiological characteristics test>

(1) Determination of growth curve

(A) pre-cultivation

A6 must be activated when entering the main culture shake flask. In order to activate the strain and reduce the lag phase, the bacteria must be cultured for 24 hours. First, a single colony was taken from the culture medium by inoculation, and inoculated into a 15 ml sterile tube containing 3 ml of LB culture solution, and cultured at 28 ° C, 150 rpm shaking incubator for 24 hours.

(B) main training

The bacteria solution was cultured for 24 hours before the culture, and 500 μl of the bacterial fermentation broth was pipetted from the aseptic console. The cells were inoculated into a 125 ml triangular conical flask containing 49.5 ml of MISP culture solution, and then placed in a shaking flask at 28 ° C and 150 rpm. The culture was carried out for 3 days. During the culture, 1 ml of the bacterial fermentation broth was taken at the different time for serial dilution, and then 100 μl of the bacterial fermentation broth was applied to the LB solid medium, and the number of colonies was calculated 24 hours later.

Total bacterial count (CFU/mL): number of dish colonies × dilution factor × 10

The total number of colonies increased significantly to the log or exponential phase after the 4th hour; the peak reached 9.44 × 10 ⁹ CFU/mL at the 20th hour, and then entered the stationary phase (Stationary phase); the number of bacteria after the 24th hour Significant decline into the death period (Decline or death phase), the total number of colonies from 48 to 72 hours did not change significantly (see Figure IX).

(2) UV UVB test:

The A6 bacteria solution cultured for 1 day was adjusted to 10 ⁸ CFU/mL, and 200 μl was uniformly coated on a glass culture dish, and then placed in an ultraviolet light box (287 nm: 512 μw/cm ² : 11 cm), respectively, to be irradiated 10 20, 30 minutes, collect the culture dish and add 1.8 ml of sterile water to wash the bacteria, collect the eluate and then serially dilute it, then take 100 μl of the plate and then place the medium in a 28 °C incubator. Calculate the number of colonies every other day. Then to calculate its survival rate. Survival Ratelog = (CFU/mL) / log (10 ⁷ CFU / mL) × 100%

The bacterial solution was cultured in LB medium (Luria-bertani agar) without additional anti-UV protective agent. The bacterial survival rate was about 43% within 10 minutes after UVB irradiation. After 20 minutes, the survival rate was over time. Growth showed a rapid downward trend, and the bacterial survival rate dropped to 19% over a 30-minute period (see Figure 10).

(3) Enzyme diffusion test:

This experiment uses mineral salt medium to add different carbon raw materials to prepare different formula plate medium, and paste the tablet in the center of the disk surface, and take the culture liquid for 24 hours to adjust to 10 ⁸ CFU/mL, then absorb 50 μl and add paper to the paper. On the tablets, the cells were incubated at 28 ° C for 2 days. After staining with 0.1% Congo red (congo red, Sigma, USA) for 10 minutes, it was defeated twice with 1 M sodium hydroxide (NaOH) for 10 minutes, and it was observed whether the enzyme diffused with a transparent or pink enzyme circle.

Test protease, β-glucanase, chitinase, and protein culture medium is 1% Anjia skim milk powder (Friend Food) Industrial Co., Ltd.), β-glucan (β-glucan) medium is MS added 1% laminin (laminarin, Sigma, USA), chitin medium is added 1% made by the invention Colloidal chitin.

Please refer to Figure 11. In the three formulations of the medium, after staining with Congo red, it was found that the enzyme could decompose the matrix. Among them, β-glucanase is more obvious in the enzymatic circle formed by laminaria, but the effect of protease on proteinase and chitinase in chitin culture is not obvious.

(4) Enzyme activity test

50 ml of the culture liquid for 1, 2, and 3 days of culture was centrifuged at 10,000 rpm and 4 ° C for 30 minutes (Thermo Legend ^TM MACH 1.6/R, Germany), and the bacterial liquid was divided into a bacterial body and a supernatant, followed by ice. In order to carry out the next enzyme test.

(A) Protease

Take 1 ml of the supernatant in a centrifuge tube (eppendorf), add 40 μl of protein substrate to the experimental group [0.4 g azocasein (Azocasein, Sigma, USA) and add 10 ml of 50 mM Trimethylol Hydrochloride buffer solution. After (Tris-HCl buffer) mixing, 40 μl of 50 mM Tris-HCl was added, and the control group was added with 80 μl of 50 mM Tris-HCl, and then placed in a 37 ° C incubator for 3 hours. The reacted sample was added to 400 μl of 10% Trichloroacetic acid (TC), placed in ice for 10 minutes, and then centrifuged at 10,000 g for 10 minutes (centrifuge 5415G, Germany) for 10 minutes with spectrophotometer 440 nm. Measure its absorbance. Among them, the enzyme activity IU = mol / L / hr / mg.

(B) β-glucan degrading enzyme (β-glucanase)

Take 1 ml of the supernatant in a centrifuge tube (eppendorf), add 300 μl of polysaccharide solution [β-glucan solution (250 mg β-glucan to 40 ml DNS buffer)], and add 300 μl of DNS buffer to the control group. Place in a 37 ° C incubator for 20 minutes. After the reaction, 100 μl of stop solution was added, and the mixture was heated in boiling water for 10 minutes, and then ice-cooled for 10 minutes. The centrifuge tube (eppendorf) was centrifuged at 13,000 g for 1 minute, and the absorbance was measured with a spectrophotometer at 550 nm. Among them, the enzyme activity IU = mmol / L / min / mg.

(C) Chitinase (chitinase)

Take 750 μl of the supernatant in a centrifuge tube (eppendorf) and add 750 μl of chitin buffer to the experimental group {chitin buffer [0.05% colloidal chitin plus 50 mM acetate buffer]} The control group was added with 750 μl of 50 mM Tris-HCl, and then reacted at room temperature for 30 minutes. After the reaction, the reaction was terminated by boiling water bath at 100 ° C for 5 minutes, and then 500 μl of the sample was added to 100 μl of 0.8 MK ₂ B ₄ O ₇ , and then placed in a boiling water bath at 100 ° C for 3 minutes, and then taken out at room temperature to cool. Take 200 μl of sample and add 1 mL of dimethylamine borane reagent {DMAB reagent [dimethylamineborane (DMAB) diluted 10 times with acetic acid before use]}, immediately mix and place at 37 °C The box was allowed to cool for 20 minutes at the end of the chamber, and then the absorbance was measured by a spectrophotometer at 544 nm. Among them, the enzyme activity IU = mol / L / min / mg.

(D) Total protein assay

Take 20 μl of ddH ₂ O as a blank group and add 1 mL of protein quantification reagent (1/5X Bradford). Add 20 μl of the above supernatant from the cultured cells to add 1 mL of protein quantitative detection reagent. /5X Bradford), the absorbance was measured by OD ₅₉₅ after 8 minutes of reaction time. After that, the data were taken into each enzyme calibration curve, and the activity of the enzyme was calculated and divided by the total protein content to quantify the specific activity of the enzyme. Among them, the enzyme activity IU = mmole / min / mg.

After three days of enzyme activity in LB medium, it can be seen that the chitinase decreases with the growth time, but the protease and β-glucanase are in the first step. There was a minimum amount in two days, but on the third day, β-glucanase showed a slight increase trend, and the protease showed a significant increase of 0.002 IU (see the twelfth map). ).

<Pseudomonas meadocina A6 on the medium for inhibition of pathogenic bacteria>

After 24 hours the culture broth was adjusted to pathogens 24 hours after taking 100μl drops ¹⁰⁸ PDA medium plate coating, the paste in the central pastilles PDA plate, 50μl bacterial suspension was adjusted to A6 added dropwise to ¹⁰⁸ pastilles Observe the results (see Figures 13 and 14). It can be seen from the thirteenth figure that a significant inhibition zone is produced around the tomato bacterial spots (Xanthomonas campestris pv. vesicatoria Dye) and Ralstonia solanacearum, especially for the inhibition of tomato bacterial wilt. The day suppression radius can reach 0.7 cm (Fig. 14), while the Pseudomonas meadocina A6 ring growth continues to expand to day 7.

<Phytopathogenic test>

In order to test whether A6 has the possibility of harming plants, it can be used as a potential assessment of biopesticide, so several common crops were selected, namely Cucurbitaceae melon var. saccharinus and large Cauliflower (Cucumis sativus), Solanaceae (Capsicam annuum var. grossum), eggplant (Solanum melongena), tomato (Solanum lycopersicum) and sacred tomato (Lycopersicon esculentum Mill), Brassicaceae (Brassicaceae) broccoli (Brassica oleracea L. var. botrytis L.) and Brassica campestris, Poaceae corn (Zea mays var. rugosa).

The plants were grown to the appropriate age for inoculation, the A6 bacterial solution was adjusted to 10 ⁸ CFU/mL, and the plants were wounded by the root stabbing method. Then, 50 ml of the bacterial liquid was uniformly poured into the soil and covered with a plastic film to keep it. Soil moisture was continuously observed and photographed within one month.

After inoculation of A6 by root pricking method for one month, Cucurisaceae melon var. saccharinus and Cucumis sativus and Solanaceae sweet peppers (Capsicam annuum var) were found. . grossum), eggplant (Solanum melongena), tomato (Solanum lycopersicum) and sacred tomato (Lycopersicon esculentum Mill), Brassica oleracea L. var. botrytis L. and Brassica campestris The plants of the Poaceae corn (Zea mays var. rugosa) showed no disease compared with the control group (see Table 2).

Table 2. Crop pathogenicity results of Pseudomonas meadocina A6 "-" means no infection (Indicated as no infection.)

<Pseudomonas meadocina A6 Greenhouse Test for Biological Control>

This experiment mainly explores whether A6 has the ability of biological control. Tomato (Solanum lycopersicum) was incubated to a suitable height for the bacterial wilt pathogen (Ralstonia solanacearum). First adjust the bacterial solution to 10 ⁸ CFU/mL, and take 50 ml of the bacterial solution evenly into the root soil, apply once every other week, and apply it three times in succession. The wound is made by the root pricking method, and the plant is allowed to stand for two angel root circles to fully contact with the A6 bacteria. Finally, the bacterial wilt pathogen (Ralstonia solanacearum) is diluted to 10 ⁸ CFU/mL, and 50 ml of the bacterial liquid is evenly poured. After continuous observation in the root soil, the control group died of onset.

After the experimental group was treated with A6, the wound was further treated with the bacterial wilt pathogen (Ralstonia solanacearum) and continuously observed. On the 10th day, the control group developed symptoms, and the leaves showed symptoms of blue withering. On the 14th day, the whole tomato shoots of the control group showed severe withering in the downward curved leaves, which was a symptom of the bacterial wilt of Solanaceae. In the experimental group treated with A6, although the wounds were made by root cutting and pathogenic bacteria were used, the normal development of the plants was not affected by the pathogens (see Figure 15 (A)). On the 20th day, the whole plant of the control group had withered and browning in the stem, but in the experimental group, there were two signs of bacterial wilt. On the 27th day, the control group had all withered, and in the experimental group, several plants showed symptoms of withering, and most of them continued to grow.

After cutting the stem of the tomato plant of the control group and inserting it into clear water, it was observed that the apparent white bacterial liquid gradually flowed out from the incision (refer to Fig. 15 (B)). The white bacterial solution was plated, genomic DNA was extracted, and the 16S rDNA sequence was amplified and sequenced, and then the alignment was found to be 100% identical to the inoculum of the present invention. In addition, after comparison with NCBI's GenBank database, it was found that all the data were Ralstonia solanacearum, and the degree of identity was 99%. It was confirmed that the isolated bacteria were bacterial wilt pathogens (Ralstonia solanacearum). .

<Stirring fermentation tank experiment>

A stirred fermentation tank culture test of Pseudomonas meadocina A6 was carried out in MISP medium at a working volume of 3 liters.

(1) Pre-cultivation

First, the A6 strain was taken from the culture medium in a single colony by inoculation, inoculated into the LB culture solution, and cultured at 28 ° C, 150 rpm shaking incubator for 24 hours.

(2) Main training

Take appropriate amount of the above-mentioned inoculum (final reaction concentration: 1 × 10 ⁶ CFU/mL) into a stirred fermentation tank, and control the aeration rate to 1 vvm during fermentation, and the agitation rate is 150 rpm. The fermentation was carried out at 28 °C. Samples were taken every 24 hours, and 1 ml of each sample was taken for 10-fold serial dilution. Then, 100 μl of the bacterial fermentation broth was applied to the LB solid medium, and the number of colonies was counted 48 hours later. Total bacterial count (CFU/mL): number of dish colonies × dilution factor × 10.

The baffle was placed in a 5 liter agitated fermentation tank at a working volume of 3 liters, and an aeration rate of 1 vvm was controlled during the fermentation, and the agitation was 150 rpm, and fermentation was carried out at 28 °C. In the sixteenth chart, the total number of colonies at the 24th hour reached 1.48 × 10 ¹⁰ CFU/mL, while at 48 hours, the total number of colonies had dropped to 4.68 × 10 ⁸ CFU/mL, and remained at 10 ⁸ CFU/72 hours. There was no significant change in mL.

<Pseudomonas meadocina A6 Root Ring Probiotic (PGPR) Test>

<<Greenhouse seedling growth impact assessment>>

The experimental crops tested in this experiment were Cucumis melo var. saccharinus, Solanum lycopersicum, Banana (Musa sapientum L.), Phalaenopsis aphrodite Reichb.f., and Carica papaya.

The plants were cultured to a size suitable for inoculation (about 20 cm high) and divided into three groups, namely H ₂ O group (ie CK group), LB group and A6 group. In the A6 group, the A6 bacteria solution should be adjusted to 10 ⁸ CFU/mL. All three groups were treated with 50 ml of uniform pouring in the root soil, applied once every seven days for 4 consecutive applications. After the third time, the cantaloupe and tomato were observed for one month and the plant height, leaf number, maximum leaf surface and dry root weight were recorded. Banana, Phalaenopsis and papaya recorded their plant height and number of leaves per week. And the maximum blade area. The data obtained from the experiment were analyzed by one-way ANOVA.

From the point of view of the melon plant, the LB was similar in height to the A6 group in height difference, but significantly higher than the CK group. As can be seen from the leaf size, the A6 group is significantly larger than CK and LB, and is darker in color. Finally, the root part is dried, and the three are not easy to judge the growth condition by the appearance, but it is known that the A6 group is relatively heavy after weighing (Fig. 17). In the statistical part, the difference in the height of melon was p = 0.443 > 0.05, which was not significant difference, while the difference in leaf number was p = 0.004 < 0.05, and the maximum leaf p = 0.027 < 0.05 was a significant difference. The dry root weight of p = 0.025 < 0.05 was a significant difference (see Table 3).

Table 3. Results of One-way ANOVA analysis of Pseudomonas meadocina A6 in the root ring of probiotics Hd= height difference Ld= leaves number difference ML= maximum leaf RW= root dry weight

From the perspective of tomato plants, the A6 group was significantly better than the LB group and the CK group, and the difference was very large. The A6 group was also significantly better than the two in terms of the number of branches. However, in the discussion of dry roots, it can be seen from the picture that the LB group is relatively good (see Figure 18). In terms of statistical values, the partial height difference of tomato is p = 0.008 < 0.05, and the number of branches is p = 0.001 < 0.05. The dry root weight is p = 0.231 > 0.05, which is not significant difference (see Table 4). ).

Table IV, Results of Pseudomonas meadocina A6 in One-way ANOVA Analysis of Tomato Probiotics in Root Zone Hd= height difference Bn= branch number RW= root dry weight

From the perspective of banana plants, there are relatively high values in the sixth and tenth weeks (see Figure 19), so the two-week data were analyzed by One-way ANOVA (one-way analysis of variance). At the sixth week, the p-values for height difference, number of leaves, and maximum leaf area were all less than 0.05 (p=0.000), which was a significant difference (see Table 5); followed by the One-way ANOVA at the tenth week. Factor variation analysis) analysis, height difference (p = 0.002), number of leaves (p = 0.008) and maximum leaf area (p = 0.001) p values were less than 0.05, a significant difference (see Table 6).

Table V. Results of the sixth week of Pseudomonas meadocina A6 in the banana root ring probiotics SPSS One-way ANOVA Hd= height difference Ld= leaves number difference ML= maximum leaf

Table 6. Results of the tenth week of Pseudomonas meadocina A6 in banana root ring probiotics SPSS One-way ANOVA Hd= height difference Ld= leaves number difference ML= maximum leaf

From the plant of Phalaenopsis, the growth of CK group, LB group and A6 group is similar in terms of number of leaves, but there is a significant increase in the tenth week (see the twenty-fifth figure), and the maximum leaf area In terms of A6's growth, it is the best. There are also obvious peaks in the sixth and tenth weeks, so the two-week data was analyzed by One-way ANOVA (single-factor variance analysis) and the number of leaves was found. Although the p value was greater than 0.05 (p=0.311) at the sixth week (see Table VII), the p value was less than 0.05 (p=0.016) at the tenth week, which was a significant difference (see Table VIII), while the largest leaf Regardless of whether the area is the sixth week or the tenth week, the p value is less than 0.000, which is a significant difference.

Table VII, Pseudomonas meadocina A6 in the Phalaenopsis root ring probiotics SPSS One-Way ANOVA sixth week analysis results Ld=leaves number difference ML= maximum leaf

Table 8. Analysis results of Pseudomonas meadocina A6 in the 10th week of SPSS One-Way ANOVA of Phalaenopsis root ring probiotics Ld=leaves number difference ML= maximum leaf

From the statistical graph of papaya, there are obvious peaks at the 3rd and 5th week (see the 21st chart), analyzed by One-way ANOVA (one-way variance analysis), regardless of height difference, The difference in leaf number or the maximum leaf area difference, p value is 0.000, which is a significant difference, confirming that A6 bacteria have a very high growth-promoting ability for papaya (see Tables IX and X).

Table IX, Pseudomonas meadocina A6 in the papaya root ring probiotics SPSS One-way ANOVA fifth week analysis results Hd= height difference Ld= leaves number difference ML= maximum leaf

Table X. Results of the ninth week of Pseudomonas meadocina A6 in the papaya root zone probiotics SPSS One-way ANOVA Hd= height difference Ld= leaves number difference ML= maximum leaf

<<Field growth impact assessment>>

The field is covered with dark plastic cloth, and the spacing is measured according to the different plants. The depth is excavated and the plant height is similar to the plant height. The CK group, LB group, A6 whole liquid group, A6 The bacteria precipitation group and the A6 supernatant group (which must be adjusted to 10 ⁸ CFU/ml first) were treated with five different reagents, 8 strains per treatment group, and the amount of reagent applied per plant was about 50 ml. The chopsticks are poked 4 to 5 holes about 5 cm deep from the plant, and are poured every 7 days for 4 times. The plant height, the number of leaves and the maximum leaf area should be recorded every week. After fruit production or harvest, the weight of the product must also be recorded. The experimentally completed data was analyzed by One-way ANOVA (single-factor variance analysis) using the SPSS program. The test crops in this experiment were eggplant (Solanum melongena) and cucumber (Cucunis anguria).

Please refer to Table 11 to Table 13. From the average height of cucumber plants, there are relatively high values at the fourth and sixth weeks. From the average number of leaves and the average maximum leaf area, there are also higher values in the fourth and fifth weeks. Trend, so the three-week data was analyzed by SPSS software for One-way ANOVA (one-way variance analysis), and found that at the fourth week, the height difference (p=0.000), the number of leaves (p=0.024), and the maximum The p-values of the leaf area (p=0.001) were all less than 0.05, which was a significant difference; the fifth week data were analyzed by One-way ANOVA (single-factor variation analysis), height difference (p=0.001) and maximum leaf area (p = 0.003) The p values are all less than 0.05, which is a significant difference, but the p value of the leaf number difference is greater than 0.05, which is not significant difference; the sixth week height difference (p=0.004), the leaf number difference (p=0.009) The p-values of the difference with the maximum leaf area (p=0.006) were all less than 0.05, which was a significant difference. It is inferred that the fourth to sixth weeks may be the best proliferative stage of A6 bacteria to cucumber. From the weekly average line chart, almost all of the A6 group had the highest value, and the sedimentation group and the supernatant group were in the second state, which proved that the A6 strain promoted the cucumber. The height part is too high to be observed, so the top bud is cut at about 140 cm, and the top leaf is not proliferated, so the height difference and the difference in the number of leaves are stagnant from the graph (see the twenty-second Figure).

The highest average weight of cucumber fruit was 125.2g in the sediment group, followed by the supernatant group, the average weight was 99.2g; the average weight was 98.3g in the A6 group; the average weight in the LB group was 94.9g, in the CK group. The average weight is 89.1 g. The production quantities of the five groups were ranked in the LB group and the supernatant group, followed by the CK group, which was 95% of the LB and supernatant groups, and the production of the A6 group accounted for only 90% of the LB and supernatant groups. %, the sedimentation group accounted for only 78% of the LB and supernatant groups.

Table XI, Pseudomonas meadocina A6 in the cucumber cucumber root ring probiotics SPSS One-way ANOVA fourth week analysis results Hd= height difference Ld= leaves number difference ML=maximum leaf

Table 12, Pseudomonas meadocina A6 in the cucumber cucumber root ring probiotics SPSS One-way ANOVA fifth week analysis results Hd= height difference Ld= leaves number difference ML= maximum leaf

Table XIII, Pseudomonas meadocina A6 in the cucumber cucumber root ring probiotics SPSS One-way ANOVA sixth week analysis results Hd= height difference Ld= leaves number difference ML= maximum leaf

Refer to Table 14 to Table 16. From the perspective of the eggplant plants, the supernatant group was the best in terms of height, number of leaves or maximum leaf area, followed by the sedimentation group, and the A6 group was very similar to the LB group. Observing the average curve, we can find that there is a relatively high trend in the seventh week, the eighth week and the tenth week. One-way ANOVA (one-way variance analysis) analysis is performed with SPSS software, and the p value is less than 0.05, which proves A6. The growth promoting effect of bacterial liquid on eggplant plants. The number of leaves showed a downward trend in the fifth week, because the lower leaves of the eggplants needed to be removed during the planting process to make the plants grow better (see Figure 23).

The highest average weight of eggplant fruit was 135.9g in the sediment group, followed by A6 group, the average weight was 135.8g; the average weight was 129.5g in the LB group; the average weight in the supernatant group was 126.9g, the average in the CK group. The weight is 121.5g. The production of the five groups above the crown group, followed by the sedimentation group, 94% of the LB and supernatant groups, the production of the A6 group only accounted for 63% of the supernatant group, the LB group accounted for 50.6% of the clear liquid group and 41% of the supernatant group.

Table 14. Results of the seventh week of Pseudomonas meadocina A6 in the eggplant root ring probiotics SPSS One-way ANOVA Hd= height difference Ld= leaves number difference ML= maximum leaf

Table 15. Results of the eighth week of Pseudomonas meadocina A6 in the eggplant root ring probiotics SPSS One-way ANOVA Hd= height difference Ld= leaves number difference ML= maximum leaf

Table 16. Results of the tenth week of Pseudomonas meadocina A6 in the eggplant root ring probiotics SPSS One-way ANOVA Hd= height difference Ld= leaves number difference ML= maximum leaf

<Ramstonia solanacearum field control trial and evaluation>

Tomato selection criteria: Tomatoes with normal appearance and no obvious infection by pests and diseases were used as test materials.

Field design: This test is a single factor test, and the Randomized Complete Block Design (RCBD). In each plot, 1 tomato was planted with a diameter of 27 cm and a height of 17 cm, and 2 rows of pots were placed in each plot, with a row spacing and a plant spacing of 40 cm each. A total of 4 treatments, 4 repetitions per treatment. 20 pots are placed in each plot, and a one-meter-long compartment is set up between the districts of each district to avoid the liquid medicine floating and polluting another treatment, and the 16 communities treated by 4 are arranged in each zone in a random manner.

Pathogen inoculation: Ralstonia solanacearum was cultured in TM medium (peptone 10 g per liter, Casein 1 g, Glucose 5 g) at 30 ° C for 24 hours. In order to meet the field infection situation, the bacterial wilt bacteria should be directly mixed into the medium before planting the tomato seedlings, and the final concentration of the pathogenic bacteria is >1×10 ⁵ CFU/g medium.

Application method: When the bacterial wilt pathogen is infected, it can damage the stalks and stems, and the roots invade the plants, causing the vascularization of the vascular bundles of the plants, causing the plants to wither rapidly. The pathogen is a soil-borne bacteria, which can be planted in non-host plants. It has survived for 10 years in the field and survived for more than 4 years in the soil of the fallow field. The soil pH value was the most serious at 6.6, and the optimum temperature was 24-36 °C.

Location: Plant roots are evenly applied.

Interval and secondary sputum: Apply 3 times in the seedling stage 3 days before planting (plant height about 10 cm); apply the first time one day after planting, apply once every seven days, each time 100ml watering per plant, follow-up Apply three times. Record the application times and the date of each application and the growth and development of the tomato.

The period of investigation of the occurrence of pests: one survey before each application after planting, every seven days after the third application, and two subsequent investigations, a total of five investigations.

Investigation and measurement method: If the plant is completely withered, it will be a rickety disease, and the rickets will be recorded, and the rickets rate and prevention rate will be calculated.

Statistical analysis: The severity of each treatment was tested after data conversion. If the level was significant, the Fisher's Least Significance Test (LSD) was used to determine 1% and 5% significant difference.

See Tables 17 and 24. The control group and the drug treatment (1X10 ⁹ CFU/mL A6) were 100 times, 200 times, 400 times and the CK tomato plants had a bacterial wilt disease of 22.5%, 45%, 51.25%, and 53.75. The results of the survey were analyzed by SPSS18.0 statistical software with 1% and 5% significant difference analysis by One-way ANOVA, and 100 times of drug treatment (1× ^{10 9} CFU/mL A6) was significantly different from the control treatment.

Table 17. Efficacy test of Pseudomonas meadocina A6 solution against tomato bacterial wilt (Ralstonia solanacearum)

<Impact of chemical agents on the survival of A6>

Single colonies were selected from LB solid medium plates, inoculated in 3 ml LB liquid medium, and cultured at 28 ° C for 24 hours at constant temperature. The concentration of the bacteria solution was adjusted to 10 ⁸ (cfu/ml), and 100 μl of bacterial solution, 100 μl of 10X was taken. The chemical agent of stock was mixed with 800 μl of sterile water, mixed and shaken evenly, and then allowed to stand for 10 minutes. In the control group, 100 μl of the bacterial solution was mixed with 900 μl of sterile water. After 10 minutes, 100 μl was serially diluted, diluted 10 ³ , 10 ⁴ , 10 ⁵ Each 100 μl was plated on an LB solid medium plate, placed in a 28 ° C incubator for 24 hours, and the number of colonies was counted. The chemical pesticides used are recommended for concentration testing according to the pesticide information service network of the Animal and Plant Epidemic and Quarantine Bureau of the Executive Yuan Agricultural Committee. There are important chemical agents such as bactericides and herbicides. Survival rate: log (CFU/ml) / log Check × 100%

Among them, the herbicide used is a phenoxy acid-based di- or tetra-sodium salt, a quaternary amine-type paraben, a hypophosphorous acesulfame, a guanamine-based phosphamethoxazole salt, and a diphenyl ether. The system is based on the complex of ruthenium and dinitroaniline.

Among them, the bactericide used is oxolinic acid of quinoline type, copper of copper, copper hydroxide, antibiotic class of tetracycline, streptomycin.

See Figure 25 for the effect of herbicides on A6. Most of the herbicides have no significant effect on A6. The phenoxy acid-based di- and tetra-sodium salts are 80% WP, dinitroaniline. The survival rate of 38.7% of CS and the sub-phosphorus of Trichoderma sinensis was almost the same as that of CK group, and the survival rate of quaternary amines with 24% SL was more than 80%. The 22.3% EC of Fulufen had a significant effect on the survival rate of A6, and the survival rate was less than 70%. Among them, A6 and guanamine-based phosphazone isopropylamine salt 41% SL are completely unable to grow.

See Figure 26 for the effect of bactericides on A6, including copper copper hydroxide 53.8% WG, antibiotic tetracycline 10% WP, antibiotic streptomycin 12.5% SL and copper. The effect of CAT on 81.3% WP on the survival rate of A6 bacteria is obvious, and the survival rate is almost 0%. The quinoline fungicide with 20% WP of oxolinic acid had no effect and the survival rate was 86%.

< Conclusion>

A6 bacteria still have 10 ⁸ CFU/mL in 10 days of culture, especially the growth period of A6 bacteria is particularly long. After continuous culture for 30 days, there are also bacteria to survive, which can be applied to biological control or promote plant growth. Have great advantages.

A6 bacteria do not produce endospores like Gram-positive bacteria, so the effect of ultraviolet light irradiation on survival rate will be obvious, and it is recommended to apply directly to soil in practical use.

A6 protease, β-glucanase and chitinase, the maximum activity of enzyme activity appeared on the first day.

A6 has a good inhibitory effect on the scale, the radius of the inhibition circle is 0.7 cm and continues to grow until the radius of the inhibition circle reaches 1.2 cm on the seventh day.

A6 on Cucumber melo var. saccharinus and Cucumis anguria, Solanaceae eggplant (Solanum melongena) and tomato (Solanum lycopersicum), Muscoaceae (Musa sapientum) L.), Phalaenopsis aphrodite Reichb.f. of Orchidaceae and Carica papaya of Caricaceae all have growth promoting effects.

A6 bacteria can indeed help the tomato to achieve biological control, which can delay the onset of tomato in the experimental group for 10 days, and slow down the severity of the disease and prolong the survival date.

The above is only a part of the embodiments of the present invention, and is not intended to limit the present invention. It is intended to be included in the scope of the present invention.

In summary, the embodiments of the present invention can achieve the expected use efficiency, and the specific technical means disclosed therein have not been seen in similar products, nor have they been disclosed before the application, and have completely complied with the patent law. The regulations and requirements, the application for invention patents in accordance with the law, and the application for review, and the grant of patents, are truly sensible.

Unordered list <110> National Kaohsiung Normal University <120> Pseudomonas monosodium strain isolated from mud volcanic mud and its use <160> 2 <210> 1 <211>640 <212>DNA <213> Mendo Sa aeruginosa strain of the 16S rRNA gene and ITS sequence <400> 1 1 CCCGGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGT 51 GGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTGGCCTTGA 101 CATGCTGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCAGACAC 151 AGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTC 201 CCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTAATGGTGGGCA 251 CTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCA 301 AGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGTCGGT 351 ACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCG 401 TAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGT 451 AATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACAC 501 CGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTAGCTAGTCTAACCT 551 TCGGGGGGACGGTTACCACGGAGTGATTCATGACTGGGGTGAAGTCGTAA 601 CAAGGTAGCCGTAGGGGAACCTGCGGCTGGATCACC TCCT <210> 2 <211> 510 <212> DNA <213> Pseudomonas mendocina strain rec A gene sequence of <400> 1 1 ATCTGGAACTCGGCCTGACGGAAAGGCGGGGCCACCTTGTTCTTGACGATCTTCACGCGG 61 GTTTCGCTGCCAACCACTTCCTCGCCTTCCTTGACCGCGCCGGTGCGGCGGATGTCCAGA 121 CGAACCGAGGAGTAGAACTTCAGCGCGTTACCGCCGGTGGTGGTTTTCGGGCTGCCGAAC 181 ATCACGCCGATCTTCATGCGGATCTGGTTGATGAAGATCACCAGGCAGTTGGCATTCTTG 241 ATGTTGCCGGTGATCTTGCGCAGCGCCTGGCTCATCAGACGCGCCTGCAGGCCGACGTGC 301 ATATCGCCCATCTCGCCTTCGATCTCCGCCTTGGGCACCAGAGCCGCCACGGAGTCGACG 361 ATGATCACGTCGATGGCATTGGAGCGCACCAGCATGTCGGTGATTTCCAGTGCCTGCTCG 421 CCGGTGTCCGGCTGCGATACCAGCAGGTCGTCGACGTTGACGCCGAGCTTGCCGGCGTAG 481 TCGGGATCCAGCGCGTGCTCGGCATCGACG

First image: Mud separation bacteria with 16S universal primer increase results Lane M: 14 kb DNA marker (promega, USA) Lane 1~8: A group bacteria were isolated from No. 5 mud.

Figure 2: Results of PCR amplification of ITS sequences Lane M: 100 bp-3 kb DNA marker (promega, USA) Lane 2: Pseudomonas meadocina

Figure 3: Results of the increase in the rec A gene sequence

Figure 4: Adding different nitrogen source components to change the pH value of Pseudomonas meadocina A6

Figure 5: Comparison of the growth of Pseudomonas meadocina A6 with different nitrogen sources

Figure 6: Adding different carbon source components to change the pH value of Pseudomonas meadocina A6

Figure 7: Comparison of the growth of Pseudomonas meadocina A6 with different carbon source components

Figure 8: Effect of different media on the growth of Pseudomonas meadocina A6

Figure 9: Growth curve of Pseudomonas meadocina A6 in MISP medium

Figure 10: CFU and survival rate of Pseudomonas meadocina A6 at different times after UVB irradiation

Figure 11: Results of Pseudomonas meadocina A6 enzyme diffusion in MS solid medium

Figure 12: Pseudomonas meadocina A6 cultured for three days of protease, β-glucanase and chitinase enzyme activity

Figure 13: Pseudomonas meadocina A6 inhibits pathogenic bacteria in LB solid plate medium A. Xanthomonas campestris pv. vesicatoria B. Ralstonia solanacearum

Figure 14: Pseudomonas meadocina A6 Four-day inhibition radius of pathogens Aac: Acidovorax avenae subsp. Citrulli Xac: Xanthomonas axonopodis pv. Citri Xcv: Tomato bacterial spots ( Xanthomonas campestris pv. vesicatoria ) Pcc: Pectobacterium carotovorum subsp. carotovorum Rs: Ralstonia solanacearum

Figure 15: Pseudomonas meadocina A6 in the biological control test of tomato crops A (left): Watering the tomato crop of Pseudomonas meadocina A6 in 14 days after inoculation of bacterial wilt pathogens A (right): not The growth of the tomato crop with Pseudomonas meadocina A6 solution after 14 days of inoculation of bacterial wilt pathogen B: The case where the stem of tomato plant of A (left) was cut and inserted into clear water, and the white bacterial liquid gradually flowed out from the incision.

Figure 16: Growth of Pseudomonas meadocina A6 in a stirred fermentation tank

Figure 17: Pseudomonas meadocina A6 in the PGPR test of cantaloupe A: plant hight B: maximum leaf C: dry root

Figure 18: Pseudomonas meadocina A6 in tomato PGPR test scenario A: plant hight B: dry root

Figure 19: Bi-annual average line graph of Pseudomonas meadocina A6 probiotics in banana root ring A. Hd height difference High difference B. Ld leaf number difference number difference C. ML maximum leaf area difference max leaf

Figure 20: Bi-annual average line graph of Pseudomonas meadocina A6 probiotics in the Phalaenopsis root ring A. Ld leaf number difference leaves number difference B. ML maximum leaf area difference max leaf

Twenty-first chart: Bi-annual average line graph of Pseudomonas meadocina A6 probiotics in papaya root ring A. Hd height difference High difference B. Ld leaf number difference number difference C. ML maximum leaf area difference max leaf

Twenty-second picture: Weekly average line chart of Pseudomonas meadocina A6 in the cucumber root ring probiotics

Twenty-third map: weekly average line chart of Pseudomonas meadocina A6 probiotics in eggplant root ring

Twenty-fourth picture: Bacterial wilt disease test situation of tomato plants A. Chemical treatment (1X10 ⁹ CFU/mL A6) 100 times B. Chemical treatment (1X10 ⁹ CFU/mL A6) 200 times C. Pharmaceutical treatment (1X10 ⁹ CFU/mL A6 ) 400 times D. Control group CK

Figure 25: shows the effect of herbicides on Pseudomonas meadocina A6

Figure 26: shows the effect of bactericides on Pseudomonas meadocina A6

Claims (9)

A strain of Pseudomonas sinensis isolated from mud, deposited in the Food Industry Development Research Institute of the consortium, registered as BCRC 910628, and named A6, with a unique gene sequence. A strain for use according to the first aspect of the patent application, for producing at least one enzyme in the following group consisting of: a protease, a beta-glucan decomposing enzyme (β- Glucanase), chitinase. A strain for use according to the first aspect of the invention is for inhibiting the activity of a phytopathogenic bacterium, wherein the phytopathogenic bacterium comprises at least one of the following: tomato bacterial spot (Xanthomonas campestris pv. vesicatoria) tomato green Pathogen (Ralstonia solanacearum). A use of the strain according to the first aspect of the invention is for promoting plant growth, wherein the genus of the plant comprises at least one of the following: Cucurbitaceae, Solanaceae, and Musa ( Musaceae), Orchidaceae, Caricaceae. The use according to claim 4, wherein the Cucurbitaceae comprises Cucumis melo var. saccharinus and Cucumis anguria. The use according to claim 4, wherein the Solanaceae comprises eggplant (Solanum melongena) and tomato (Solanum lycopersicum). The use of the invention of claim 4, wherein the Orchidaceae comprises Phalaenopsis aphrodite Reichb.f. The use of the invention of claim 4, wherein the Musaceae is a banana (Musa sapientum L.). The use of the invention of claim 4, wherein the Caricaceae is Carica papaya.