TW201234971A - Bacillus amyloliquefaciens and application of the same - Google Patents

Abstract

This invention relates to Bacillus amyloliquefaciens BF-1 (Food Industry Research and Development Institute, the patent organisms in number BCRC910498) and the application of the same, which has a unique gene sequence of 16S rRNA, Tet (L), ItuA, ItuB, ItuC, and ItuD. BF-1 and its antibiotics can inhibit the growth of plant pathogenic fungi and plant pathogenic bacteria. It can be used with fungicides, insecticides, herbicides, bactericides, and Bacillus thuringiensis to be a plant protecting composition. BF-1 has productive amylase, protease, cellulase and lipase activity. BF-1 and its products can be applied to plant protection, sewage treatment in agriculture and aquaculture, food and chemical industries.

Description

201234971 VI. Description of the Invention: [Technical Field of the Invention] The present invention relates to a strain of liquefied Bacillus amyloliquefaciens, in particular to a type of BF-1 having unique i6SrRNA, Te1: (L), ItuA, ItuB, A strain of Bacillus amyloliquefaciens of the gene sequence of ItuC and ItuD (the Institute of Food Industry Development, the patented microbial deposit number BCRC910498) is used to produce a variety of enzymes and various antibiotics. [Prior Art] It is known that various microorganisms can be used to control the biological activity of plant diseases, which has been helpful in the field of 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). 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 Environmental Protection Agency (EPA) has approved a number of species of Bacillus (your 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 house powder). Bacillus (rar. amyloliquefaciens, you k palmate % { Bacillus cereus), also table * each release instrument { Baci 1 lus 1 icheniformis) '201234971 short and other notes {Baci 1 lus pumi lus), good effort {Baci 1 lus thuringiensis), H Bacijjus popi 11lae) 舆Q Baci 11 us sphaericus). 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 lentus.

Many important plant diseases are caused by soil or rhizosphere fungi. Researchers use the antagonism between soil microorganisms to develop biocontrol 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 and application of biotechnology industries such as seed protection agents have been used for many years as 'beneficial microorganisms that are generally recognized as safe. Recently, it has been applied to the roots of the soil and the pathogens to compete for the nutrients in the root system to become the dominant species, thereby reducing the risk of pathogenic bacteria. It is also possible to directly spray the plant leaves to protect the leaf fungus, such as the bean disease or the seed mix. (4) Prevention of soil and disease] The growth of the crops, especially the obvious promotion of the roots; can also be used as antibacterial agents after harvesting fruits and vegetables, such as peach brown rot and citrus blue mold. For the liquefied Bacillus amyloliquefaciens, it was found in 194 scholar Fukomoto, which can produce Daping from Japan [alpha-amylase] and protease [protease], and also cellulolytic enzymes, so it is used for biological feed. The meat exchange rate and breeding rate of Force 4 can be promoted to the camp: raw; 201234971 Increase disease resistance, reduce the use of chemical pesticides and fertilizers; also cooperate with organic cultivation to improve chemical pesticides and fertilizer residues Problems with fruits and vegetables, soil and water, and reducing barriers to continuous cropping. SUMMARY OF THE INVENTION One main object of the present invention is to provide a strain which is a novel liquefied Bacillus amyloliquefaciens having the code BF-1, which produces a specific and beneficial enzyme such as a lipolytic enzyme. It is used to break down lipids, amylolytic enzymes to hydrolyze starch, cellulolytic enzymes to hydrolyze cellulose, and proteolytic enzymes to hydrolyze proteins. The novel 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 anti-biomass 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 stand (A), sunflower lily (B), flax leaf (C), Pestalotia (D), Pestalotia (gua) (E), papaya rot (F) , lemon fruit rot (G), lemon fruit anthrax (H), papaya anthrax (I), lotus aphid (J), jujube anthrax (K), onion purple root (L), verticillium wilt (M), papaya disease (N), cabbage black spots (0). The three objects of the present invention are to propose a novel Bacillus amyloliquefaciens 201234971 BF-1 and/or an anti-biomass produced by the strain as an antibacterial agent for inhibiting the growth of at least one bacterial infection source in the following composition groups' § Mysterious bacteria include: Bianyou bacterial wilt, Fanzhuang bacterial spot (Q), sweet orange ulcer disease (R), melon bacterial fruit spot (S). 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 (stupid imidazole precursors), kekemin (amine amides (materials), to Klee (three, three yuan = copper (copper), chlorpheniramine (diphenyl phthalimide) ), Deandi (dithiocarbamate), free of rot (polymeric dithiocarbamate), poker ramie), forsythia (organophosphorus), chloramine (bite type), fenremore (♦ class), fast, fen (quinoline), nitrile sulfonium (indole), edeli (thiazole) , Binbin (urea), Sansaiwa (triterpene benzopyrene). The five purposes of the present invention are to propose a novel plant protection composition, which is the novel liquid B. oxysporum Bp proposed by the present invention. In the group = at least one insecticide is used in combination, the insecticide package, the surname 1 is eliminated (three nitrogen wells), the edaramine (organic nitrogen and heterocyclics), organic nitrogen and heterocyclics ), Na Naid (ammonia methanate), 4 & machine acid salt), _ pine (_organic thio-squaric acid 201234971 salt), new snail (Lianfujing s, shunning (salmon), Bifendan (organic gas), class), _organic dish) sitting class...Xinni" Kefanpai sitting class), big] killing pine (organophosphorus), class), carbamide (antibiotic) The linear amine amine thioester salt 2 case of the six-purpose system proposes a new plant protection composition, which is the liquid cut material L of the (4) paste of the present invention: at least one herbicide combined with the bacteria in the group The herbicide package = tetradiazepine (phenoxy acid), chlorpyrifos (phosphoric acid), Jia = isopropylamine salt (bristled amine), complex rifampine (biphenyl) Shouting), 圃 圃 (二丝笨胺系), 巴(四)(四级胺类). The seven purposes of this case are to propose a new plant protection composition, which is the liquefaction powder of the (4) paste of the present invention. Bacillus (4) is combined with at least one of the following bactericides, such as copper hydroxide (copper), chain tetracycline (antibiotics), gentamicin (antibiotics). Oxalolinic acid (quinoline fungicide). [Embodiment] The present invention relates to a novel strain of Bacillus amyloliquefaciens deposited in the Institute of Food Industry Development of the Corporation [Accession No.: BCRC910498] Bp-ι has a unique gene sequence of 16S rRNA, Tet (L), ItuA, ItuB, ItuC, ItuD. The 16SrRNA sequence of the Bacillus amyloliquefaciens BF-1 is:

AGACCGACTTGGCGCCTCCCCAATAATGCAAGTCGAGCGGACAGATGGGAGC

TTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCC

TGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTT 201234971

GAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGG

ACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGAT

GCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCC

AGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCT

GACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGT

TGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCT

AACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGT

GGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTT

AAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGG

GGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATG

CGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAAC

TGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTA

GTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAG

TGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACT

GAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTT

AATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATC

CTAGAGATAGGACGTCCCCTTCGGGGCAGAGTGACAGTGTGCATGGTTGTCG

TCAGCCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT

GATCTAGTGCCAGCATTCAGTGGGCACTCTAAGGTGACTGCGTGACAACCGG

AGAGGTGGGATGACGTCAATCATCATGGCCCTATGACCTGGCTCAACAAGTG

CTACATGGCCGAACAAGGGCAAGAAACCGCAGAGGTTTAGCACTCCACGAGT

CTGGTTTCCAATTCGAGATCGA The gene sequence of Tet (L) of the liquefied Bacillus amyloliquefaciens BF-1 is:

AGCGTTTCTGACCAGATATTAATGTTTCAGCTTGTCATTTTTATGCCAAAAA

TAACTTTGTCATAGGAGCGGTCGATACAATGCTTTATTCCAATTTTTCAACA 9 201234971

GCAAATGAAATTGCGGTTTTTTGGTACTGTAAATTTTAAAGATAACAGTATT

TGATGTTTCGTCATTTAGCGAGTGTGTGTCGCAAAGTATTCAAAACATCGGC

TTGAGGATTTATAATAAGGTATATCAACCAATGCAGGGGGAGTTAAGTATGA

AGTGCAAAAAAATGAACAGGGTACAATTAAAAGAGGGAAGCGTATCAATGGC

CCTATAAACTGCGTCTGCCCTCATTAATGGAGGGGAAATTGTGAATACGCCT

TATTCACAATCAACTTTACGGCACAACCAAGTGTTCATTTGGCTTTGTGTTC

TGTCATTTTTCAGCGTATTAAATGAAATGGTTTTGAACGTCTCGTTACCTGA

TATCGCCAACGACTTTCATAAACTGCCAGCGAGTGCAAACTGGGTGAACACG

GCCTTTATGCTCACCTTCTCCATTGGAACAGCTCTATATGG The sequences of ItuA, ItuB, ItuC, ItuD of Bacillus licheniformis BF-1 are: <ItuA>

CCGGAAGATGACATAAGCTGAGGTCTGGCGCATTTGGAATATATTAAATCAT

CCGTTTCTGATTGCCTCTGAAAAAGTATTGGACAAAATAAAGAAATACGCTG

CAGAACACGATTTACAGGATTTCCATCATCAATTAAACGAAAAATCTGACAT

CATTCAAGATCAAACCTACGATTACCCCGCTTCATTTTATGAACCTGATGCG

GATGAACTCGCCTTTATCCAATTTTCTTCTGGATCAACAGGAGATCCAAAAG

GAGTCATGTTAACGCATCACAACTTAATACATAACACGTGCGCCATTGGGAC

TGCCCTACACGTTCATTCGAAAGACTCTTTCTTATCATGGATGCCTTTAACG

CATGATATGGGGCTCATCGCCTGCCACCTTGTTCCCTTCATAACCGGAATCA

ATCAAAATCTAATGCCTACAGAGTTATTTATTCGAAGACCTATTCTTTGGAT

GAAAAAAGCTCATGAACACAAAGCCAGTATTCTATCATCTCCTAATTTCGGG 201234971

TACAACTACTTCCTTAAATTTCTGAAAAACGAACCAGACTGGGATTTATCAC

ACATCAAGGTCATCGCAAACGGTGCAGAACCGATATTGCCGGAGCTCA <ItuB>

TGGGGAATTTAGGCATTCTCCGGGCGGATCGCGTGCTTGTCTTGTACCCGTA

TGTTTTTGATGCGTTCATTTTAAACTTCTTCGGCCCGCTCATCTCAGGAGCA

ACTTTGCACCTTTTGCCTAATGAAGAGAATAAAGAAACTTTCGCCATTCAAA

ATGCCATAAAACAAGAGAGAATTACTCATTTTTCAACTTCTCCCCGTCTTCT

GAAAACGATGATTGAACAGATGAACGCAGAGGATTTCATCCATGTTCAGCAT

GTTGTTGTAGGCGGTGAGCAGCTGGAAACAGATACAGTTGAGAAGCTGCATT

CTCTGCAGCCTCGCATTCGGATCAATAATGAATACGGACCGACAGAAAACAG

CGTGGTATCTACATTTCACCCTGTTCAGTCCGCTGATGAACAGATCACAATC

GGCAGCCCGGTGGCCAACCATCAAGCTTATATTTTAGGAGCGCACCATCAAA

TCCAGCCGATCGGCGTACCGGGAGAGCTGTATGCGGGAAGGGAGCA <ItuC>

AAGTTCCTAGATATAAGATCGCTTCGTCCAGCGTATGTTTCAATTCATGGAA

AATTGATTTAAACGTACTGCCGGAATTGATATGCTGTTTGATTAACATGAGC

TGGTCTGAACGAAGATCTTCATCAGTTTCGTCTTCAAAGGTTGGAACCCCTG

TAATCACGCCCTCTTCCCCCGTGTATTTGTATAAAAGGGATTCAATTCCGAT

CAGGAGAACCAGATAGACGGCCATTGGCGTTTGGTTCGTCATCGTCAGGATG

CGCTGTGATACATCCGCACTCAATGAACTGTGAATACAGTCAGATCCGCTTG

TTATATCACGCTTTACTGTTGAGTCGGACATTTGAA <ItuD> 11 201234971

CCTAAATTTTTTCCGGGGGATATGAATGATTGACAAAGACAATGAACGCGCA

GCCCGCTATTTTAACGGTCAGTGTCATTGCTTTTCAAGTGTATATGCAGGAA

ATAGGGGTGAAGCCACGCTTCCTGGCAGGCCATAGCTTAGGCGAATATTCAG

CGCTTGTCTGTGCCGGCGCCCTTTCTTTTCAGGATGCCGTTACACTTGTAAG

GGAGCGGGGAATTCTTATGCAAAATGCGGATCCTCAGCAGCAGGGGGCGATG

GCCGCCGTGACTCACCTCTCTCTCCAAACCTTGCAGGAA 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 characteristic test results. &lt;Method of colony screening&gt;, take the powder of the strain 〇.5 g, add 4 5 mL of sterile water to the 14-inch bacterial culture tube (when the powder of the right g strain is insufficient, take the actual weight of 9 times the volume of sterile water) 'Oscillate at 303⁄4, 200 rpm for 1 hour. 2 Transfer the bacterial culture tube to a water bath of 6 ° 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 is diluted with 1 〇n in sterile water (1 〇〇 bacteria liquid + 卯〇 ML sterile water). Take a large, scoop solution of 1G5-7 strain of the strain, take (10) #L coating> </ br> each medium is placed in 3 (rc, overnight culture. Out 翌Λ', each strain of colony type, if More than one type of colony, it is necessary to fully test the liquid culture of the two cockroaches (in addition, each plan - please, used to do Na Na 201234971 &lt; identification of strains] (a) bacterial 16S rRNA gene sequence polymerase chain reaction using universal primer pair 16S-F and 16S-R (refer to Table 1) are nucleic acid primers, and the polymerase chain reaction is carried out using GenomicDNA of the strain as a template. The reaction solution includes 1 Genomic DNA, 2 &quot;1 ι〇χ pcr buffer (TaKaRa, Japan) , 1 //12. 5 mMdNTP, l forward primer and 1 reverse primer, and TaKaRa TaqTM (5 units//zl, TaKaRa, Japan), and 13·8 //1 ddIM) The volume was 20^1 and the PCR reaction was carried out. The reaction conditions were as follows: 94 °C for 5 minutes; (94 〇C, 30 seconds for DNA denaturation; 52 °C, 45 seconds for primer bonding; π °C, 45 seconds for extension) Synthesis) Repeat 30 cycles; 72 ° C, 7 minutes; 4 t reaction is completed. After the completion of the PCR reaction, the product is 1.5% Agarose gel / 0.5X TAE buffer for nucleic acid electrophoresis analysis 'take 1 βΐ 6X loading dye and 5 //1 PCR product mix evenly 'first 50 volts for i〇 minutes, then adjust the voltage to volts for minutes' and then through the desertification key (ethidium Bromide 0. 5 mg/ml) staining and deionization of distilled water, and then placing the film under uv light, and comparing the size of the PCR product with 100 bp DNA ladder (100-3, 000 bp; Protech, Taiwan) The first figure is the result of sequence amplification, where ' Lanel: 100-3,000 bp DNA markers (Gene Mark, Taiwan); Lane2: This strain. (II) Purification and nucleotide sequence determination of bacterial 16S rRNA gene sequence amplification products Order comparison 13 201234971

The PCR amplified product was purified using GeneMark®'s DNA clean/Extraction Kit. The purification step is as follows: firstly, the amplified PCR product is transferred to a 1.5 ml microcentrifuge tube and an equal volume of Binding solution is added to the PCR product and shaken to uniformly mix. Then move the mixed liquid to Spin

Column ’ and combined with the Collection tube, centrifuged at 12 000 rpm for 2 minutes. After removing the filtrate, add 7 〇〇 //1 Wash solution and centrifuge at 1 000 rpm for 1 minute. The filtrate was removed and centrifuged at 12, rpm for 5 minutes. Move the Spin Column to a new 1.5 mU drum centrifuge and place the oven for 15 minutes to remove the alcohol completely. Further, 3 〇 E: iuti〇n solution was centrifuged at 12,000 rpm. After 1 minute, the liquid in the microcentrifuge tube was subjected to electrophoresis analysis using a 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 autosequencing analyzer were used. The sequenced results are stored and then searched into the NCBI website's nucleotide sequence database (nucle〇tide blast) for search alignment to confirm that the sequence fragment of this strain is a bacterium of the genus Bacillus licheniformis. (3) Tet (L) gene sequence polymerase chain reaction West R subt mountain sm sequence cis β. Qing j〇h. Quefac]. The ITS sequence of ens is extremely high, so it is impossible to identify the strain by this fragment. According to the literature (Reva Heart/., 2004), although the two strains have many similar or identical fragments, but they have some records, they have designed three different primers according to this characteristic. Form two groups of bow scorpion pairs, respectively 201234971

YyaO-F/TetB-R, YyaR-F/TetB-R [Refer to Table 1]. The YyaR-F/TetB-R was used as a nucleic acid primer pair, and the Genomic DNA of the strain was used as a template to carry out a polymerase chain reaction. The reaction solution included 1 //1 Genomic DNA, 2 # 1 10X PCR buffer (TaKaRa, Japan), 1 //1 2. 5 mM dNTP, 1 /zl forward primer and 1 //1 reverse primer, and TaKaRaTaqTM (5 units/#l, TaKaRa, Japan), and 13. 8 /zl of ddM The final reaction volume was 20 //1 and the PCR reaction was carried out under the following conditions: 94 ° C for 5 minutes; (94 ° C, 30 seconds for DNA denaturation; 50 ° 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 #1 6X loading dye and 5 &quot; 1 PCR products were mixed uniformly, first for 10 minutes at 50 volts, and then Adjust the voltage to 1 volt for 20 minutes' and then de-stain through the deserted bromide (5 mg/ml) dyeing and distilled water, then place the film under UV light and observe with 100 bp DNA ^ ladder (100-3, 〇〇〇bp; Protech, Taiwan) Align the size of the PCR product, see Figure 2, for the amplification of the Tet(L) gene. Among them, Lanel: 100-3, 000 bp DNA markers (Gene Mark, Taiwan) i Lane2 · This strain. (4) Purification of the Tet (L) gene sequence amplification product and nucleotide sequence sequencing ratio

The PCR amplified product was purified using GeneMark®'s DNA clean/Extraction Kit. The purification step is as follows: firstly, the amplified PCR product is transferred to an L 5 ml microcentrifuge tube and added with an equal volume of the PCR product iBinding 15 201234971 solution and shaken to uniformly mix. The mixed liquid was then transferred to a Spin Column' and combined with a Colection tube at 21,000 Torr for 2 minutes. After the mash and liquid were removed, the addition was carried out twice. Wash solution was centrifuged at 12 000 rpm for 1 minute. The filtrate was removed and centrifuged at 12,000 rpm for 5 minutes. Move the Spin Column to a new 1.5 ml microcentrifuge tube and place the oven for 15 minutes to remove the alcohol completely. Further, 3 〇 μ 1 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 a 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 autosequencing analyzer were used. The sequenced results were searched into the nucleotide blast library of the NCBI website for comparison to confirm that the sequence fragment of this strain was a bacterium of the bacterium A. licheniformis. (5) The Inturin A gene sequence polymerase chain reaction utilizes the Iturin A four-group nucleic acid primer designed by the predecessor (Shi, 2007) to ituA, ituB, ituC, and ituD [Table 2], and then the Genomic DNA of the strain is used as a template. 'Perform a polymerase chain reaction, including 1 Genomic DNA '2 μΙ 10Χ PCR buffer (TaKaRa, Japan) '1 //1 2. 5 mM dNTP, l forward and i reverse primer, and TaKaRaTaqTM (; 5 units///1, TaKaRa, Japan) 'There are also 13.8 with 1 (MH2O to make the final volume of the reaction 20 / zl and then carry out the PCR reaction, the reaction conditions are as follows: 94 ° C for 5 minutes; (94 ° C '30 seconds for DNA denaturation; 52 t, 45 seconds for primer adhesion 201234971 for '72 C ' 45 seconds for extension synthesis) Repeat 3 cycles; π °c, 7 deduction, 4 C reaction is completed. PCR reaction After completion, the product was analyzed by nucleic acid electrophoresis using 1. Agarose gel / 〇 5X TAE buffer, and the ii 6X loading dye and 5 #1 PCR products were uniformly mixed, firstly subjected to 5 volts for 10 minutes, and then the voltage was adjusted. 2 volts to 1 volt, then dyed with ethidium bromide (0.5 mg/ml) Color and distilled water were deproteinized, and the film was placed under a UV lamp. The size of the PCR product was compared with the 1 bp DNA ladder (100-3, 000 bp; Protech, Taiwan).

The result of the increase in the IturinA gene. Among them, Lanel: 1〇〇-3, 〇〇〇bp DNA markers (Gene Mark, Taiwan); Lane2: strain ituA gene; Lane3: strain ituB gene; Lane4: strain ituC gene; Lane5: strain ituD gene . (VI) Purification and nucleotide sequence sequencing ratio of the amplification product of the Iturin A gene sequence The gel product of the PCR product was purified by GeneMark® Gel Elution kit. The purification procedure is as follows: firstly, 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 then transferred to a Spin Column' and combined with a Colection tube and centrifuged at 12 000 rpm for 2 minutes. After the filtrate was removed, two additions of 7 〇〇 #1 Wash solution were centrifuged at 12 000 rpm for 1 minute. The tears were removed and centrifuged at 12,000 rpm for 5 minutes. Move the Spin Column to a new ι 5 ml microcentrifuge tube and place the oven for 5 minutes to remove the alcohol completely. Add 3〇 17 201234971

Elution solution After centrifugation at 12,000 rpm, 1 minute later, the liquid in the microcentrifuge tube was analyzed by electrophoresis with 1.5% Agarose Gel to confirm the purified PCR product.

The purified product is subjected to sequencing and sequencing. Use Αβί prismtM

The 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 performed. The sequenced results were entered into the nucleotide sequence of the NCBI website for search alignment to confirm that the sequence fragment of this strain was a bacterium of the bacterium A. bacillus. &lt;Strain Character Test&gt; (I) Pathogenic Fungi Inhibition Test A single colony was selected from the LA medium plate, inoculated into 3 mi of LB, and cultured at 28 ° C for 24 hours under constant temperature and then subjected to inhibition test. Use a hole cutter with a hole diameter of 0.4 cm to cut the tip of the cultured fungal mycelium. Place the cut hyphae in the center of the PDA plate, and attach a radius of 两端. 4 cm to both ends of the hyphae. After the cm ingot, the cultured bacteria solution was adjusted by spectrometer (spectrophotometer ultrospec 2000, Pharmacia biotech, USA) to adjust the absorption value (~illusion, adjust the concentration of the bacteria solution to ι〇8 colony-forming units (Cfu/mi) Take 30 to the paper key, and then incubated at room temperature for five days to observe the competition for the pathogen. Please refer to the fourth figure, which is the result of the experiment of inhibiting the activity of phytopathogenic fungi by using BF-1 strain. Among them, the tested pathogenic fungi include: (A) guava blight fungus you 5·/ 幻娜 as/J// Sawada et Kurosawa, (B) K. sylvestris L. sinensis and// milk/7 .. (c) Flax leaf blight® 汾&gt;&lt;9^9/7'5· o/yzae (Breda de Haan) 57 coffee (D) lotus 201234971 haze fruit disease Pestalotiopsis euginae, gangue Su 1 disease Pestalotiopsis psidii (Pat) Mordue Pestalotia psidii, every disease 鲁Botyodiplodia theobromaeht. (^a^ Ays^'iS^^^^J^^i^BotryodiplocUatiieobroinae Pat. (Mango), (H) eoeos/^riWifes (Penz. &amp; Sace.), (I) Colletotrichum gloeosporioides Yenzig, {], Lianwu H disease Colletotrichum gloeosporioides i?enz·, hcc., (X) HBM Lu Colletotrichum gloeosporioides, (X) H H 旅 亀 Fusarium oxysporum f· sp. cepae, 〇〇令奏隹 Fusarium sp . Phytophthora palmivora (Butler) Butler., (〇) Brassica oleracea from (BERK) Sacc. Among them, the left (A) ~ (〇) left culture dish is the control group, the right side culture The dish is an experimental group, and it can be observed that the BF-丨 strain has an effect of inhibiting the activity of the pathogenic fungus. (2) Pathogenic bacteria inhibition test A single colony is selected from the LA and KB medium plates, and inoculated into 3 ml LB and In the KB liquid culture solution, the inhibition test was carried out after being placed in a 28 t: constant temperature shaking culture for 24 hours. Adjust the absorption value (")' of the pathogen bacterial solution and the BF-1 bacterial solution spectrometer (spectrophotometer ultrospec 2000, pharmacia biotech, USA) for 24 hours to adjust the bacterial concentration to 1〇8 colony-forming units (cfu/ After the filter disc diffusion method (paper disc diffusi0n meth〇d), the pathogenic bacteria inhibition activity test was carried out, and the pathogen of 100 #1 was applied to the LA and KB plate, and the radius was 201234971 0. 4 cm. Paper key 'retake 50 # 1 bacterial liquid and add it to the paper, then put it into the 28 t incubator for 24 hours to observe the size of the inhibition circle. The pathogenic bacteria used in this study are melon bacteria. Fruit spot disease. Also avenae sxibsp. citmlli, 祜栲 ulcer disease 蛰Xanth〇m〇nas axonopodis pv. citri, tomato fine spot disease 蛰Xanth〇m〇ms (10) ρβίτπρν.yang sic you r/a (Doidge) Dye , bacterial soft rot disease PectoBActerium carotovorum subs, carotovorum, acne 'sister disease Ralstonia solanacearum, 螂绿圣故病病

Pectobacteriwn chrysanthemU slip, E. chusantfiemO, U earned blue brown spot disease did not follow subsp. New &quot; Please refer to the fifth figure for the use of BF-1 strain to inhibit the growth of phytopathogenic bacteria. The phytopathogenic bacteria tested are: (Q) tomato bacterial spots, (R) citrus canker, (S Cucumber bacterial fruit spot; (q) ~ (s) left culture fox is the control group, and the right culture dish is the experimental group, so it can be observed that the strain _1_1 does have an effect of inhibiting the activity of the pathogenic bacteria. The BF-1 strain of the invention can be used together with a chemical pesticide, and the chemical pesticide can be: killing the scorpion, killing the smear, the insecticide, the herbicide and the different dilution factors. The murder includes: ke jiejing (known alkane nitrogen), poker pull (melamine), chlordane (amine phthalic acid), leucoside (benzamide), gram rot (benzoquinone) Aniline), Saimin (renewed amines), gibberellin (antibiotics), atornamine (spheroids), tetra-isophthalonitrile (aromatic), free benzo (benzo ° rice bran), methyl multi-protective (benzene 201234971, galenamine fine salt), Yi Pu Xiao stupid A dimethyl - ° sitting class), copper sulphate (copper), fenning (two Chlorine rotten; -=, Degen (dithiocarbamate), lest ^ = class), poker her (° meter saliva), Fuchsing (^, gibberamine (screaming), fen Remo (physical), Kunotin (), nitrile (_), eduli (face), bin clone (urinary h _ second 赛 赛 (three ° benzopyrene). —]. The chemistry ^ To / comprises: copper hydroxide (copper-based), chain tetracycline (antibiotics),, antibiotics), Ousuo Lin acid (quinolin-type fungicide). [See Appendix II]. = academic insecticides include: sent out of the net (money (four)), edamine (organic nitrogen) heterocyclics, Datnam (organic nitrogen and heterocyclics), Na Naid (ammonia methanum) plus insurance Fu (ammonia methanate), Taosson (bite organic thioguanate), new acaricidal (biphenyl), Pedan (organic gas), Dinningbu sitting, Bifening (wow class ), cotinine (new nicotine), fentanyl (organic turn), acesulfame (organophosphorus), kefanpai (indole), dassam (straight bond amine organothiosteanate) ), 密密〉,丁(Antibiotics). [See Appendix III]. The chemical herbicides include ··············································· Phosphorus isopropylamine salt (melamine), valprofen (biphenyl test), sedative (dinitroaniline), barragel (quaternary amine). [Ref. Appendix IV]. The different dilution factors The spreading agent includes: Trit〇n χι〇〇. &lt;Example 1&gt; This embodiment is mainly for the survival rate of the test strain in chemical pesticides. Single colonies were selected from the LA medium plate, inoculated into 3 ml LB, and placed in a 28 t ten-temperature incubator for 24 hours. The absorbance was measured by Spectra 21 201234971 (spectrophotometer ultrospec2000, pharmacia biotech, USA). (Heart 2.), adjust the concentration of the bacteria solution to colony-f〇rmingunits (cfu/(4), take 1〇〇#丨菌液, 1〇〇10X stock chemical agent and _ ^丨_2〇 after mixing Shake evenly and place on the table for 1 minute, while the control group is 1〇〇#丨菌液 mixed with 900 # 1 ddmo, after 10 minutes, take 1〇〇# 1 for serial dilution, and then for dilution 10 To 10 5, each 1Q0 was applied to the LA plate, and then placed in a 28 ° C incubator for 24 hours. After 24 hours, the number of colonies was counted, and the survival rate was obtained. Among them, the fungicide, the bactericide, Insecticides, herbicides and spreading agents with different dilution factors are tested in the above manner, the difference is only the difference in the concentration of the drug. Please refer to the sixth figure for the use of the BJM strain of the present invention and the above-mentioned W species of fungicides. Sputum strain when used together Survival rate chart. It is shown in the graph of the sixth figure that the BF_i strain of the present invention is used in combination with the above-mentioned 27 kinds of fungal agents, and the survival rate of the BF-1 strain is at least 7〇% or more. The β ΙΜ strain of the present invention is combined with the above 12 types of insecticides to make (4) the test chart of the strain. The BF] strain of the present invention and the above 12 types of insecticides are shown in the chart of the seventh figure. After the combination, the survival of the BF-1 g strain was less than that of Na. The eighth figure is the BF of the present invention, and the survival rate of the strain is used when the strain is used in combination with the above six herbicides. The graph of Fig. 8 shows that the BF-lg strain of the present invention has a survival rate of at least 7 (%) or more after being used in combination with the above-mentioned six herbicides. 22 201234971 No, she issued the above three types of fine _ collocation system, su] _ survival rate chart. The heart of the present invention is shown in the ninth: chart. After use in combination with the above three types of bactericides, the survival rate of the BIM strain is at least fine % or more. Further, the BIM strain of the present invention can be further used in combination with S. faecalis. 0 &lt;Example 2&gt; • The purpose of the field test station is to investigate whether the .1 strain has the ability to prevent and treat. First, sell the eggplant (Solanum lycopersi, CUJJ) to a single colony suitable for the age of 'u-culture medium, inoculate it in 3 ml LB, and place it in a 28 ° constant temperature culture for 24 hours. In a LB of 200 m!, incubate at 28 C for 24 hours at a constant temperature, and adjust the absorption value (Aw.) with a spectrometer (spectrophotometer ultrospec 2000, pharmacia biotech, USA) to adjust the concentration of the bacteria to 1 colon 8 colony-forming. Units (cfu/ml), 50 ml of the bacterial solution was uniformly poured into the root soil, and the Check (control group) was watered for a total of five groups. The bacterial solution 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 the seven angel root ring to fully contact the BF-1 bacteria. Paistoni'a soianacearum single colony was selected from IA medium and plate, inoculated into 3 ml LB, and cultured at 28 °C for 24 hours under constant temperature. It was placed in 200 ml of LB and shaken at 28 °C. After culturing for 24 hours, the absorption value (A62 〇 was adjusted by spectrophotometer ultrospec 2000, Pharmacia biotech, USA), and the concentration of the bacterial liquid 23 201234971 was reduced to 1 colon 8 colony-forming units (cfu/ml), and 5 〇 ml was taken. The evenly poured portion of the bacterial liquid was observed in the root soil for three weeks. 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 there was a white cloud-like liquid. From the incision, the red plant of the female group will be removed from the base of the stem: the stem will be cut off, the stem will be cleaned, and the stem will be cleaned with the alcohol disinfectant of (10). The sterilized stem is placed on a non-bacteria table to be dried. After drying, the surface of the stem of the na-small section is removed, and after being shaken in ddM, the suspension is taken with a heterocyclic ring and streaked into κβ. Culture #基平板, The medium plate was placed at 28. (10) Warm culture was carried out for 2 days, and single-colon colonies were picked for four-zone scribing, repeated three times, and then transferred to a fairy medium plate to pick a single colony and cultured in 3 ml of Κβ lyric medium. Perform genomic DMA extraction, and then use the universal primer to perform 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. 'De-sequence and sequence the purified product. After the sequenced results, the nucleotide blast of the website is searched and compared to confirm the sequence and fragment of the strain and compare with the original 16s sequence. 'Yes = the original strain. Please refer to Dilai, where the control group is #

The growth condition of the disease-causing bacteria of B. sinensis was treated as (4) the growth of the pathogen of the inoculum of the inoculum after inoculation of the fermented product of the young plant, and the BF-1 of the present invention was compared by the control group and the _ group. The strain is solid and has the ability to control plant diseases. &lt;Example 3&gt; 24 201234971 The BF-1 strain of the present invention has a potent ability to produce a proteolytic enzyme. 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. Place 'L hair miscellaneous on lem (10) on the _ filter paper, and then place the paper on a skim milk agar (SMA) plate, which contains 1.5 xia, h 5% nutrition Culture medium (nutri shirt broth, NB) and 1.5% agar (agar). Place the plate at 3 inches. 〇 culture 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 (_); (2) η! (deleted) (3) 12. 21 (mm), (4) 12. 28 (mm); average distance: 12. 4 (mm ), where 'the distance from the silk shows its decomposition effect is better. This protein knife enzyme can be added to the secret feed, food processing added, agricultural aquaculture sewage treatment. Xuan - map, fine display of this (four) bm strain The ability to decompose proteins. &lt;Example 4&gt;

The ability of the BF-1 (protease) of the present invention. The purpose of preparing BF-1 fermentation broth, the steps. Will 5 a L be slit? 25 201234971 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 decompose lipids is shown. &lt;Example 5&gt; The BF-1 strain of the present invention has the ability to strongly produce cellulolytic enzymes. The cellulose 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. Place the 5G&quot;L fermentation broth on the rounded paper on the sterilized (10) and place the filter paper on a Mandel-Reese, MR) vegetable plate containing 1% carboxy me1:hyl cellui〇se, 绫Methylcellulose, 〇. 1°/◦ protein gland, 0.03% urea, 0.15% ammonium sulphate, etc. and 15% agar (agar) 'PH6. 0. The plate was placed in 3 (rc culture for 2 days, 3. 5 mi ° Congo red cover was immersed in the medium for 30 minutes, and then washed with sodium sulphate as a red part. This part of the cellulose was decomposed by the enzyme. Observe the solution around the colony. (1) 4. 55 coffee; (2) 4. 80 delete; (3) 4. 68 face; w) 4 account for coffee; average distance of 4. 6 faces. Among them, the larger the dissolution circle, the better the decomposition effect. Please refer to the thirteenth figure for showing the ability of the BF] strain of the present invention to decompose cellulose. &lt;Example 6&gt; The BF-1 strain of the present invention has a strong ability to produce (iv) a protease protease. The starch decomposition test method is as follows: Preparation of BF-1 fermentation broth The preparation steps are as follows: The 5G 4 fermentation broth was placed on a lem (10) sterilized circular paper. The paper was placed on an aquaculture plate, which contained 1% yeast, 1% starch and 1.5% amaranth. After the plate was placed in 3 叱 for 2 days, 3.5 ml of Wei (0.3% 峨 and 3% potassium hydride) was covered with the immersion: the nutrient 'completed in 5 minutes' and its surroundings were not dyed. Black part of the temple powder is 26 201234971 by the enzyme knife solution. Observe the dissolution circle around the colony: (1) ι 〇ι legs; (2) 〇 99 mm ’(3) 1.03 mm; (4) l.Oi mm; average distance 1 〇 _. Among them, the larger the melting circle, the better the decomposition effect. Referring to Figure 14, the ability of the BF-1 strain of the present invention to break down starch is shown. The embodiment or the drawings are not intended to limit the scope of the invention, and any suitable variations or modifications may be made without departing from the scope of the invention. &amp; described above, 'the embodiment of the present invention can achieve the expected use · f effect' and its disclosed structure, not only has not been seen in the same kind of mouth: nor has it been disclosed before the application, Cheng has fully complied with the patent law The provisions and requirements, 提出 legally filed an application for an invention patent, pleaded for review, and granted a patent, it is really sensible. [Simplified description of the figure] First figure: the result of the increase of the 16S rRNA sequence of the BIM strain of the present invention. Figure 2: Results of the increase in the Tet (L) gene. Figure 3: Results of the increase in the IturinA gene. • Figure 4: The plant that uses the fermentation product of the strain-1 to inhibit the fungal disease of the crop—# Dendrobium sinensis (A), K. sylvestris L. (8), F. sphaeroides (〇, Pestalotia (9), Pestalotia (Panshiquan) (E) Papaya rot (7), citrus rot (6), fruit charcoal Symptoms (8), papaya anthrax (1), lotus aphid (1), jujube anthrax (1), onion purple root (L), verticillium (M), papaya disease (N), cabbage black spot (〇) fifth picture: the old fermentation The phytopathogenic bacteria whose products have inhibitory activity against crops are Susu bacterial spots (9), citrus occidentalis (8), 27 201234971 cucurbit bacterial fruit spots (s). Figure 6: Utilization of BF-1 bacteria Cell viability with the following 27 types of fungicides; 1. gram heat (alkane nitrogen), 2. poker pull (melamine), 3. halal (amine phthalic acid), 4 . 白克列 (benzoamide), 5. gram of rot (stupid aniline), 6 赛赛灭 (sulfonamides), 7. carnitrol (antibiotics), 8. Tomin (spheroids), 9. tetrachloroisomeric nitrile (aromatic), 10. free of bismuth (benzimidazoles), 11. thiodoprene (stupid imidazole precursors) 12. Hundreds of sensitizers (amino amides), 13. espresso (imidazoles), 14 s. Kelly (triazoles), 15 · ternary copper sulphate (steel), 16 · chlorhexidine Phenyl phenyl diimine), 17. Deandi (dithiocarbamic acid brinic acid), 丨 8. Free side (polymerized dithiofluoride f acid salt), poker extinguished (° rice pin) 2〇·福赛得(有_类), 21. 焦吉胺(吼咬), 22. 芬里莫 (money), 23. 快诺芬 (♦琳), 24. nitrile thio (酉酉), 25. Yideli (sigh), Shenbin clone (urea), 27. Sansai saliva (three-salbenone-sitting). The seventh picture is the use of BF] The following bacteria used in combination with insecticides; U/xiao (three _,), 2. fiber (organic gas and miscellaneous class), 3. Datnan t ' (organic nitrogen and heterocyclic) 4, Na Naide (ammonia machine support (虱 machine formate), 6· Taosson (pyridine organic thiophosphate) ), 7, new killing mr, worm (combined), &amp; Pedan (organochlorine), 9. dinning (azole), 10, . Bifen > (azole), 11. Knitting (new nicotine), 12. killing pine (with organic phosphorus), 13. love pine (organophosphorus), 28 201234971, (° azoles), 15. big succulent (straight Chain amine organic thiophosphate di-negative; Ding (antibiotics) ^ is the bacterial survival rate of the use of 1 strain of bacteria combined with the following 6 types of herbicides.]_acid system,, mono- and tetra-sodium Salt (phenoxy acid system), 2. Gutocao (secondary phosphorus 3. Jiahe isodecylamine salt (__), 4. Fulufen (biphenyl listens to 5. Shidezhen (di-diphenylene)), 6 Barra 4 (quaternary amines)

The body I system is the use of BIM bacteria in combination with the following three types of bactericides to use the bacteria μ rate ' 1. Copper hydroxide (copper), 2. Chain tetracycline (antibiotics), ''numbers (k Biotin), 4. Oxalic acid (material fungicide) The tenth figure is the control group for the young plant inoculated with the pathogen of the blight of the buckwheat disease, and the 'Bai vaccination BF- 1 The growth of the pathogen of tomato bacterial wilt after inoculation of the fermentation product. Figure 11: This is the test of the invention. (4) The test of the protein decomposition of the strain is isolated. == Fig.: It is the test of the invention. Cultivating a solitary diagram. This is a test dish for the decomposition of cellulose. The fourteenth figure is the test of the invention _ decomposition of starch [refer to the attached table]. Table 1, exemption / 7 / your spp. Identification of the primer used in the polymerase chain reaction table 2, primers for the amplification of the Iturin 々 gene sequence used in the polymerase chain reaction [refer to the appendix] Appendix I, list of fungicide species 29 201234971 Appendix II, List of Bactericide Types Appendix III, List of Insecticide Types Appendix IV, List of Herbicide Types [Key Component Symbol Description]

30

Claims (1)

201234971 VII. Scope of application for patent: The liquid buds of the larvae of the larvae are deposited in the food industry of the Judiciary [registered number: BCRC91_], which is named BF-1 with 3 ships, Tet (L), ItuA, ItuB, ItuC, Ituk· • The species used in the liquefaction of Bacillus licheniformis strains as described in claim 1 is used to decompose at least one of the following components, the group consisting of starch and starch , protein, cellulose and lipids. \ Kind of use as described in the scope of claim 1 of the liquefaction of Bacillus subtilis strains are used for feed addition or agricultural aquaculture and sewage treatment. For example, the use of the strain of Bacillus licheniformis described in Item 1 of the patent application scope is used to inhibit the activity of the plant pathogen, and the towel of the plant pathogenic fungus is included in the following species: Blight, B. sinensis, Rhizoctonia solani, Rhizoctonia solani, guava sore, Papaya, Phytophthora, Phytophthora, Prunus, Papaya, Pistachio Bacteria, jujube anthracnose g, foreign purple root disease g, verticillium g, papaya disease, Ganhuang, black spot disease. 5. The use of the liquid granule spore rod as described in the scope of the patent application category is for inhibiting the activity of the plant pathogen, wherein the plant pathogenic bacteria includes one of the following: Panyou bacteria Sex spots, citrus-like bacterial fruit spots. The species of the liquefied Bacillus licheniformis strain described in the first paragraph of the patent scope is used in combination with a fungicide to form a plant protection composition, wherein the sterilization includes, among other things, the following: Hot (in the case of nitrogen), poker (mineral amines), chlordane (amine phthalic acid), white gram (benzamide), rotten (stupid aniline), racer (category) , caricillin (anti-31 201234971 biotin), atropin (spheroids), four-gas isomeric nitrile (aromatic free Lai (Bin Lin °), methyl multi-safe (benzene) And the saliva precursors), Baikemin (amines), Yiputong (Miso), Keli (three saliva), copper bismuth (copper), chlorhexidine Dimethyl carbonitrile imine (dithiomethane fine), free _ (polymerized diweiramine, salt), poker pull (sit), forsythia (organophosphorus), support吉amine (吼钱), 芬瑞莫定类), 快诺芬 (喧琳), 猜ς :":), 依得利(°塞销), 宾克隆(尿细,三赛4 (two ° sit benzene and sigh wow class). 7 two kinds of applications such as the application of the patent system 丨 丨 之 之 液 液 液 液 液 液 液 液 液 液 液 液 液 液 液 液 液 液 液 液 液 丨 丨 丨 丨 丨 丨 丨 丨 丨 丨 丨 丨Towels, the killing includes the following ones: the genus (three nitrogen wells), idadamine (organic nitrogen and heterocyclics), dardon (organic nitrogen and heterocyclics), and nadine (ammonia) = acid salt), plus Baofu (ammonia methanate), Taosson (Calding organic thioformation = class) = acaricidal (biphenyl), Pedan (organic gas), Dihenning (jun class II ) 'Knitidine (new nicotine), fumicide (J, acesulfame (organophosphorus), kefanpai (sales), big pine (linear amine organothiophosphate), Mi Mijiang (antibiotics). 8 kinds of strains of Bacillus licheniformis as described in the scope of the patent scope, 'the system and the herbicides are used to protect the composition of the ribs, the towel, the weeding = To include the following: - two, four kinds of sodium salt (phenoxy acid, solid kill, (- human phosphate), Jia Phosin isopropylamine salt (melamine), complex Lufen (biphenyl) Shi Derived from bismuth (di-diphenylene) and paraben (quaternary amine). • The use of the strain of Bacillus licheniformis as described in the scope of the patent application scope is used in combination with killing and bactericidal agents. The plant protection composition, wherein the killing agent includes one of the following: copper hydroxide (10), key four-ring emblem 32 201234971 (antibiotics), streptomycin (antibiotics), Linic acid (Glutinous fungicide) 10. The use of the strain of Bacillus aeruginosa as described in claim 1 of the patent application is used in combination with S. cerevisiae to form a biopesticide composition. 33