67 results about "Ralstonia" patented technology

The invention relates to microorganism plant vaccine capable of controlling continuous tomato bacterial wilt disease, and belongs to the agricultural intensive production technology. According to the microorganism plant vaccine capable of controlling the tomato bacterial wilt disease, a ralstonia solanacearum sibling bacterium pearson ralstonia eutropha (Ralstoniapickettii) NJQL-A6 is isolated from the rhizosphere of a healthy tomato, and the ralstonia eutropha has a weaking inhibited impact on a bacterial wilt pathogenic bacteria ralstonia eutropha (Ralstonia solanacearum) panel. However, the ralstonia eutropha possesses powerful colonization capability on the tomato rhizosphere and inside a plant, and can inhibit the infection of the ralstonia eutropha to a tomato plant. Experiments show that after the vaccine is irrigated to be applied to soil, the vaccine can breed rapidly so that an advantage group is formed in the soil, and the biocontrol efficiency can be reached up to more than 70% in the soil where the tomato bacterial wilt disease is severe. By inoculating the biocontrol bateria on the tomato plant with the root being infected by the bacterial wilt on the overground part, the further development of the disease can be effectively controlled.

The invention discloses a biocontrol agent for preventing and curing bacterial wilt and club roots of a plant as well as preparation method and application thereof. The preparation method of the biocontrol agent comprises the following steps: inoculating activated bacillus amyloliquefaciens ZJUB2008 into a culture solution, and fermenting and culturing at 25-45 degree C for 6-120 hours, wherein the culture solution comprises the following components by taking the volume of 1L as the basis: 3-15g of peptone, 0-3g of beef extract, 0-5g of glucose, 0-5g of yeast powder, 0-8g of NaCl, and the balance of water; and the pH value of the culture solution is 5.5-9.0. According to the invention, the bacillus amyloliquefaciens ZJUB2008 is used to prepare the biocontrol agent on / under the appropriate fermentation medium and culture conditions by means of microbial fermentation; in addition, the prepared biocontrol agent has a better prevention effect to different chemotyped ralstonia and plasmodiophora, can remarkably lowering the occurrence rate and severity of the bacterial wilt and the club roots, and is suitable for large-scale production.

The invention discloses method for preventing and treating Pseudomonas solanacearum, comprising the following steps: culture and purification of ralstonia dolaanacearum: utilizing sterilized water to soak tobacco leaf vein with Pseudomonas solanacearum, screening on a culture medium, selecting single bacterial colonies with good fluidity, continuing to culture the single bacterial colonies with good fluidity, and storing purified ralstonia dolaanacearum; screening of bacteriophage: acquiring tobacca planting soil subjected to Pseudomonas solanacearum, sieving, removing impurities, acquiring fine particles, soaking the fine particles with sterilized water, centrifuging, acquiring a supernatant liquid, filtering, acquiring a filtered solution, culturing the filtered solution and a ralstonia dolaanacearum solution together, and storing screened bacteriophage; and enrichment culture and collection of the bacteriophage: adding the culture medium and sterilized water containing ralstonia dolaanacearum into a triangular flask, carrying out shake culture, then adding sterilized water containing bacteriophage, continuing to culture, centrifuging a culture solution, acquiring a supernatant liquid, and filtering to obtain a filtered solution for later use; soaking a toothpick with sterilized water, flushing, sterilizing, drying, cooling to room temperature, soaking in the filtered solution, taking out, sub-packaging, and storing for later use; and after tobacco is transplanted, inserting the toothpick to the stem base of a tobacco plant so as to prevent and treat the Pseudomonas solanacearum.

The invention relates to two ralstonia-solanacearum-resistant endophytic bacillus velezensis strains and application thereof. By separation, screening and purification in roots of healthy tobacco plants in a ralstonia solanacearum occurrence area of the Enshi Prefecture, two biocontrol bacillus endophyticus strains R-3 and R-9 are obtained, have evident resistance effects on ralstonia solanacearumand are both capable of generating high-temperature-resistant ralstonia-solanacearum-resistant substances. By gyrB gene sequence identification and phylogenetic tree construction, results show that the R-3 and R-9 are both bacillus velezensis; by potted plant root irrigation experimental verification, the bacilli R-3 and R-9 have great colonization performance in tobacco roots and can enter stemsthrough root transmission; by field experimental verification, bacillus velezensis R-3 and R-9 are effective in ralstonia solanacearum prevention and treatment, remarkably superior to chemical agentkasugamycin and free of pollution. The biocontrol endophyte research field is further enriched by researching on the bacilli R-3 and R-9, and a guiding significance to ralstonia solanacearum prevention and treatment is achieved.

The invention relates to bacillus subtilis. The preservation number of the strain is CGMCC NO: 9269. The strain is obtained by deep fermentation, ceramic membrane concentration and spray-drying. A shaking culture liquid of the strain has an obvious effect of inhibiting the target germ Xanthomonas oryzae pv. oryzae, the target germ ralstonia solannacearum, the target germ Pseudomonas syringae pv. tabaci, the target germ Xanthomonas axonopodis pv. citri and the target germ cercosporella albomaculans. The bacillus subtilis provided by the invention has advantages of fast growth and propagation, short fermentation period, high filling spore number, good control efficiency and the like. Meanwhile, a preparation method of the strain has simple steps, is convenient to operate, comprises steps of deep fermentation, ceramic membrane concentration and spray-drying to obtain the bacillus subtilis, has high preparation efficiency and has good effects. The strain can control various bacterial diseases and now has been registered on cercosporella albomaculans, Xanthomonas axonopodis pv. citri, Pseudomonas syringae pv. tabaci, ralstonia solannacearum and Xanthomonas oryzae pv. oryzae.

The invention relates to the detection of ralstonia solancearum in tobacco, in particular to a time resolution fluorescent quantitative test strip for detecting ralstonia solancearum in tobacco leavesas well as a preparation method and application thereof. The test strip successively comprises a sample absorption mat, a conjugate release mat, a nitrocellulose membrane, a water absorption mat anda bottom plate, and is characterized in that the conjugate release mat is coated with a time resolution fluorescent microsphere marked detection antibody; and the nitrocellulose membrane is coated with a detection line for capturing the antibody and a quality control line of goat-anti-mice secondary antibody. The rapid immunity analysis for the ralstonia solancearum in the tobacco leaves can be realized by adopting the time resolution fluorescent microsphere marked detection antibody and the immunochromatography assay. The invention also provides a method for detecting the content of ralstoniasolancearum in the tobacco leaves by using the test strip. The time resolution fluorescent quantitative test strip has the advantages of high sensitivity, accuracy in quantification, rapid in detection, convenience in operation, economical performance and practicability, and capability of performing rapid and on-site detection on a great amount of tobacco leaf samples.

The invention provides Trichoderma asperellum TD3104 and application thereof in preparation of microbial agent for inhibiting plant pathogenic bacteria. The preservation number of the Trichoderma asperellum TD3104 is CGMCC 13161. The invention further provides application of the Trichoderma asperellum in the controlling of the plant pathogenic bacteria of tobacco and apples. The antagonistic Trichoderma asperellum can effectively inhibit the growth of Phytophthora parasitica var. nicotianae, ralstonia solannacearum, rhizoctonia solani, tobacco root rot pathogenic fusarium, alternaria alternata and the pathogenic bacteria of apple rot and apple ring rot, can effectively control related diseases, can reduce the use amount of chemical pesticides and accumulation of the chemical pesticides in soil, is beneficial to the increasing of quality and yield of the economic crops of China and is evident in economic and social benefits.

The invention relates to the field of microorganism application and environment protection and in particular relates to a phosphorus-solubilizing and cadmium-reducing microorganism bacterium agent anda preparation method thereof. The invention provides the microorganism bacterium agent with phosphorus-solubilizing and cadmium-reducing effects and the preparation method thereof; the microorganismbacterium agent is prepared from the following components: 15 to 30 parts of a compound bacterium agent and 70 to 85 parts of a carrier, wherein the compound bacterium agent contains 0.25 to 1.5 billion cfu / g of bacillus methylotrophicus, 0.10 to 0.50 billion cfu / g of ralstonia eutropha and 0.05 to 0.40 billion cfu / g of paecilomyces lilacinus; the carrier is powder obtained by drying and sieving turf and diatomite, wherein the turf has the advantages of improving water retention, ventilation and fertilizer retention capabilities of soil; the diatomite has the advantages of strong adsorption performance, neutral pH (Potential of Hydrogen) and no toxin, good mixing uniformity and the like. The phosphorus-solubilizing and cadmium-reducing microorganism bacterium agent provided by the invention is powdery; when being applied in a land preparation process, the phosphorus-solubilizing and cadmium-reducing microorganism bacterium agent can be used for effectively improving the fertility of the soil and increasing the content of effective phosphorus capable of being absorbed by crops in the soil; meanwhile, the mobility of cadmium ions is reduced through the metabolic activity of microorganisms and the absorption of the crops on the cadmium ions is reduced.

The invention relates to the agricultural application of Echinopsis diffusa. Both the ethanol extract and water extract of the whole herb of G. radix had good antagonistic activity against the pathogen of R. solanacearum. The extract is a natural plant source component, which can replace chemical pesticides, reduce environmental pollution and residues of chemical pesticides, and is an ideal biological fungicide in agriculture. The use of the whole plant as green manure has good control effect on the occurrence of tobacco bacterial wilt and can promote the growth of tobacco plants, which has good economic, social and ecological benefits.

The invention discloses a loop-mediated isothermal amplification detection primer for ralstonia solanacearu and a quick detection method for the ralstonia solanacearu, which can be used for detecting the specificity of the ralstonia solanacearu. An LAMP (loop-mediated isothermal amplification) detection primer for the ralstonia solanacearu is designed and comprises outer side primers F3:5'-TCTTGATAAGGCGGGGGT-3' and B3:5'-AAACACCGATCTCTCGATGC-3' and inner side primers FIP:5'-CCAGTTCACGGCAAGATCGCTTTCAAGTCCTACCAGACCCA-3' and BIP:5'-ACCTGCTTTGCAAGCAGGGG-GCGTTTGGCTACCACAAGG-3'. After the ralstonia solanacearu is subjected to isothermal amplification, green fluorescence can be observed or a trapezoid strip of the LAMP characteristic emerges through color development of a developing agent SYBRgreenI or electrophoresis detection of agarose gel. The LAMP primer and the detection method which are disclosed by the invention can be used for quickly, sensitively and accurately detecting the ralstonia solanacearu in plants infected with the ralstonia solanacearu during production practice or detecting the ralstonia solanacearu in the plants in the morbidity latent period and can be also used for performing early diagnosis on field diseases, and monitoring and identifying germs; a reliable technical and theoretical basis can be provided for prevention and treatment of diseases caused by the ralstonia solanacearu.

Provided are a recombinant Ralstonia eutropha capable of producing polylactate or a hydroxyalkanoate-lactate copolymer, and a method of preparing polylactate or a hydroxyalkanoate-lactate copolymer using the same. The recombinant Ralstonia eutropha, which is prepared by introducing a gene of an enzyme converting lactate into lactyl-CoA and a gene of a polyhydroxyalkanoate (PHA) synthase using lactyl-CoA as a substrate thereto, may be cultured, thereby efficiently preparing a lactate polymer and a lactate copolymer.

The invention provides a preparation method of organic fertilizer special for corn. The preparation method includes following steps: 1), exposing and drying chicken manure under the sun, and well mixing with green tea powder, artemisia apiacea residue, garlic seedling and oyster shell powder to obtain a material A; 2), using a microbial agent E to ferment seafood leftover and selenium mineral powder for 30-35d, ultrasonically smashing, and adding zeolite powder to obtain a material C; 3), using to serratia rubidaea, ralstonia entropha, paenibacillus macquariensis, sphingomonas, azospirillumlipoferum, alcaligenes faecalis, bacillus thuringiensis and photosynthetic bacterium to ferment decomposed corn straw, the material A and the material C to obtain a material D; 4), putting the material Dinto a stirrer to be mixed with urea, calcium superphosphate, potassium chloride, borax and soil conditioner, granulating, drying, and coating to obtain the organic fertilizer. The organic fertilizerprepared by the method has the advantages of stable dissolution process and good prevention effect on various common corn diseases.

The invention relates to a microbial agent, in particular to a biochar-based microbial agent as well as a preparation method and application thereof. The biochar-based microbial agent comprises biochar and (Ralstonia sp.) Ralstonia bcul-1 adsorbed on the biochar. The preparation method comprises the following steps: (1), preparing a biochar-(Ralstonia sp.) Ralstonia bcul-1 mixed solution containing biochar, sodium alginate and a (Ralstonia sp.) Ralstonia bcul-1 seed solution, and performing standing overnight at a room temperature; (2), slowly dropwise adding the mixed solution into a calciumchloride solution with the mass concentration of 1%, continuously performing stirring, performing crosslinking to obtain a product, and cleaning the product with deionized water to obtain a microbialagent. The microbial agent is used for repairing heavy metal polluted water. According to the invention, zinc pollution is repaired by using physical adsorption and bioanalysis; the preparation methodhas advantages of simple operation, high remediation efficiency, no secondary pollution and the like; and the biochar-based microbial agent as well as a preparation method and application thereof have great application potential.

The invention discloses a method for producing mevalonic acid by using grease as a raw material, and a gene engineering bacterium constructed by the same. The method comprises the step of introducing foreign hydroxymethylglutaryl-CoA synthetase gene and hydroxymethylglutaryl-CoA reductase gene into ralstonia eutropha with phaC1 gene deleted. The bacterial strain produces mevalonic acid by fermenting the grease as a substrate, and has the advantages of relatively high conversion rate. Production cost is effectively reduced. The method provides a solid foundation for industrial application of biological synthesis of mevalonic acid.

The invention relates to a Ralstonia strain as well as an application, a preparation method and a control effect evaluation method thereof. The Ralstonia strain is Ralstonia sp.56D2, and the strain is preserved in the China Center for Type Culture Collection on November 12, 2019; the preservation address is Wuhan University, Bayi road No.299, Wuchang District, Wuhan City, Hubei Province; the accession number of the strain is CCTCC No: M 2019921. The invention provides Ralstonia sp. 56D2 capable of preventing and treating tobacco bacterial wilt; a preparation method of the Ralstonia sp. 56D2; and an application of the Ralstonia sp. 56D2 in tobacco bacterial wilt disease.

This invention relates to an application of fermented Aspergillus niger xj to prevention and control of Ralstonia solannacearum of tobacco. The antagonistic bacteria is Aspergillus niger xj which is separated by Institute of Fungus Resource, Guizhou University and now preserved in the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China) with a preservation number of CCTCCNO: M206021. The Ralstonia solannacearum of tobacco can be effectively prevented and controlled by fermented Aspergillus niger xj disclosed by the invention.

A method for synthesizing polyhydroxyalkanoate using a microorganism includes providing a Ralstonia eutropha strain; providing a fermentation tank and adding crude glycerol into the fermentation tank; adding a nitrogen source into the fermentation tank to adjust a carbon to nitrogen (C / N) ratio to 20:1 to 150:1; controlling temperature of the fermentation tank to a temperature ranging from 20 to 45° C.; adding 5 to 20% by volume of the Ralstonia eutropha strain into the fermentation tank using a mixing speed of from 100 to 500 rpm, an aeration quantity of 0.5-2 vvm and a concentration of dissolved oxygen of 30 to 80%; fermenting contents of the fermentation tank for 48 to 96 hours to allow the Ralstonia eutropha strain to proliferate and to allow polyhydroxyalkanoate to be synthesized from the crude glycerol within cells of the Ralstonia eutropha strain; and disrupting the cells of the Ralstonia eutropha strain and extracting polyhydroxyalkanoate.

The invention discloses an RPA (Recombinase polymerase amplification) primer, a probe, a kit and a detection method for detecting ralstonia solanaceraum and belongs to the technical field of biological safety. The RPA primer is designed based on Rip TALI-9 gene of the ralstonia solanaceraum, and a sequence of the RPA primer is shown as SEQ ID No:1 and 2; the PRA probe is designed according to an amplification area of the RPA primer, and a sequence size of the PRA probe is 45 bp to 54 bp; the 5' end is labeled by FAM, and the 3' end is modified by C3-Spacer; furthermore, THF modification is performed at distance to the 34 bp of the 5' end at the middle of the sequence of the RPA probe. The invention further provides the kit containing the RPA primer and the probe. The invention further provides the detection which is constructed based on the RPA primer and the probe. According to the detection method, DNA extracted from a sample is utilized as a template to perform RPA amplification reaction. According to the detection method disclosed by the invention, an RPA technology is applied to molecular detection of the ralstonia solanaceraum for the first time. The detection method has thecharacteristics of strong specificity, high flexibility, good practicability, lower requirement for instruments and equipment; a novel method is provided for diagnosing pseudomonas solanacearum, preventing and treating diseases and reducing pesticide use amount.

The invention relates to heterocyclic substituted 1, 3, 4-oxadiazole hydrazide compounds and a preparation method and application thereof. The compound has a structure shown as a general formula (I). The compound has a relatively good inhibition effect on pathogenic fungi and bacteria; and the compound has good inhibition effects onfusarium graminearum, potato late blight bacteria, blueberry root rot bacteria, pepper fusarium wilt, sclerotinia sclerotiorum, colletotrichum gloeosporioides, botryosphaeria dothidea, rhizoctonia solani, xanthomonas oryzae pv., ralstonia solannacearum, Xanthomonas citri, kiwi canker bacteria, Xanthomonas oryzae cucumber, Xanthomonas oryza konjak, Botryosphaeria dothidea, tomato canker pathogens, apple canker pathogens and the like.

The invention belongs to the technical field of microbial prevention and control and particularly relates to a biological fungicide, as well as a preparation method and application thereof. The biological fungicide comprises pseudomonas koreensis CLP-23 which has a biological preservation number of CGMMCC No.13203. The biological fungicide can inhibit ralstonia solannacearum bacteria, can improvethe prevention and control effect on the ralstonia solannacearum under an acid soil condition, has a biologically soil repairing effect, and can promote growth of solanaceae.

The invention relates to the field of pollution management and discloses a method for digesting bottom mud of black and odorous river channel through a microorganism preparation. The microorganism preparation is added into a black and odorous river channel; the microorganism preparation is prepared by mixing CQ1, CQ2 and CQ3 according to the volume ratio of (1 to 3) to (1 to 3) to 1; the CQ1 is amixed bacterium agent of bacillus subtilis, bacillus aureus, acinetobacter, psychrobacter, acinetobacter and pseudomonas strains; the CQ2 is a mixed bacterium agent of arcobacter, acinetobacter and shewanella oneidensis strains; and the CQ3 is a mixed bacterium agent of aeromonas, ralstonia eutropha and ralstonia eutropha strains. By adopting the method disclosed by the invention, the problems that original biological population structures and living conditions of rivers and lakes are damaged in a traditional dredging manner, a self-cleaning function is weakened so that negative influences onecological restoration are caused and secondary pollution is easy to cause, principal and secondary aspects cannot be treated at the same time, people are likely to be disturbed and the management price is high are solved.

The invention relates to a method for improving yield of PHB produced by Ralstonia eutropha by optical driving. The method comprises the following specific steps: 1) microbial culture: activating theRalstonia eutropha stored in a freeze-drying tube in a seed culture medium, and after the Ralstonia eutropha grows for 24h, inoculating the obtained bacterial solution into a basal culture medium to perform enlarging cultivation until the od value is 1; 2) passaging the Ralstonia eutropha subjected to enlarging cultivation to a basal culture medium II containing a photocatalyst and an electron donor; 3) collecting the bacterial solution in the basal culture medium II and extracting PHB. The method provided by the invention only needs to directly add an appropriate amount of preferred kind of photocatalyst and electron donor to a suitable microbial culture medium, and can induce the microorganism to generate more metabolites. The method has the advantages of simple operation, low cost, mildcondition, environmental friendliness and the like.

The invention belongs to the technical field of microbial remediation, and specifically relates to heavy metal resistant bacteria and an application thereof. The heavy metal resistant bacteria provided by the invention is Ralstonia Bcul-1, which is preserved in China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, West Beichen Road, Chaoyang District, Beijing on April 12, 2019, with an accession number being CGMCC NO. 17565. The heavy metal resistant bacteria provided by the invention are used for biological remediation of a heavy metal contaminated water body or heavy metal contaminated soil. According to the invention, microorganisms in the soil environment are used for passivating and adsorbing heavy metalpollutants to reduce toxicity of heavy metals in soil, so the advantages of low cost, high efficiency, no secondary pollution and the like are achieved.

The invention discloses a method for expanding reproduction of ralstonia solanacearium bacteriophage. An avirulent ralstonia solanacearium strain RSsw326-2 disclosed by the invention is preserved in CCTCC (China Center for Type Culture Collection) and has the preservation number of CCTCC NO. M 2018197. The avirulent ralstonia solanacearium disclosed by the invention is ralstonia solanacearium obtained by separating from naturally infected tobacco plants; by subculture, dissociant is obtained; by pathogenicity determination, the dissociant losing pathogenicity for tobacco seedlings is the ralstonia solanacearium avirulent strain; when the avirulent ralstonia solanacearium avirulent is utilized to reproduce the ralstonia solanacearium bacteriophage, the ralstonia solanacearium bacteriophagewith a high titer can be obtained. The method is expected to be used as a carrier for greatly expanding reproduction of the bacteriophage in the ralstonia solanacearium bacteriophage biological pesticide development so as to avoid secondary damage to prevention and control objects due to the toxicity of a host strain.

The invention discloses an application of a fermentation product of ralstonia eutropha in the field of animal feed. PHB (poly-Beta-hydroxybutyrate) is a biosynthetic thermoplastic high molecular material, and is used for the field of plastics processing as a completely biodegradable material before. The contents of protein, PHB, coenzyme Q10 in fermentation liquid or fermentation liquid dry powder of the ralstonia eutropha are measured firstly, a certain proportion of fermentation liquid or fermentation liquid dry powder is uniformly mixed with the animal feed as an additive after measurement and calculation, mixed feed with the PHB content of 0.5-5% is obtained, and then an animal is fed according to the conventional feed adding method to achieve the purpose of improving nutrition. Since the fermentation liquid or the fermentation liquid dry powder of the ralstonia eutropha is added to the common feed, substances such as the protein, PHB and the coenzyme Q10 contained in the fermentation liquid or the fermentation liquid dry powder can greatly promote animal growth and improve an immunologic function of the animal.

The invention relates to a compost decomposition additive. The additive comprises, by mass, 100 parts of fresh brewer's grains, 5-15 parts of bran, 2-4 parts of ammonium sulfate, 10-20 parts of urea,0.5-1 part of potassium dihydrogen phosphate, 0.1-0.5 part of NaCl and a strain seed solution with the total volume 10-20% of the mass of the fresh brewer's grains, wherein the strain seed solution isa mixture of seed solutions of aspergillus niger, penicillium, ralstonia and candida utilis in the volume ratio of (2-3):(2-3):(1-2):(1-2). According to the additive, the fresh brewer's grains serveas a culture medium for primary fermentation under the symbiotic action of various microorganisms, the number of bacteria is increased, and cellulase is also generated for further decomposing cellulose; meanwhile, the cellulose can be effectively degraded through the symbiotic action of various kinds of cellulase, and organic decomposition of feces of livestock and poultry can be better promoted.

The invention discloses a method for producing isoprene by adopting a cell fusion technology and a fusant constructed by the same. The method comprises the following steps: screening by utilizing a protoplast fusion technology, taking escherichia coli and ralstonia eutropha as parents and taking lipid as a unique carbon source and a resistance maker plasmid to obtain the reconstituted cell of high-efficiency transformed lipid; and fermenting by utilizing the fusant to produce the isoprene. The method disclosed by the invention can increase the velocity of lipid metabolism, enhance the yield of the isoprene and reduce the fermentation cost.

The invention provides a primer for detecting potato ralstonia solancearum and a PCR detection method adopted by the primer, relating to a primer and a PCR detection method adopted by the primer, and aiming at solving the problems that when the existing methods are adopted for detecting pathogenic bacteria of potato ralstonia solancearum, the detection range is narrow, the specificity is poor, the sensitivity is poor, and the detection efficiency is low. The primer has the sequence as follows: QK5-1: 5'-GCTAATACCGCATACGAC-3'; QK3-1: 5'-GAGCGTCAGTGTTATCCC-3'. The PCR detection method comprises the following steps: extracting the total genomic DNA, carrying out PCR detection, verifying the detection primer, and if an amplification band appears at the 591bp position, indicating that the sample is positive. According to the primer and the PCR detection method, the detection sensitivity can achieve 1pg of template DNA, the detection efficiency is high, the detected sample can be potato plant DNA and tuber DNA, even PCR detection can be carried out directly by taking bacterial liquid as a template.