TWI417052B - Bacillus amyloliquefaciens and application of the same - Google Patents

Description

Bacillus amyloliquefaciens strain and use thereof

The invention relates to a strain of liquefied Bacillus amyloliquefaciens, in particular to a gene sequence named BF-1 having unique 16S rRNA, Tet(L), ItuA, ItuB, ItuC, ItuD (the development of the food industry) The patented microbial deposit number BCRC910498) is used to produce a variety of enzymes and various antibiotics.

According to the fact that various microorganisms can be used to control the biological activity of plant diseases as a known means, which has been helpful in the field of controlling plant diseases and developing insecticides, but most of the applied insecticides on the market are still synthesized. 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).

That is, biological control provides another method of controlling plant diseases and insecticides other than synthetic chemical fungicides, and this method is safer and biodegradable.

The US EPA (USEnvironmental Protection Agency) has approved a number of species of Bacillus spp. microbial pesticide products to be marketed as an agent for controlling plant diseases and insect pests, in addition to the commonly known Bacillus subtilis , including liquefied starch. Bacillus subtilis var . amyloliquefaciens, Bacillus cereus , Bacillus licheniformis , Bacillus pumilus , Bacillus thuringiensis , Bacillus popilliae and Bacillus popilliae Bacillus sphaericus and the like. Among them, Bacillus has been developed and applied as a commercial preparation, and there are Bacillus subtilis, Bacillus amyloliquefaciens, and Cactus bacillus in disease prevention and control; in the prevention and control of insect pests, it is S. cerevisiae, Bacillus cerevisiae and Bacillus sphaericus.

Many important plant diseases are caused by soil or rhizosphere fungi. Researchers use the antagonism between soil microorganisms to develop biological control agents that inhibit fungal growth and apply them to field control. Bacillus subtilis is Gram-positive, aerobic rod-shaped bacteria with perennial flagella and endospores as its main morphological features. These bacteria are common in soil and plant surface, in food, feed additives, enzymes, The development of biotechnology industry such as seed protection agents has been used for many years, and it is a beneficial microbial species that is generally considered to be safe. Recently, it has been applied to soil roots and pathogens to compete for the nutrients in the root system to become the dominant species, thereby reducing the harm of pathogenic bacteria; it can also directly spray plant leaves to protect leaf fungal diseases, such as bean rust; it can also be applied to soil or Seed dressing treatment to prevent soil diseases; it has a very obvious promoting effect on the growth of various crops, especially the development of roots; it can also be used as an antifungal agent to prevent spoilage after harvesting fruits and vegetables, such as peach brown rot and Citrus Penicillium, etc.

In the case of liquefied Bacillus amyloliquefaciens, it was discovered in 1943 by Japanese scholar Fukomoto, which produces a large amount of α-amylase [α-amylase] and protease [protease], and also has lipid and cellulolytic enzymes. Therefore, it can be used for biological feed additives to improve the meat exchange rate and breeding rate of livestock, and it can promote vegetative growth when applied to plants. Increase disease resistance, reduce the use of chemical pesticides and fertilizers; also cooperate with organic cultivation to improve the problem of chemical pesticides and fertilizer residues in fruits and vegetables, soil and water, and reduce continuous cropping obstacles.

A main object of the present invention is to provide a strain which is a novel Bacillus amyloliquefaciens having the code BF-1, which produces a specific and beneficial enzyme such as a lipolytic enzyme for decomposing lipids. Amylolytic enzymes are used to hydrolyze starch, cellulolytic enzymes to hydrolyze cellulose, and proteolytic enzymes to hydrolyze proteins.

The novel liquefied Bacillus amyloliquefaciens codenamed BF-1 of the invention can be applied to treat agricultural aquaculture sewage, water pipeline system, animal feed, kitchen waste treatment, to improve the decomposition of organic substances in sewage and the quality of sewage and garbage treatment processes. And efficiency. Therefore, the novel strain of the present invention, code name BF-1, and its enzyme can be made into a detergent and applied to decontamination and food manufacturing.

The second object of the present invention is to propose a novel Bacillus amyloliquefaciens BF-1 and/or an antibiotic produced by the strain as an antifungal agent for inhibiting the growth of at least one fungal infection source in the following composition group, the fungus Including: guava rot (A), Phytophthora nigra (B), Phytophthora leaf blight (C), Pestalotia euginae (D), guava pustophila [Pestalotia psidii] ( E), Papaya rot fungus (F), Phytophthora rot fungi (G), Phytophthora anthracis (H), Papaya anthracis (I), Anemone anthracis (J), Anchovies anthracis (K), Rhizopus oryzae (L), Verticillium dahliae (M), Papaya Phytophthora (N), and Black Spotted Cabbage (O).

The three objects of the present invention are to propose a novel Bacillus amyloliquefaciens BF-1 and/or an anti-biomass produced by the same as an antibacterial agent for inhibiting the growth of at least one bacterial infection source in the following composition group, The bacteria include: tomato bacterial wilt pathogen, tomato bacterial spot pathogen, citrus ulcer pathogen, melon bacterial fruit spot pathogen.

The four objects of the present invention are to propose a novel plant protection composition, which is to use the novel Bacillus amyloliquefaciens BF-1 proposed by the present invention in combination with at least one fungicide in the following group, the fungicide comprises: Net (alkane nitrogen), poker (melamine), chlordane (amine phthalic acid), leucoside (benzamide), gram rot (benzidine), race (sulfonamides), carnitamics (antibiotics), atorenos (spheroids), tetrachloroisophthalonitrile (aromatics), linoleum (benzimidazoles), methylpoly Net (benzimidazole precursors), kekemin (amino amides), eppyz (imidazoles), chlorpyrifos (triazoles), ternary copper sulphate (copper), chlorpheniramine (dichloro Phenyldimethyl quinone imine), Deandi (dithiocarbamate), free of rot (polymeric dithiocarbamate), Poker Mn (imidazole), Forsyth (organic Phosphorus), chlordiazepine (pyridine), fenrimyl (pyrimidine), vebufen (quinoline), nitrile sulfonium (indole), edetril (thiazole), bun clone (urea) , trimethoxazole (triazole benzothiazole).

The fifth object of the present invention is to propose a novel plant protection composition, which is to use the novel Bacillus amyloliquefaciens BF-1 proposed by the present invention in combination with at least one insecticide in the following group, the insecticide including: Net (three nitrogen well systems), idadines (organic nitrogen and heterocyclics), Datnam (organic nitrogen and heterocyclics), and naphthalene (ammonia methanate), Plus Baofu (ammonia mesylate), Taosson (pyridine organic thiophosphates), new acaricidal (biphenyl), Pedan (organochlorine), chlorhexidine (azole), bifenin (azole) ), nicotine (new nicotine), fumicide (organophosphorus), acesulfame (organophosphorus), kefanpai (pyrazoles), chlorhexidine (linear amine thiophosphate) Class), thiophene (antibiotics).

The six objects of the present invention are to propose a novel plant protection composition, which is to use the novel Bacillus amyloliquefaciens BF-1 proposed by the present invention in combination with at least one herbicide in the following group, the herbicide includes: two or four Sodium salt (phenoxy acid system), chlorpyrifos (phosphoric acid), jiaphosphine isopropylamine salt (melamine), valprofen (diphenyl ether), sedative (dinitroaniline), bar Pulling (four-grade amines).

The seven objects of the present invention are to propose a novel plant protection composition, which is to use the novel Bacillus amyloliquefaciens BF-1 proposed by the present invention in combination with at least one bactericide in the following group, the bactericide comprising: Copper (copper), chain tetracycline (antibiotics), oxolinic acid (quinoline fungicide).

The invention relates to a novel strain of Bacillus aeruginosa which is deposited in the Food Industry Development Research Institute of the Corporation, and has the registration number BCRC910498, which is named as BF-1 with unique 16S rRNA, Tet(L), ItuA, ItuB, The gene sequence of ItuC and ItuD.

The novel Bacillus amyloliquefaciens, codenamed BF-1, produces specific and beneficial enzymes such as lipolytic enzymes to break down lipids, amylolytic enzymes to hydrolyze starch, cellulolytic enzymes to hydrolyze cellulose, and Proteolytic enzymes are used to hydrolyze proteins.

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.

<Method of colony screening>

1. Take 0.5 g of the strain powder, add 4.5 mL of sterile water to 14 mL of the bacterial culture tube (if the powder amount of the strain is insufficient, take 9 times the actual weight of the sterile water), and shake at 30 ° C, 200 rpm for 1 hour.

2. Move the bacterial culture tube into a water bath at 60 ° C. The water surface height must be higher than the liquid level of the bacterial culture tube and heated for 35 minutes.

3. The strain culture solution was diluted with 10 n of sterile water (100 μL bacterial solution + 900 μL sterile water).

4. Take about 105-7 concentration of the strain dilution, apply 100 μL of each tube to NA (or LA) medium, place at 30 ° C, and incubate overnight.

5, the next day, observe the colony type of each strain, if more than one colony, you need to pick all out, and then do liquid culture separately (in addition to each piece of a NA, for the preparation of subsequent tests).

<Identification of strains> (1) Bacterial 16S rRNA gene sequence polymerase chain reaction

The 16S-F and 16S-R (Table 1) were used as primers for nucleic acid primers, and the Genomic DNA of the strain was used as a template for polymerase chain reaction. The reaction solution included 1 μl of Genomic DNA and 2 μl of 10X PCR buffer (TaKaRa, Japan). , 1μl 2.5mM dNTP, 1μl [mu] l reverse primer and forward primers, and ^{TaKaRa Taq TM (5units / μl,} TaKaRa, Japan), as well ddH ₂ O 13.8μl of the reaction the final volume of 20μl PCR reaction was further carried out, The reaction conditions were as follows: 94 ° C for 5 minutes; (94 ° C, 30 seconds for DNA denaturation; 52 ° C, 45 seconds for primer bonding; 72 ° C, 45 seconds for extension synthesis) repeated 30 cycles; 72 ° C, 7 minutes; The °C reaction is completed. After the completion of the PCR reaction, the product was analyzed by nucleic acid electrophoresis in 1.5% Agarose gel/0.5X TAE buffer, and 1 μl of 6X loading dye and 5 μl of PCR product were mixed uniformly, firstly at 50 volts for 10 minutes, and then the voltage was adjusted to 100. Volt was carried out for 20 minutes, then stained with ethidium bromide (0.5 mg/ml) and distilled water, and the film was observed under UV light, compared with 100 bp DNA ladder (100-3,000 bp; Protech, Taiwan). For the size of the PCR product, please refer to the first figure, which is the result of the amplification of the 16S sequence, wherein Lane1: 100-3,000 bp DNA markers (Gene Mark, Taiwan); Lane 2: the strain.

(2) Purification of nucleotide 16S rRNA gene sequence amplification product and nucleotide sequence alignment

GeneMark ^® using the DNA clean / Extraction Kit purification of the PCR product was increased. The purification procedure is as follows: firstly, the amplified PCR product is transferred to a 1.5 ml microcentrifuge tube, and an equal volume of Binding solution with the PCR product is added and shaken to uniformly mix. The mixed liquid was transferred to a Spin Column and combined with a Collection tube and centrifuged at 12,000 rpm for 2 minutes. After the filtrate was removed, the addition of 700 μl of Wash solution was carried out by centrifugation at 12,000 rpm for 1 minute. The filtrate was decanted and centrifuged at 12,000 rpm for 5 minutes. Move the Spin Column to a new 1.5 ml microcentrifuge tube and place in the oven for 15 minutes to remove the alcohol completely. Further, 30 μl of Elution solution was added to centrifuge at 12,000 rpm. After 1 minute, the liquid in the microcentrifuge tube was subjected to electrophoresis analysis using 1.5% Agarose Gel to confirm the purified PCR product.

The purified product is subjected to sequencing and sequencing. The ABI PRISMTM BigDyeTM Terminator Cycle Sequencing Ready Reaction kit and the ABI PRISMTM 3730 DNA Sequence (Perkin Elmer, U.S.A.) nucleic acid automated sequencing analyzer were used. The sequenced results were stored and then searched into the nucleotide sequence of the NCBI website for a search alignment to confirm that the sequence fragment of this strain was a bacterium of the bacterium A. licheniformis.

(C) Tet (L) gene sequence polymerase chain reaction

B.subti.lis ITS ITS sequence due to sequence similarity with B.amyloliquefaciens high, whereby it can not be identified bacteria fragments, according to the literature (Reva et al., 2004) , although this strain has a number of two similar Or the same fragment, but some of the genes are arranged in a different order. Therefore, according to this characteristic, three different primers are designed to form two sets of primer pairs, namely YyaO-F/TetB-R and YyaR-F/. TetB-R [Refer to Table 1]. Using YyaR-F/TetB-R as a nucleic acid primer pair, the polymerase chain reaction was carried out using Genomic DNA of the strain as a template. The reaction solution included 1 μl of Genomic DNA, 2 μl of 10X PCR buffer (TaKaRa, Japan), 1 μl of 2.5 mM dNTP, and 1 μl. 1μl forward primer and reverse primers, and ^{TaKaRa Taq TM (5units / μl,} TaKaRa, Japan), as well ddH ₂ O 13.8μl of the reaction the final volume of 20μl PCR reaction was further carried out, the reaction conditions were as follows: 94 ℃ 5 minutes; (94 ° C, 30 seconds for DNA denaturation; 50 ° C, 45 seconds for primer bonding; 72 ° C, 45 seconds for extension synthesis) repeated 30 cycles; 72 ° C, 7 minutes; 4 ° C reaction is completed. After the completion of the PCR reaction, the product was analyzed by nucleic acid electrophoresis in 1.5% Agarose gel/0.5X TAE buffer. 1 μl of 6X loadingdye and 5 μl of PCR product were mixed uniformly, firstly at 50 volts for 10 minutes, and then the voltage was adjusted to 100 volts. After 20 minutes, it was stained with ethidium bromide (0.5 mg/ml) and distilled water, and the film was observed under a UV lamp, and compared with a 100 bp DNA ladder (100-3,000 bp; Protech, Taiwan). The size of the PCR product, see Figure 2, is the result of the increase in the Tet (L) gene. Among them, Lane1: 100-3,000 bp DNA markers (Gene Mark, Taiwan); Lane 2: this strain.

(4) Purification of nucleotide sequence sequencing of Tet(L) gene sequence amplification

The GeneMark ^® using a DNA clean / Extraction Kit of purified PCR product was increased. The purification procedure is as follows: firstly, the amplified PCR product is transferred to a 1.5 ml microcentrifuge tube, and an equal volume of Binding solution with the PCR product is added and shaken to uniformly mix. The mixed liquid was transferred to a Spin Column and combined with a Collection tube and centrifuged at 12,000 rpm for 2 minutes. After the filtrate was removed, the addition of 700 μl of Wash solution was carried out by centrifugation at 12,000 rpm for 1 minute. The filtrate was decanted and centrifuged at 12,000 rpm for 5 minutes. Move the Spin Column to a new 1.5 ml microcentrifuge tube and place in the oven for 15 minutes to remove the alcohol completely. Further, 30 μl of Elution solution was added to centrifuge at 12,000 rpm. After 1 minute, the liquid in the microcentrifuge tube was subjected to electrophoresis analysis using 1.5% Agarose Gel to confirm the purified PCR product.

The purified product is subjected to sequencing and sequencing. Use ABI PRISMTM BigDyeTM Terminator Cycle Sequencing Ready Reaction kit This was carried out with an ABI PRISMTM 3730 DNA Sequence (Perkin Elmer, U.S.A.) nucleic acid autosequencing analyzer. The sequenced results were searched into the nucleotide sequence of the NCBI website for nucleotide alignment to confirm that the sequence fragment of this strain was a bacterium of the bacterium A. bacillus.

(5) Iturin A gene sequence polymerase chain reaction

Using the four sets of Iturin A primers designed by the predecessors (Stone, 2007), the ituA, ituB, ituC, and ituD [Table 2], and the Genomic DNA of the strain as a template, the polymerase chain reaction, including 1 μl of the reaction solution. Genomic DNA, 2μl 10X PCR buffer ( TaKaRa, Japan), 1μl 2.5mM dNTP, 1μl [mu] l reverse primer and forward primers, and ^{TaKaRa Taq TM (5units / μl,} TaKaRa, Japan), as well as of ddH ₂ O 13.8μl The final reaction volume was 20 μl and the PCR reaction was carried out under the following conditions: 94 ° C for 5 minutes; (94 ° C, 30 seconds for DNA denaturation; 52 ° C, 45 seconds for primer bonding; 72 ° C, 45 seconds for extension synthesis) Repeat 30 cycles; 72 ° C, 7 minutes; 4 ° C reaction is completed. After the completion of the PCR reaction, the product was analyzed by nucleic acid electrophoresis in 1.5% Agarose gel/0.5X TAE buffer, and 1 μl of 6X loading dye and 5 μl of PCR product were mixed uniformly, firstly at 50 volts for 10 minutes, and then the voltage was adjusted to 100. Volt was carried out for 20 minutes, then stained with ethidium bromide (0.5 mg/ml) and distilled water, and the film was observed under UV light, compared with 100 bp DNA ladder (100-3,000 bp; Protech, Taiwan). For the size of the PCR product, please refer to the third figure, which is the result of the increase of the IturinA gene. Among them, Lane1: 100-3,000 bp DNA markers (Gene Mark, Taiwan); Lane 2: the strain ituA gene; Lane3: the strain ituB gene; Lane4: the strain ituC gene; Lane5: the strain ituD gene.

(VI) Purification of the amplification product of the Iturin A gene sequence and nucleotide sequence alignment

The use GeneMark ^® Gel Elution kit for gel purification of PCR products were cut. The purification steps are as follows: first, the desired fragment is cut out from the nucleic acid electrophoresis film, and the cut colloid is transferred to a 1.5 ml microcentrifuge tube, and an equal volume of Binding solution with the PCR product is added and shaken to uniformly mix. The mixed liquid was transferred to a Spin Column and combined with a Collection tube and centrifuged at 12,000 rpm for 2 minutes. After the filtrate was removed, the addition of 700 μl of Wash solution was carried out by centrifugation at 12,000 rpm for 1 minute. The filtrate was decanted and centrifuged at 12,000 rpm for 5 minutes. Move the Spin Column to a new 1.5 ml microcentrifuge tube and place in the oven for 15 min to remove the alcohol completely. Further, 30 μl of Elution solution was added to centrifuge at 12,000 rpm. After 1 minute, the liquid in the microcentrifuge tube was subjected to electrophoresis analysis using 1.5% Agarose Gel to confirm the purified PCR product.

The purified product is subjected to sequencing and sequencing. The ABI PRISMTM BigDyeTM Terminator Cycle Sequencing Ready Reaction kit and the ABI PRISMTM 3730 DNA Sequence (Perkin Elmer, U.S.A.) nucleic acid automated sequencing analyzer were used. The sequenced results were searched into the nucleotide sequence of the NCBI website for nucleotide alignment to confirm that the sequence fragment of this strain was a bacterium of the bacterium A. bacillus.

<Strain characteristic test> (a) pathogenic fungi inhibition test

A single colony was selected from the LA medium plate, inoculated into 3 ml of LB, and cultured at 28 ° C for 24 hours with constant temperature shaking before the inhibition test. Using a hole cutter with a hole diameter of 0.4 cm, cut the tip of the cultured fungal mycelium, place the cut hyphae in the center of the PDA plate, and affix a paper ingot with a radius of 0.4 cm from both ends of the 2.5 cm of the hyphae. After that, the cultured bacteria liquid was adjusted by spectrometer (spectrophotometer ultrospec 2000, pharmacia biotech, USA) to adjust the absorption value (A ₆₂₀ ), and the concentration of the bacterial liquid was adjusted to 10 ⁸ colony-forming units (cfu/ml), and 30 μl was added. On the paper ingots, they were incubated at room temperature for five days to observe their competition for pathogens.

Please refer to the fourth figure, which is the result of the test for inhibiting the activity of phytopathogenic fungi by the BF-1 strain of the present invention, wherein the pathogenic fungi tested include: (A) Myxosporium psidii Sawada et Kurosawa, (B) Rhizoctonia solani , (C) Bipolaris oryzae (Breda de Haan) Shoemaker , (D) Pestalotiopsis euginae , (E) Pestalotiopsis psidii (Pat.) Mordue Pestalotia psidii , (F) Botyodiplodia theobromae Pat. (Papaya), (G) Botryodiplodia theobromae Pat. (Mango), (H) Colletotrichum gloeosporioides (Penz. & Sace. (I) Colletotrichum gloeosporioides Penzig, (J) Colletotrichum gloeosporioides (Penz.) Sacc., (K) Colletotrichum gloeosporioides , (L) Fusarium oxysporum f.sp.cepae (M) Verticillium sp. Fusarium sp., (N) Phytophthora palmivora (Butler) Butler., (O) Alternaria brassicae (BERK) Sacc. Among them, the left culture dish of (A)~(O) in the first figure is the control group, and the culture dish on the right side is the experimental group, thereby observing that the BF-1 strain has an effect of inhibiting the activity of the pathogenic fungus.

(2) Pathogenic bacteria inhibition test

A single colony was selected from the LA and KB medium plates, and inoculated into 3 ml of LB and KB liquid culture solution, and cultured at 28 ° C for 24 hours under constant temperature shaking, and then subjected to inhibition test. Adjust the absorption value (A ₆₂₀ ) of the pathogen bacterial solution and the BF-1 bacterial solution spectrometer (spectrophotometer ultrospec 2000, pharmacia biotech, USA) for 24 hours, and adjust the bacterial concentration to 10 ⁸ colony-forming units (cfu/). After ml), the pathogenic bacteria inhibition activity test was carried out by using a paper disc diffusion method. 100 μl of the pathogenic bacteria was applied to the plate coated with LA and KB plates, and a paper ingot having a radius of 0.4 cm was attached, and 50 μl of the bacteria was taken. The liquid was applied to the paper ingot, and then placed in a 28 ° C incubator for 24 hours to observe the size of the inhibition ring radius. The pathogenic bacteria used in this study were Acidovorax avenae subsp. citrulli , Xanthomonas axonopodis pv. citri , tomato Xanthomonas campestris pv. vesicatoria (Doidge) Dye, bacterial soft rot PectoBActerium carotovorum subsp. carotovorum , Ralstonia solanacearum , Pectobacterium chrysanthemi (formerly E. chrysanthemi ), Actidovorax avenae subsp. cattleyae . Please refer to the fifth figure for the results of experiments using BF-1 strain to inhibit the activity of phytopathogenic bacteria. The tested plant pathogenic bacteria are: (Q) tomato bacterial spot pathogen, (R) citrus canker pathogen, (S Cucumber bacterial fruit spot pathogen; the left culture dish of (Q)~(S) is the control group, and the right culture dish is the experimental group. It can be observed that the BF-1 strain has the effect of inhibiting the activity of the pathogenic bacteria. .

The BF-1 strain of the present invention can be used in combination with a chemical pesticide, which can be: a fungicide, a bactericide, an insecticide, a herbicide, and a spreading agent of different dilution ratios. Among them, the chemical fungicides used include: gram heat (aliphatic nitrogen), poker (melamine), chlordane (amine phthalic acid), leucoside (benzamide), gram Rotten (benzaldehyde aniline), racer (sulfonamide), carnitrol (antibiotics), atorine (squamosin), tetrachloroisophthalonitrile (aromatic), free Obtained (benzimidazoles), methylpolyproton (benzimidazole precursors), kekemin (aminoformate), yiputong (imidazoles), kylate (triazoles), ternary Copper sulphate (copper), chlorfenapyr (dichlorophenyl dimethyl quinone imide), dernedi (dithiocarbamate), free of rot (polymeric dithiocarbamate), poker Manganese (imidazole), forsylate (organophosphorus), chlorpromazine (pyridine), fenrimyl (pyrimidine), vebufen (quinoline), nitrile sulfonium (indole), Lithium (thiazoles), Binclones (urea), trioxazole (triazole benzothiazoles). [See Appendix I]. The chemical bactericide includes: copper hydroxide (copper), chain tetracycline (antibiotics), streptomycin (antibiotics), and oxolinic acid (quinoline fungicide). [See Appendix II]. The chemical insecticides include: Pyrene (three nitrogen wells), edamine (organic nitrogen) And heterocyclics), Datnam (organic nitrogen and heterocyclics), nanode (ammonia methanate), Jiabao (ammonia methanate), Taosson (pyridine organic thiophosphates), new Killing (biphenyl), Pedan (organochlorine), chlorhexidine (azole), bifenin (azole), cotinine (new nicotine), fumicide (organophosphorus), killing Pine (organophosphorus), kefanpai (pyrazole), macrosomia (straight-chain amine thiophosphates), methiophene (antibiotics). [See Appendix III]. The chemical herbicide includes: di- or tetra-sodium salt (phenoxy acid system), chlorpyrifos (phosphoric acid), phosphazone isopropylamine salt (melamine), and phloem (diphenyl ether) , applied bismuth (dinitroaniline), paraben (quaternary amine). [See Appendix IV]. The different dilution factor spreaders include: Triton X100.

<Example 1>

This embodiment is mainly for the survival rate of the test strain in chemical pesticides. Among them, the experimental group was selected from a single colony on the LA medium plate, inoculated into 3 ml LB, and cultured at 28 ° C for 24 hours under constant temperature, and the absorption value was adjusted by spectrometer (spectrophotometer ultrospec 2000, pharmacia biotech, USA) (A _620). ), the concentration of the bacterial solution was adjusted to 10 ⁸ colony-forming units (cfu/ml), 100 μl of the bacterial solution, 100 μl of 10X stock of the chemical agent and 800 μl of ddH ₂ O were added and mixed, and then shaken evenly, and placed on the table to stand 10 minute. In the control group, 100 μl of the bacterial solution was mixed with 900 μl of ddH ₂ O. After 10 minutes, 100 μl of the bacterial solution was serially diluted, and then 100 μl of each of the dilutions of 10 ^-3 to 10 ^-5 was applied to the LA plate for coating, and then placed at 28 The incubator was cultured for 24 hours in a °C incubator, and the number of colonies was counted after 24 hours, thereby obtaining the survival rate. Among them, fungicides, bactericides, insecticides, herbicides and spreading agents of different dilution factors are tested in the above manner, the difference is only the concentration of the drug is different.

Please refer to the sixth figure for the survival rate of the BF-1 strain when using the BF-1 strain of the present invention in combination with the above 27 types of fungicides. It is shown in the graph of the sixth figure that the BF-1 strain of the present invention is used in combination with the above-mentioned 27 kinds of fungicides, and the survival rate of the BF-1 strain is at least 70% or more.

Please refer to the seventh figure for the survival rate of the BF-1 strain when using the BF-1 strain of the present invention in combination with the above 12 types of insecticides. It is shown in the graph of the seventh figure that the BF-1 strain of the present invention is used in combination with the above 12 types of insecticides, and the survival rate of the BF-1 strain is at least 70% or more.

Referring again to the eighth figure, the survival rate of the BF-1 strain is used when the BF-1 strain of the present invention is used in combination with the above-mentioned six types of herbicides. It is shown in the graph of the eighth figure that the BF-1 strain of the present invention is used in combination with the above-mentioned six types of herbicides, and the survival rate of the BF-1 strain is at least 70% or more.

Referring again to the ninth figure, the survival rate of the BF-1 strain is used when the BF-1 strain of the present invention is used in combination with the above three types of bactericides. It is shown in the graph of the ninth figure that the BF-1 strain of the present invention is used in combination with the above three types of bactericides, and the survival rate of the BF-1 strain is at least 80% or more.

Furthermore, the BF-1 strain of the present invention can be further used in combination with Suri strain.

<Embodiment 2>

The main purpose of the field trial was to investigate whether the BF-1 strain has the ability to control. The tomato ( Solanum lycopersicum ) was firstly bred to the appropriate age for inoculation. A single colony was selected from the LA medium plate, inoculated into 3 ml of LB, and cultured at 28 ° C for 24 hours under constant temperature, and then placed in 200 ml of LB. Incubate at 28 ° C for 24 hours with constant temperature, adjust the absorption value (A ₆₂₀ ) by spectrometer (spectrophotometer ultrospec 2000, pharmacia biotech, USA), adjust the concentration of the bacteria to 10 ⁸ colony-forming units (cfu/ml), and take 50 ml of bacteria. The liquid was evenly poured in the root soil, and the Check (control group) was watered. A total of five groups were tested. The bacterial liquid is applied once every other week, and after three consecutive administrations, the wound is made by the rooting method, and the plant is allowed to stand in contact with the BF-1 bacteria by standing on the seven angel root ring.

A single colony of Ralstonia solanacearum was selected from the LA medium plate, inoculated into 3 ml of LB, and cultured at 28 ° C for 24 hours under constant temperature, placed in 200 ml of LB, and cultured at 28 ° C for 24 hours under constant temperature, using a spectrometer ( Spectrophotometer ultrospec 2000, pharmacia biotech, USA) adjust the absorption value (A ₆₂₀ ), adjust the concentration of the bacterial liquid to 10 ⁸ colony-forming units (cfu / ml), and take 50 ml of the bacterial liquid evenly poured into the root soil and observe continuously. week.

The tomato plants of the control group in which the symptoms appeared were cut out from the browning of the stem base, and the incision was placed in clear water to observe whether or not a white cloud-like liquid flowed out from the incision. Then, the tomato plants in the control group with the symptoms are cut out from the browning of the stem base, the stems are cleaned, and the stems are cleaned with 70% alcohol to clean the stems, and then the stems are disinfected. Place it on the aseptic table for drying. After drying, cut a small section of stem to remove the surface. After shaking in ddH ₂ O, the suspension is taken up with a transplanting ring and streaked onto the KB medium plate. The plate was placed in a 28 ° C incubator for 1-2 days, and a single colony was picked for four-zone scribing, repeated three times, and then transferred to a KB medium plate, and a single colony was picked and cultured in 3 ml KB liquid medium for genomic DNA extraction, and then use the universal primer to carry out PCR amplification and nucleic acid electrophoresis analysis on 16S-F/16S-R (Table 1), confirm the size of the amplified product, and then purify the amplified product, and then disassemble the purified product. Sequence, the results of the sequencing, and then enter the nucleotide sequence of the NCBI website (nucleotide blast) for search alignment to confirm which strain of this strain is the genus, and the 16S sequence of the original strain Than to confirm whether the original strain. Please refer to the tenth figure, in which the control group is the growth of tomato bacterial wilt pathogen inoculated with tomato young plants, and the treatment group is the growth of tomato bacterial wilt pathogen after inoculation of the BF-1 fermentation product, and the control group and the control group Under the comparison of the treatment groups, the strain of BF-1 of the present invention does have the ability to control plant diseases.

<Example 3>

The BF-1 strain of the present invention has the ability to strongly produce a protease. The protein decomposition test method is as follows: A BF-1 fermentation broth is prepared, and the preparation steps are as follows: a pathogenic fungi inhibition test implementation step. 50 μL of the fermentation broth was placed on a 1 cm (ID) sterilized round filter paper, and the filter paper was placed in a skim milk agar (SMA) plate containing 1.5% SMA, 1.5%. Nutrient broth (NB) and 1.5% agar. The plate was incubated at 30 ° C for 2 to 4 days. The size of the more transparent circle (protein is hydrolyzed) around the colony is measured. Four times of experiment, the decomposition distance is: (1) 12.89 (mm); (2) 12.32 (mm); (3) 12.21 (mm); (4) 12.28 (mm); the average distance is 12.4 (mm); wherein the greater the distance, the better the decomposition effect. This proteolytic enzyme can be used for feed addition, food processing addition, agricultural aquaculture wastewater treatment, and the like. Referring to Figure 11, the ability of the BF-1 strain of the present invention to decompose proteins is shown.

<Embodiment 4>

The BF-1 strain of the present invention has a potent ability to produce a lipase. The lipid decomposition test method is as follows: A BF-1 fermentation broth is prepared, and the preparation steps are as follows: a pathogenic fungi inhibition test implementation step. 5 μL of the fermentation broth was placed on a Rhodoamine B culture plate containing 0.2 to 1% vegetable oil, 0.001% 1 rose red B, and 1.5% amaranth. The plate was incubated at 30 ° C for 5 to 7 days, and observed under a fluorescent microscope, and the size of the lipid-free (hydrolyzed) transparent circle was recorded. Observe the dissolution circle around the colony (1) 6.56 mm; (2) 6.22 mm; (3) 6.57 mm; (4) 6.43 mm; average distance 6.45 mm. Among them, the larger the dissolution circle, the better the decomposition effect. Referring to Figure 12, the ability of the BF-1 strain of the present invention to break down lipids is shown.

<Embodiment 5>

The BF-1 strain of the present invention has a strong ability to produce cellulase. The cellulolytic test method is as follows: A BF-1 fermentation broth is prepared, and the preparation steps are as follows: a pathogenic fungi inhibition test implementation step. 50 μL of the fermentation broth was placed on a 1 cm (ID) sterilized round filter paper, and the filter paper was placed on a Mandel-Reese, MR) vegetable culture plate containing 1% carboxy methyl cellulose, carboxymethyl cellulose. , 0.1% peptone, 0.03% urea, 0.15% ammonium sulfate, etc. and 1.5% amaranth (agar), pH 6.0. After the plate was incubated at 30 ° C for 2 days, 3.5 ml of 0.1% Congo red was overlaid on the medium for 30 minutes, and then washed with 1 M sodium chloride as a reddish portion, and this portion of the cellulose was decomposed by the enzyme. The dissolution circle around the colony was observed: (1) 4.55 mm; (2) 4.80 mm; (3) 4.68 mm; (4) 4.49 mm; average distance 4.6 mm. Among them, the larger the dissolution circle, the better the decomposition effect. Referring to Figure 13, the ability of the BF-1 strain of the present invention to decompose cellulose is shown.

<Embodiment 6>

The BF-1 strain of the present invention has an ability to strongly produce amylase. The starch decomposition test method is as follows: The BF-1 fermentation broth is prepared, and the preparation steps thereof are the steps of the pathogenic fungi inhibition test. 50 μL of the fermentation broth was placed on a 1 cm (ID) sterilized round filter paper, and the filter paper was placed on a canola culture plate containing 1% yeast extract, 1% soluble starch and 1.5% amaranth ( Agar). After the plate was incubated at 30 ° C for 2 days, 3.5 ml of iodine solution (0.3% iodine and 3% potassium hydride) was covered to cover the medium, and the observation was completed within 5 minutes, and the starch was not blackened around the part. For decomposition by enzymes. The dissolution circle around the colony was observed: (1) 1.01 mm; (2) 0.99 mm; (3) 1.03 mm; (4) 1.01 mm; average distance 1.0 mm. Among them, the larger the dissolution circle, the better the decomposition effect. Please refer to Figure 14 for the ability to decompose starch of the BF-1 strain of the present invention.

The above-described embodiments or the drawings are not intended to limit the scope of the invention, and any suitable variations or modifications of the invention will be apparent to those skilled in the art.

In summary, the embodiments of the present invention can achieve the expected use efficiency, and the specific structure disclosed therein has not been seen in the same product, nor has it been disclosed before the application, and has 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.

First panel: Results of the amplification of the 16S rRNA sequence of the BF-1 strain of the present invention.

Figure 2: Results of the increase in Tet(L) gene

Figure 3: Results of the increase in the IturinA gene

Figure 4: The phytopathogenic fungi which are inhibited by the fermentation products of BF-1 against crop fungal diseases are guava (A), R. solani (B), and F. fuliginea (C). , Pestalotia euginae (D), Pestalotia psidii (E), Papaya rot fungi (F), Phytophthora rot fungi (G), Anthracis sinensis (H) , Papaya anthracis (I), anthracnose (J), anthracnose (K), Rhizopus aureum (L), Verticillium dahliae (M), Papaya (N), Black spot (B.) )

Fig. 5: The phytopathogenic bacteria which are inhibited by the fermentation products of BF-1 bacteria against bacterial diseases of plants are tomato bacterial spots (Q), citrus canker (R), melon bacterial fruit spot bacteria (S )

Figure 6: The cell viability using BF-1 bacteria in combination with the following 27 types of fungicides; 1. gram heat (alkane nitrogen), 2. poker pull (melamine), 3 .Indole (amine phthalic acid), 4. leucoside (benzamide), 5. gram rot (benzamide), 6. 赛 灭 (sulfonamides), 7. Neomycin (antibiotics), 8. Atomomin (spheroids), 9. tetrachloroisophthalonitrile (aromatic), 10. free (benzimidazole), 11. methyl Safe (benzo Imidazole precursors), 12. 100 grams of amide (amino amides), 13. espresso (imidazoles), 14. to Klee (triazoles), 15. ternary copper sulphate (copper), 16 .Phen Ning (dichlorophenyldimethylimine), 17. Dedi (dithiocarbamate), 18. Free of rotten (polymeric dithiocarbamate), 19. Poker Manganese (imidazole), 20. Forsyth (organophosphorus), 21. chlorinated (pyridine), 22. fenrimyl (pyrimidine), 23. vebufen (quinoline), 24. Nitrile sulfonium (indole), 25. edeli (thiazole), 26. bin clone (urea), 27. trixazole (triazole benzothiazole).

Figure 7: The cell viability using BF-1 bacteria in combination with the following 12 classes of insecticides; 1. Destructive net (trinitrogen system), 2. Yida amine (organic nitrogen and heterocyclic ring) Class), 3. Datnam (organic nitrogen and heterocyclic), 4. nanode (ammonia methanate), 5. plus Baofu (ammonia mesylate), 6. Taosson (pyridine organic thiophosphate) Salt), 7. New acaricidal (biphenyl), 8. Pedan (organochlorine), 9. dying (azole), 10. Bifenin (azole), 11. Cotinidine (new Nicotine), 12. Fenicide (organophosphorus), 13. Ai pine (organophosphorus), 14. Kfanti (pyrazole), 15. Dasong (straight-chain amine organophosphoric acid) Salt), 16. Mistidine (antibiotics)

Figure 8: The cell viability using BF-1 bacteria in combination with the following six classes of herbicides; 1. Two or four sodium salts (phenoxy acid), 2. Guticide (phosphoric acid) ), 3. Phosphate isopropylamine salt (melamine), 4. Fulufen (diphenyl ether), 5. Shit (dinitroaniline), 6. Barragel (tetramine)

Figure 9: Using BF-1 bacteria in combination with the following three types of bactericides Body survival rate; 1. Copper hydroxide (copper), 2. Chain tetracycline (antibiotics), 3. Streptomycin (antibiotics), 4. Oxalic acid (quinoline fungicide)

The tenth picture shows the growth of tomato bacterial wilt pathogens in tomato plants in the control group, and the growth of tomato bacterial wilt pathogens inoculated with tomato BF-1 fermentation products in the treatment group.

Figure 11: Test dish for decomposing protein of BF-1 strain of the present invention

Twelfth figure: a test dish for decomposing lipids of the BF-1 strain of the present invention

Thirteenth figure: a test dish for decomposing cellulose of the BF-1 strain of the present invention

Figure 14: Test dish for decomposing starch of BF-1 strain of the present invention

[Refer to the attached table]

Table 1. Bacillus spp. Identification of primer pairs used for polymerase chain reaction

Table 2. Primer pairs of the amplified Iturin A gene sequence used in the polymerase chain reaction

[refer to the appendix]

Appendix I, list of fungicide types

Appendix II, List of bactericides

Appendix III, List of Insecticides

Appendix IV, List of herbicide species

<110> National Kaohsiung Normal University

<120> Bacillus aeruginosa strain and use thereof

<140> TW100105877

<141> 2011-02-22

<160> 1

<210> 1

<211> 1270

<212> DNA

<213> 16S rRNA sequence of Bacillus amyloliquefaciens BF-1

<400> 1

<110> National Kaohsiung Normal University

<160> 1

<210> 1

<211> 561

<212> DNA

<213> The gene sequence of YyaR of Bacillus amyloliquefaciens BF-1

<400> 1

<110> National Kaohsiung Normal University

<160> 4

<210> 1

<211> 620

<212> DNA

<213> ItuA gene sequence of iturin of Bacillus amyloliquefaciens BF-1

<400> 1 (ItuA)

<210> 2

<211> 514

<212> DNA

<213> ItuB gene sequence of Bacillus amyloliquefaciens BF-1 iturin

<400> 2 (ItuB)

<210> 3

<211> 348

<212> DNA

<213> ItuC gene sequence of Bacillus amyloliquefaciens BF-1 iturin

<400> 3 (ItuC)

<210> 4

<211> 299

<212> DNA

<213> ItuD gene sequence of Bacillus amyloliquefaciens BF-1 iturin

<400> 4 (ItuD)

Claims (10)

A novel strain of Bacillus aeruginosa liquefied, deposited in the Food Industry Development Institute of the consortium, registered number: BCRC910498, named BF-1 with unique 16S rRNA, Tet (L), ItuA, ItuB, ItuC, ItuD genes sequence. A use of the strain of Bacillus algolyticus according to claim 1 for the decomposition of at least one component of the group consisting of starch, protein, cellulose and lipids. A use of the strain of Bacillus algolyticus according to claim 1 of the patent application is for use in feed addition or agricultural aquaculture wastewater treatment. A use of the strain of Bacillus amyloliquefaciens according to claim 1 for inhibiting the activity of a phytopathogenic fungus, wherein the phytopathogenic fungus comprises at least one of the following: guava blight, sunflower lily Rhizoctonia solani, flax leaf blight, lotus leaf rot, guava scab, papa rot, citrus rot, lemon anthracnose, papaya anthracnose, lotus aphid, anthracnose, Purple onion, Verticillium, Papaya, and Brassica. A use of the strain of Bacillus amyloliquefaciens according to claim 1 for inhibiting the activity of a phytopathogenic bacterium, wherein the phytopathogenic bacterium comprises at least one of the following: tomato bacterial spot pathogen, citrus ulcer Pathogenic bacteria, melon bacterial fruit spot pathogens. A use of the strain of Bacillus algolyticus according to claim 1 of the invention, which is used in combination with a fungicide to form a plant protection composition, wherein the fungicide comprises at least one of the following: Alkane nitrogen), poker (melamine), chlordane (amine phthalic acid), leucoside (benzamide), gram rot (benzamide), cyanobacteria (sulfonamides) ), carbachol (anti- Biotin), Atmomin (Cicumin), Tetrachloroisocyanide (Aromatic), Freeride (Benzimidazole), Methyl-Pure (Benzimidazole Precursor) , Baikemin (Aminoformate), Yiputong (Imidazole), Klein (Triazole), Ternary Sulfate (Copper), Phthaline (Dichlorophenyldimethylimine) , Dessert (dithiocarbamate), free of rot (polymeric dithiocarbamate), poker pull manganese (imidazole), forsythia (organophosphorus), chloramine (pyridine Class), fenrimyl (pyrimidine), vafenfen (quinoline), nitrile sulfonium (indole), edeli (thiazole), bun clone (urea), trimethoprim (triazole benzo) Thiazoles). A use of the strain of Bacillus algolyticus according to claim 1 of the patent application, which is used in combination with an insecticide to form a plant protection composition, wherein the insecticide comprises at least one of the following: Nitrogen wells, edamines (organic nitrogen and heterocyclics), Datnam (organic nitrogen and heterocyclics), nadine (ammonia methanate), plus Baofu (ammonia methanate), Taosson (pyridine organic thiophosphates), new acaricidal (biphenyl), Pedan (organochlorine), chlorhexidine (azole), bifenin (azole), cotinine (new nicotine), Fenicide (organophosphorus), acesulfame (organophosphorus), kefanpai (pyrazole), chlorfenapyr (linear amine thiophosphate), dexamethasone (antibiotics). A use of the strain of Bacillus algolyticus according to claim 1 of the patent application, which is used in combination with a herbicide to form a plant protection composition, wherein the herbicide comprises at least one of the following: di- and tetrasodium salt ( Phenoxy acid system), chlorpyrifos (phosphoric acid), phosphamethoxazole isopropylamine (melamine), valprofen (diphenyl ether), sedative (dinitroaniline), barrageen ( Tertiary amines). A use of the strain of Bacillus algolyticus according to claim 1 of the patent application, which is used in combination with a bactericide to form a plant protection composition, wherein the bactericide comprises at least one of the following: copper hydroxide (copper) Tetrahymena (antibiotics), oxolinic acid (quinoline fungicides). A use of the strain of Bacillus licheniformis according to claim 1 of the patent application, which is used in combination with Suri strain to form a biological pesticide composition.