CN103146600B - Antagonistic bacteria for prevention and treatment of tobacco bacterial wilt and application thereof - Google Patents

Abstract

The invention discloses antagonistic bacteria for prevention and treatment of tobacco bacterial wilt and application thereof. The antagonistic bacteria are Bacillus cereus QJ-1 and Aspergillus niger Ty-3. Specifically, the preservation unit of QJ-1 is China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, the preservation date is July 5, 2012, and the preservation number is: CCTCC, NO:M2012271; and the preservation unit of Ty-3 is China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, the preservation date is July 8, 2011, and the preservation number is CCTCC NO:M2011241. The antagonistic bacteria, i.e. the Bacillus cereus QJ-1 and Aspergillus niger Ty-3 provided in the invention can be combined into a microbial agent. The microbial agent and an organic material can be fermented into a microbial organic fertilizer, which can provide effective nutrition and functional bacteria, and realize effective prevention and treatment of tobacco bacterial wilt.

Description

Antagonistic bacteria for controlling tobacco bacterial wilt and its application

technical field

The invention relates to antagonistic bacteria for preventing and treating tobacco bacterial wilt, and also relates to the application of the antagonistic bacteria in preventing and treating tobacco soil-borne diseases, belonging to the field of biological control.

Background technique

Tobacco bacterial wilt is an important soil-borne disease of tobacco, which usually infects the roots of tobacco, causes diseases of crop roots and even the whole plant, and causes significant economic losses. Tobacco bacterial wilt is a systemic infectious disease caused by Ralstonia solanacearum. Once the tobacco plant is infected, the whole plant often dies. The damage is often devastating, so it often causes significant economic losses to tobacco production. . The pathogenic bacteria of tobacco bacterial wilt mainly survives the winter in the soil along with plant residues, and can also survive the winter in seeds or other hosts in the field. The soil with bacteria, diseased tissues and organic fertilizers containing bacteria are the main primary infection of the disease. The source of the disease, the spread of the disease mainly depends on irrigation water, rain, diseased seedlings, farm tools, diseased soil, and the activities of humans and animals.

At present, agricultural measures such as disease-resistant varieties and crop rotation are mainly used in production to prevent and control tobacco soil-borne diseases. However, because disease-resistant varieties require diversification of disease-resistant genes and are greatly affected by the environment, the control effect is not ideal. Crop rotation is also one of the effective methods to prevent and control tobacco bacterial wilt, but due to the large number of people in our country and the lack of land, normal crop rotation measures in production cannot be realized. Phytopathologists believe that plant pathogenesis is closely related to three factors: host, pathogenic bacteria and environment. When pathogenic bacteria and susceptible hosts meet in a suitable environment, plants will begin to develop disease. If any of these three factors are modified or eradicated , can reduce or control plant diseases.

A large number of studies have shown that there are not only microorganisms that cause tobacco diseases but also a variety of non-pathogenic beneficial microorganisms that improve the vitality of tobacco in the soil of tobacco fields. Therefore, the biological control and ecological control of tobacco have been paid more and more attention. Biological control measures are to use beneficial microorganisms to reduce the harm of plant pathogens to plants, and ecological control is to introduce antagonistic microorganisms into the soil, fertilize the soil, and improve the biodiversity in the soil, so as to balance the soil micro-ecology, and finally through the soil. The antagonism and competition between different organisms in soil can realize the control of soil diseases.

Contents of the invention

The object of the present invention is to provide antagonistic bacteria for preventing and treating tobacco bacterial wilt.

In order to achieve the above object, the technical scheme adopted in the present invention is to provide an antagonistic bacteria for preventing and treating tobacco bacterial wilt, and said antagonistic bacteria are Bacillus cereus (Bacillus cereus) QJ-1 and Aspergillus niger (Aspergillus niger) Ty-3; wherein , Bacillus cereus (Bacillus cereus) QJ-1, deposit unit: China Center for Type Culture Collection, deposit address: Wuhan University, Wuhan, China, deposit date: July 5, 2012, deposit number: CCTCC NO: M2012271; black Aspergillus niger Ty-3, deposit unit: China Center for Type Culture Collection, deposit address: Wuhan University, Wuhan, China, deposit date: July 8, 2011, deposit number: CCTCC NO: M2011241.

The screening method of antagonistic bacteria for preventing and treating tobacco bacterial wilt of the present invention comprises the following steps:

1) Isolation of pathogenic bacteria

Select the tobacco strains with tobacco bacterial wilt disease, clean and disinfect the tobacco stems, take the lesion spots on the tobacco stems, cut the vascular tissue of the susceptible parts into pieces, and put them into beef extract peptone bacteria plates for culture; wait until the bacterial wilt pathogen grows When it is mycelium, pick the colony to purify, cultivate, inoculate on the slope of beef extract peptone and store it;

2) Pathogenicity identification of pathogenic bacteria

Sow tobacco seeds, and when the tobacco plants grow to have vascular tissue, destroy the tobacco roots and pour the cultured bacteria suspension of bacterial wilt pathogen into the roots, keep high temperature and high humidity, and observe the disease after 30 days;

3) Isolation of soil microorganisms

Take the soil around the rhizosphere of the infected tobacco plant, add sterile water, shake it and let it stand still, carry out the concentration gradient dilution of the soil solution, and take the diluted solution in the beef extract peptone medium, Gao's No. 1 medium and Bengal red medium respectively. Spread evenly on the surface, culture, pick a single colony to purify and inoculate to the corresponding slant medium for storage;

4) Antagonist screening

The cultured different soil microorganisms and the pathogenic bacteria of bacterial wilt were cultured in confrontation on NA medium to determine whether there was antagonism;

5) Antagonism assay

Wash out the pathogenic bacteria of bacterial wilt with sterile water, prepare a bacterial suspension, mix the bacterial suspension with NA medium, place it to solidify into a flat plate, and inoculate the soil microorganisms with antagonistic effects measured by confrontation culture individually or in pairs Cultivate on a flat plate to measure the size of the antibacterial plaque or zone of soil microorganisms against pathogenic bacteria;

6) Morphological observation of antagonistic bacteria;

7) Identification and classification of antagonistic bacteria.

The purpose of the present invention is also to provide the application of antagonistic bacteria for preventing and treating tobacco bacterial wilt in the aspect of microbial inoculation.

The technical solution adopted by the invention is also to provide the application of the antagonistic bacteria for preventing and treating tobacco bacterial wilt in the aspect of microbial inoculation.

The production method of the antagonistic antibacterial agent for preventing and treating tobacco bacterial wilt of the present invention: inoculate the purified antagonistic bacteria QJ-1 and Ty-3 respectively on the NA medium, cultivate in an incubator at 32°C for 24 hours, and take 5 mL of sterile water Invert the inclined plane, shake gently, and then accurately draw 1 mL of the bacterial suspension into a 250 mL Erlenmeyer flask containing 150 mL of sterile NA liquid medium, place it in a water bath shaker at 32 ° C for 1 day, and then transfer the cultured bacterial suspension to The solution was added to the sterilized wheat bran according to the inoculation amount of 10% by mass fraction, cultivated at 32°C for 3 days, and air-dried to obtain the single-bacteria agent of QJ-1 and Ty-3. The QJ-1 and Ty-3 in the single bacterial agent reach 10 ⁸ cfu/g respectively, and then the two single bacterial agents are mixed according to the mass ratio of 1:1, which is the antagonistic bacterial agent.

The object of the present invention is also to provide the application of antagonistic bacteria for preventing and treating tobacco bacterial wilt in microbial organic fertilizer.

The technical solution adopted by the invention is also to provide the application of antagonistic bacteria for preventing and treating tobacco bacterial wilt in microbial organic fertilizer.

The production method of the antagonistic microbial organic fertilizer for preventing and treating tobacco bacterial wilt of the present invention: add the inoculant to the decomposed organic fertilizer in an amount of 8 to 12% by mass fraction and evenly mix it, adjust the moisture to 50 to 60%, and pile it up to 50 to 60%. 80cm of stacking, stacking 5 ~ 7d that is.

The invention proceeds from the improvement of soil microorganisms, screens antagonistic bacteria from in situ soil, and studies the application of antagonistic bacteria in microbial agent and microbial organic fertilizer for preventing and treating tobacco bacterial wilt. The microbial organic fertilizer prepared by mixing antagonistic bacteria and organic fertilizer can significantly reduce tobacco bacterial wilt. The fertilizer provides effective nutrition and functional bacteria, and enables antagonistic bacteria to colonize rapidly in the soil, regulates soil micro-ecological balance and inhibits bacterial wilt. Applying this functional organic fertilizer to the tobacco field can achieve the purpose of improving the ability of the tobacco plant itself to resist bacterial wilt, improving the quality of the tobacco leaves, and reducing the amount of chemical fertilizers and pesticides.

Description of drawings

Fig. 1 is the Gram staining of the bacterial strain QJ-1 that is isolated from in situ soil;

Figure 2 is the fungal strain Ty-3 isolated from the soil in situ;

Figure 3 shows the confrontation between QJ-1 and Ty-3 and R. solanacearum growth for 1 day;

Fig. 4 is a micromorphological diagram of Ty-3 spores.

Detailed ways

The pathogenic bacteria of tobacco bacterial wilt were collected from the tobacco plants with typical symptoms in the smoking area of Shaowu City, Fujian Province, and the soil around the rhizosphere of the diseased plants was collected.

Preparation of medium:

Beef extract peptone medium: NaCl 5g, beef extract 3g, peptone 10g, agar 20g, add water to 1L, pH7.4～7.6, sterilize at 121℃ for 20min.

Gaoshi No. 1 medium: soluble starch 20g, KNO ₃ 1g, K ₂ HPO ₄ 0.5g, MgSO ₄ 7H ₂ O 0.5g, NaCl 0.5g, FeSO ₄ 7H ₂ O 0.01g, agar 20g, pH7.4 ~7.6. When preparing, first use a small amount of cold water to make the starch into a paste, pour it into boiling water less than the required amount of water, heat it on the fire, and dissolve other ingredients one by one while stirring. After melting, add water to 1000mL and adjust the pH , sterilized at 121°C for 20 minutes.

Bengal red medium: 5 g of peptone, 10 g of glucose, 1 g of KH ₂ PO ₄ , 0.5 g of MgSO ₄ 7H ₂ O, 20 g of agar, 100 mL of 1/3000 Bengal red solution, and 0.1 g of chloramphenicol. After adding the above ingredients into distilled water to dissolve, add Bengal red solution. Separately dissolve chloramphenicol with a small amount of ethanol, add to the culture medium, and after aliquoting, sterilize at 121°C for 20 minutes.

NA medium: 12g yeast extract, 20g sucrose, 4g K ₂ HPO ₄ , 20g agar, pH 7.0-7.2, add water to 1L, sterilize at 121°C for 20min. The difference between NA liquid medium and NA medium is that NA liquid medium does not add agar.

Malt juice agar medium (MEA): 150mL wort juice, 3g agar, natural pH (about 6.4), sterilized at 121°C for 20min.

Embodiment 1, isolation and screening of tobacco bacterial wilt antagonistic bacteria

1) Isolation of pathogenic bacteria

Before isolating the bacteria, rinse the tobacco rods clean, then use 75% alcohol to disinfect the tobacco rods and the knife, and then use a knife to cut open the lesion on the stem of the tobacco plant under aseptic conditions, and remove the vascular tissue of the susceptible part. Cut into several small pieces, pick up the cigarette pieces with sterilized tweezers and put them into the beef extract peptone bacterial plate culture dish for cultivation, put 5 pieces in each dish, repeat 3 dishes, the cultivation temperature is 32°C, and the cultivation time is 48h. White fluid colonies grow and disperse fine bacteria outwards when the bacterial wilt cigarette block contacts with the medium, and inoculate it in beef extract peptone for preservation.

The micrographs showed that bacterial wilt pathogen was rod-shaped, blunt at both ends, with flagella, without capsule, and Gram staining was negative.

2) Identification of pathogenicity

Tobacco K326 was selected as the experimental variety. After sowing the tobacco seeds in pots, when the tobacco plants grow to have vascular tissue, destroy the roots of the tobacco and dilute the cultured bacterial wilt pathogenic bacteria to make a bacterial suspension, and pour it into the root circumference of the tobacco 50mL bacterial suspension, and keep high temperature and high humidity. The incidence was observed after 30 days.

According to Koch's law, the pathogenic strains isolated from bacterial wilt diseased tobacco strains were inoculated into healthy tobacco plants to determine their pathogenicity. The isolated bacterial wilt pathogen test strain was inoculated into 20 healthy tobacco plants, and after 30 days, 15 tobacco plants showed symptoms of bacterial wilt disease.

3) Isolation of soil microorganisms

Obtain 10g of soil around the rhizosphere of tobacco plants with bacterial wilt, put it into a conical flask with 90mL of sterile water, shake it for 30min, let it stand for 20min, and then dilute it to 10 ^-4 , 10 ^-5 and 10 ^-6 g/mL, respectively. Draw 0.1 mL of solutions of different concentrations and spread evenly on beef extract peptone medium, Gao’s No. Colonies were purified and transferred to corresponding slant media for preservation.

A total of 5 kinds of bacteria, 4 kinds of fungi and 4 kinds of actinomycetes were obtained from the soil by dilution and spreading. Bacteria were numbered QJ-1 to QJ-5, fungi were numbered Ty-1 to Ty-4, and actinomycetes were numbered QF-1 to QF-4. Among the bacteria, QJ-1 is a Gram-positive bacteria, and the other four strains are all Gram-negative bacteria. The Gram-stained photo of QJ-1 is shown in Figure 1. The colony morphology of Ty-3 is shown in Figure 2.

4) Screening of antagonistic strains

The isolated soil microorganisms and the bacterial wilt pathogen were cultured in confrontation on NA medium. Spread bacterial wilt pathogenic bacteria on the medium, inoculate the isolated soil microorganisms in the middle, and the distance between the two bacteria is 3mm. Check the culture results after 2 days, and judge the isolated soil microorganisms according to the growth rate of the colonies and whether there is an inhibition zone between the colonies. Has no antagonistic effect on pathogenic bacteria.

Through confrontation culture, three strains with antagonistic effect on tobacco bacterial wilt were screened out from the isolated soil microorganisms, namely QJ-1, QJ-3 and Ty-3.

5) Antagonism assay

Wash out the pathogenic bacteria of bacterial wilt that has been cultured for 24 hours with sterile water, make a bacterial suspension, draw 1mL of the bacterial suspension into a sterilized Petri dish, then pour 15mL of NA medium at 45-50°C, and place After 2 hours of solidification, form a flat plate, inoculate the soil microorganisms with antagonistic effects measured by the confrontation culture individually or in pairs on the plate, and repeat 3 dishes for each type, and observe the growth of the pathogenic bacteria after culturing at 32°C for 2 days, and determine The size of the antibacterial plaque or antibacterial zone of soil microorganisms on the pathogen, among which the mixed bacteria of QJ-1 and Ty-3 have the largest antibacterial zone, reaching 9mm, which has a good effect on inhibiting the pathogenic bacteria of bacterial wilt, as shown in Figure 3 .

6) Antagonism mechanism

Antagonistic mechanism of QJ-1 and Ty-3 against R. solanacearum:

QJ-1 and Ty-3 can produce certain metabolites, which can effectively inhibit the growth of pathogenic bacteria of bacterial wilt, and the mixture of the two can promote the production of substances that inhibit pathogenic bacteria and enhance the antibacterial ability.

Embodiment 2, morphological observation

The initial colony of QJ-1 bacteria is flat, milky white, smooth and opaque.

The initial stage of Ty-3 bacteria is white hyphae, and then produces dark brown or dark brown spores, which are abundant.

Embodiment 3, identification and classification of antagonistic bacteria

The results identified by the Institute of Microbiology, Chinese Academy of Sciences are:

1. QJ-1 is Bacillus cereus.

1) Cell morphology and physical and chemical test results

Gram-positive, the cells are rod-shaped, the cell diameter is greater than 1 μm, spores are formed, and the spores do not expand; VP test, methyl red test, starch hydrolysis, casein decomposition, gelatin hydrolysis, catalase, oxidase, nitrate reduction reaction , Utilization of citrate were positive; acid production reaction performance: glucose positive, xylose, L-arabinose, mannitol, lactose negative.

2) 16S rRNA gene sequence determination results

GCAGTCGAGC GATGGATTAA GAGCTTGCTC TTATGAAGTT AGCGGCGGAC

GGGTGAGTAA CACGTGGGTA ACCTGCCCAT AAGACTGGGA TAACTCCGGG

AAACCGGGGC TAATACCGGA TAACATTTTG AACTGCATGG TTCGAAATTG

AAAGGCGGCT TCGGCTGCCA CTTATGGATG GACCCGCGTC GCATTAGCTA

GTTGGTGAGG TAACGGCTCA CCAAGGCAAC GATGCGTAGC CGACCTGAGA

GGGTGATCGG CCACACTGGG ACTGAGACAC GGCCCAGACT CCTACGGGAG

GCAGCAGTAG GGAATCTTCC GCAATGGACG AAAGTCTGAC GGAGCAACGC

CGCGTGAGTG ATGAAGGCTT TCGGGTCGTA AAACTCTGTT GTTAGGGAAG

AACAAGTGCT AGTTGAATAA GCTGGCACCT TGACGGTACC TAACCAGAAA

GCCACGGCTA ACTACGTGCC AGCAGCCGCG GTAATACGTA GGTGGCAAGC

GTTATCCGGA ATTATTGGGC GTAAAGCGCG CGCAGGTGGTTTCTTAAGTC

TGATGTGAAA GCCCACGGCT CAACCGTGGA GGGTCATTGG AAACTGGGAG

ACTTGAGTGC AGAAGAGGAA AGTGGAATTC CATGTGTAGC GGTGAAATGC

GTAGAGATAT GGAGGAACAC CAGTGGCGAA GGCGACTTTC TGGTCTGTAA

CTGACACTGA GGCGCGAAAG CGTGGGGAGC AAACAGGATT AGATACCCTG

GTAGTCCACG CCGTAAACGA TGAGTGCTAA GTGTTAGAGG GTTTCCGCCC

TTTAGTGCTG AAGTTAACGC ATTAAGCACT CCGCCTGGGG AGTACGGCCG

CAAGGCTGAA ACTCAAAGGA ATTGACGGGG GCCCGCACAA GCGGTGGAGC

ATGTGGTTTA ATTCGAAGCA ACGCGAAGAA CCTTACCAGG TCTTGACATC

CTCTGAAAAC CCTAGAGATA GGGCTTCTCC TTCGGGAGCA GAGTGACAGG

TGGTGCATGG TTGTCGTCAG CTCGTGTCGT GAGATGTTGG GTTAAGTCCC

GCAACGAGCG CAACCCTTGA TCTTAGTTGC CATCATTAAG TTGGGCACTC

TAAGGTGACT GCCGGTGACA AACCGGAGGA AGGTGGGGAT GACGTCAAAT

CATCATGCCC CTTATGACCT GGGCTACACA CGTGCTACAA TGGACGGTAC

AAAGAGCTGC AAGACCGCGA GGTGGAGCTA ATCTCATAAA ACCGTTCTCA

GTTCGGATTG TAGGCTGCAA CTCGCCTACA TGAAGCTGGA ATCGCTAGTA

ATCGCGGATC AGCATGCCGC GGTGAATACG TTCCCGGGCC TTGTACACAC

CGCCCGTCAC ACCACGAGAG TTTGTAACAC CCGAAGTCGG TGGGGTAACC

TTTTCAAAAT

3) gyrB gene sequence determination results

TGACGGCGGC GGTTATAAAG TTTCTGGTGG TTTGCATGGT GTTGGGGCAT

CTGTAGTAAA TGCTCTATCA ACAGAACTAG AGGTATTTGT ACATCGTGAA

GGTAAAATCC ATTATCAAAA ATACGAAAGA GGTATTCCAG TTGCGGATTT

AAAAGTCATT GGTGACACAG ATCAAACAGG AACGATAACT CGATTTAAAC

CAGATCCAGA AATTTTTTCAG GAAACAACAG TATACGAATT CGATACGCTA

GCAACTCGTA TGCGTGAATT AGCATTTTAA ATCGTAATAT TAAATTGACG

ATTGAAGATA AACGTGAACA TAAGCAAAAG AAAGAATTCC ATTACGAAGG

TGGAATTAAA TCATATGTTG AGCATTTAAA CCGCTCAAAA CAACCAATCC

ATGAAGAACC TGTATATGTA GAAGGATCAA AAGATGGTAT TCAAGTTGAG

GTTTCCTTAC AGTATAACGA AGGATATACA AATAATATTT ACTCATTTAC

GAATAACATT CATACGTATG AAGGTGGAAC ACATGAAGTA GGTTTTAAAA

CAGCTTTAAC TCGTGTGATT AACGATTACG GTCGTAAAAA TAGTATTTA

AAAGATGCAG ACAGTAACTT AACTGGCGAG GATGTTCGTG AAGGTTTAAC

TGCAATCGTA TCAATTAAAC ATCCAAATCC ACAATTTGAA GGACAAACGA

AGACGAAACT TGGGAATAGT GAAGCGAGAA CGATTACAGA GTCTGTGTTT

TCAGAGGCAT TTGAAAAGTT CTTACTAGAA AACCCAAACG TTGCACGAAA

AATTGTGGAA AAAGGTACGA TGGCAGCACG TGCGCGTGTT GCAGCGAAAA

AAGCACGTGA ATTAACACGC CGTAAGAGTG CGTTAGAAGTTTCAAGCTTA

CCTGGTAAAT TAGCAGATTG CTCTTCAAAA GATCCAGCAA TTAGCGAAAT

TTATATTGTA GAGGGTGATT CTGCCGGCGG ATCAGCAAAG CAAGGTCGTG

ACCGTCACTT CCAGGCGATT TTACCGCTAA AGGGTAAAAT TATTAACGTT

GAAAAAGCAA GATTAGATAA AATTTATCT AACGATGAAG TGCGTACAAT

TATTACTGCA ATTGGTACGA ACATTGGCGG AGATTTTGAT ATTGAGAAAG

CTCGTTATCA TAAAGTTATT ATTATGACGG ACGCCGACGT CGACGGCTCG

CACATCCG

4) Fermentation conditions

The seed medium is NA liquid medium, the initial pH of the fermentation is 7.5, the volume of the 250mL Erlenmeyer flask is 150mL, the rotation speed of the shaking table at 32°C is 150r/min, the fermentation time is 24h, and the inoculation amount of 10% by mass fraction is inoculated into the bran After being cultured in the skin for 72 hours, it was naturally dried until the moisture content was less than 30%.

2. Ty-3 is Aspergillus niger

1) Colony morphology and microscopic characteristics

The colonies on the malt juice agar medium (MEA) grow rapidly, and the diameter of the colonies is 50-63mm in 7 days under dark conditions at 25°C, and the texture is velvety; a large number of sporulation structures are formed, and the conidia heads are dark brown to dark brown. Split into several columnar structures; the back of the colony is light brown, without water-soluble pigment.

Conidiophores tall, 6-15 μm wide, smooth-walled, straight or curved; apical capsule spherical, 22-30 μm in diameter, fertile on the entire surface; conidia double-layered; conidia subspherical, light to brown, rough-walled Or with small spines, 3.0-4.8μm. No sexual spores were seen, as shown in Figure 4.

2) rRNA gene sequence sequencing results:

(Including 18S rRNA fragments, complete sequences of ITS1, 5.8S rRNA, ITS2 regions and 28S rRNA sequence fragments)

GATCCGAGGTCACCTGGAAAGAATGGTTGGAAAACGTCGGCAGGCGCCGGCCAATCCTACAGAGCATGTGACAAAGCCCCATACGCTCGAGGATCGGACGCGGTGCCGCCGCTGCCTTTCGGGCCCGTCCCCCCGGAGAGGGGGACGGCGACCCAACACACAAGCCGGGCTTGAGGGCAGCAATGACGCTCGGACAGGCATGCCCCCCGGAATACCAGGGGGCGCAATGTGCGTTCAAAGACTCGATGATTCACTGAATTCTGCAATTCACATTAGTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAACCAAGAGATCCATTGTTGAAAGTTTTAACTGATTGCATTCAATCAACTCAGACTGCACGCTTTCAGACAGTGTTCGTGTTGGGGTCTCCGGCGGGCACGGGCCCGGGGGGCAGAGGCGCCCCCCCGGCGGCCCACAAGCGGCGGGCCCGCCGAAGCAACAGGGTACAATAGACACGGATGGGAGGTTGGGCCCAAAGGACCCGCACTCGGTAATGATCCTTCCGCAGGTTCACCTACGGAAACCTTGTTAC

4) Fermentation conditions

The seed medium is NA liquid medium, the initial pH of fermentation is 7.5, the volume of the 250mL Erlenmeyer flask is 150mL, the rotation speed of the shaking table at 32°C is 150r/min, the fermentation time is 24h, and the inoculation amount of 10% by mass fraction is inoculated into the bran After being cultured in the skin for 72 hours, it was naturally dried until the moisture content was less than 30%.

Embodiment 4, the production of antagonistic antibacterial agent

Inoculate the purified antagonistic bacteria QJ-1 and Ty-3 on the NA slant medium respectively, culture them in a 32°C incubator for 24 hours, take 5mL of sterile water to pour the slant, shake gently, and then accurately suck 1mL of the bacterial suspension Add it into a 250mL Erlenmeyer flask containing 150mL of sterile NA liquid medium, place it in a water bath shaker at 32°C for 1d, and then add the cultured bacterial suspension to the sterilized In the wheat bran, cultivated at 32°C for 3 days, then air-dried to become the single bacterial agent of QJ-1 and Ty-3. The QJ-1 and Ty-3 in the single bacterial agent reach 10 ⁸ cfu/g respectively, and then the two single bacterial agents are mixed according to the mass ratio of 1:1, which is the antagonistic bacterial agent.

Embodiment 5, the production of microbial organic fertilizer

According to the inoculum amount of 10% by mass fraction, add the antagonistic antimicrobial agent into the decomposed organic fertilizer and mix evenly, adjust the water content to 50-60%, pile up into a stack of 80 cm, and stack it for 5 days.

The preparation method of decomposed organic fertilizer: adjust the water content of livestock and poultry manure to 50-60%, pile it up to a height of 1.1m, turn the compost every day, and gradually increase the compost temperature. When the temperature drops to close to room temperature, the fermentation ends, which is decomposed organic Fat.

Experimental example 1, analysis of the effect of preventing and treating tobacco bacterial wilt

1. In 2012, a field test was carried out in the smoking area of Shaowu City, Fujian Province

1) Fertilization period:

The present invention achieves the purpose of inhibiting bacterial wilt by twice applying the microbial organic fertilizer. For the first time, the microbial organic fertilizer was applied in the form of base fertilizer, so that the beneficial bacteria in the soil could increase in a large amount in the early stage of tobacco plant growth, thereby inhibiting the growth of pathogenic bacteria in the soil and greatly reducing the number of them. The second time, when the local bacterial wilt is about to occur, it is applied in the form of topdressing (high concentration bacterial fertilizer: water = 1:10), and the large-scale reproduction of beneficial bacteria is used to resist the growth and reproduction of bacterial wilt.

2) Test soil and block design:

The soil is sandy loam with flat terrain and convenient drainage and irrigation. The basic fertility is: organic matter 40.6g/kg, alkaline nitrogen 198.5mg/kg, available phosphorus (P ₂ O ₅ ) 20.52mg/kg, available potassium (K ₂ O) 80.43 mg/kg, pH 5.52. The variety of flue-cured tobacco tested was K326. The seedlings are raised by floating seedling raising technology, transplanted by the seedling transplanting technology under the mulch, and managed according to the technical specifications for high-quality tobacco production after transplanting. Each treatment was repeated three times, and the cultivation technical measures and management operation requirements of each plot were basically consistent.

A total of four treatments were set up in the experiment:

Treatment A: control, local conventional fertilization and management;

Treatment B: microbial organic fertilizer was applied in the form of base fertilizer (250g/plant);

Treatment C: Microbial organic fertilizer was applied in the form of base fertilizer (200g/plant) + topdressing (root irrigation 50g/plant);

Treatment D: Microbial organic fertilizer was applied in the form of topdressing (root irrigation 50g/plant).

3) Investigation and measurement items

During the onset of bacterial wilt in the local area, observe and record the date, incidence and speed of the first onset of bacterial wilt in different treatments, and calculate the disease index. From the onset, record once a week; the disease grading standard is based on the national tobacco industry tobacco disease survey. Conducted under the standard Yc/T39-1996, bacterial wilt investigation method: Grade 0, the whole plant is disease-free; grade 1, there are occasional chlorotic spots on the stem, or (and) the lower leaves or top leaves are slightly withered, or the lower part Lesions appear on a few leaves; grade 2, there are obvious black streaks on the stem but not reaching the top of the tobacco plant, or (and) more than half of the diseased side or some leaves above the waist are withered; grade 3, black stripes on the stem reach the top of the tobacco plant The top of the plant, or (and) all the leaves on the diseased side are withered, or most of the leaves of the whole plant are withered; grade 4, the diseased plant is dead. Disease index and control effect are calculated according to tobacco disease drug efficacy test method Yc/T40-1996:

Table 1 shows the results of the field test in the smoking area of Shaowu City, Fujian Province. As can be seen from Table 1, using the microbial organic fertilizer of the present invention, the incidence of tobacco bacterial wilt has reduced by more than 98%, and the disease index obviously descends, illustrating that the microbial organic fertilizer of the present invention has a good effect on preventing and treating tobacco bacterial wilt Effect.

Table 1 Field experiment in Shaowu City, Fujian Province in 2012 to control the effect of bacterial wilt

deal with A B C D. Morbidity 71.95% 3.11% 1.21% 4.34% Disease index 33.83% 0.98% 0.67% 1.34%

Experimental Example 2, Tobacco Leaf Yield and Quality Analysis of Tobacco

Tobacco leaf yield and quality were analyzed in the field test in Shaowu City, Fujian Province in 2012 in Experimental Example 1, and the results are shown in Table 2.

Table 2 Yield and quality analysis of tobacco leaves in field trials in Shaowu City, Fujian Province in 2012

As can be seen from Table 2, after using the microbial organic fertilizer of the present invention, the quality and output of tobacco leaves are greatly improved.

Claims (3)

1. Antagonistic bacteria for preventing and treating tobacco bacterial wilt, characterized in that the antagonistic bacteria are Bacillus cereus ( Bacillus cereus ) QJ-1 and Aspergillus niger ( Aspergillus niger ) Ty-3; wherein, Bacillus cereus ( Bacillus cereus) cereus ) QJ-1, deposit unit: China Type Culture Collection Center, deposit address: Wuhan University, Wuhan, China, deposit date: July 5, 2012, deposit number: CCTCC NO: M2012271; said Aspergillus niger Ty-3, deposit unit: China Center for Type Culture Collection, deposit address: Wuhan University, Wuhan, China, deposit date: July 8, 2011, deposit number: CCTCC NO: M2011241. 2. the application of the antagonistic bacteria for preventing and treating tobacco bacterial wilt as claimed in claim 1 in the aspect of microbial inoculum. the 3. the application of the antagonistic bacteria for preventing and treating tobacco bacterial wilt as claimed in claim 1 in microbial organic fertilizer. the