CN103146600B - Antagonistic bacteria for prevention and treatment of tobacco bacterial wilt and application thereof - Google Patents
Antagonistic bacteria for prevention and treatment of tobacco bacterial wilt and application thereof Download PDFInfo
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- CN103146600B CN103146600B CN201310046171.3A CN201310046171A CN103146600B CN 103146600 B CN103146600 B CN 103146600B CN 201310046171 A CN201310046171 A CN 201310046171A CN 103146600 B CN103146600 B CN 103146600B
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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
Technical field
The present invention relates to prevent and treat tobacco bacterial wilt Antagonistic Fungi, also relate to the application of this Antagonistic Fungi aspect control soil-borne disease of tobacco simultaneously, belong to biological control field.
Background technology
Tobacco bacterial wilt is the important soil-borne disease of tobacco of a class, and it infects tobacco root conventionally, causes the disease of crop root and even complete stool, causes great financial loss.Tobacco bacterial wilt is that a kind of systematicness being caused by cloth Ke Shi bacillus (Ralstonia solanacearum) infects disease, often causes whole strain death once cigarette strain is caught an illness, and its harm is destructive often, therefore usually produces and causes heavy economic losses to tobacco.Tobacco bacterial wilt pathogenic bacteria is mainly lost to fall within soil with plant residue and survives the winter, also can in seed or in other pin main body of field, survive the winter, the soil carrying disease germs, invalid tissue and the organic fertilizer that contains germ etc. are the main sources of just dying of this disease, and the propagation of disease is mainly by irrigation water, rainwater, seedling, farm implements, sick soil and people and animals' activity etc. in spite of illness.
At present, in production, the main agricultural measures such as disease-resistant variety and shift of crops that adopt are prevented and treated soil-borne disease of tobacco disease, but because disease-resistant variety needs disease-resistant gene variation, and being subject to the impact of environment larger, prevention effect is undesirable.Shift of crops is also one of effective ways of control tobacco bacterial wilt, but because China has a large population and a few land, in production, normal crop rotation measure cannot realize.Phytopathologist thinks that these three of plant morbidity and host, pathogenic bacteria and environment are because have substantial connection, when pathogenic bacteria and susceptible host meet in adapt circumstance, plant just starts morbidity, if revise or eradicate any one in this three, just can alleviate or control Plant diseases.
Large quantity research shows, not only exist and cause the microorganism of tobacco diseases but also have beneficial microorganism diversified non-virulent, that improve tobacco vitality, thereby the biological control of tobacco and bionomic control comes into one's own day by day in vega soil.Bio-control method is to utilize beneficial microorganism to reduce the harm of phytopathogen to plant, bionomic control is by measures such as species diversities in soil introducing antagonistic microbe, fertilizing soil and raising soil, and then make soil micro-ecosystem reach balance, finally realize the control to soil disease by the antagonism between different biologies in soil and competition.
Summary of the invention
The object of this invention is to provide control tobacco bacterial wilt Antagonistic Fungi.
In order to realize above object, the technical solution adopted in the present invention is to provide control tobacco bacterial wilt Antagonistic Fungi, and described Antagonistic Fungi is bacillus cereus (Bacillus cereus) QJ-1 and aspergillus niger (Aspergillusniger) Ty-3; Wherein, bacillus cereus (Bacillus cereus) QJ-1, depositary institution: Chinese Typical Representative culture collection center, preservation address: Wuhan, China Wuhan University, preservation date: on July 5th, 2012, preserving number: CCTCC NO:M2012271; Aspergillus niger (Aspergillus niger) Ty-3, depositary institution: Chinese Typical Representative culture collection center, preservation address: Wuhan, China Wuhan University, preservation date: on July 8th, 2011, preserving number: CCTCC NO:M2011241.
The present invention prevents and treats the screening method of tobacco bacterial wilt Antagonistic Fungi, comprises the following steps:
1) pathogenicbacteria separation
Choose the cigarette strain of tobacco bacterial wilt morbidity, tobacco rod is cleaned, sterilized, get scab on tobacco rod, susceptible position vascular bundle is organized and put into that beef extract-peptone bacterium is dull and stereotyped to be cultivated after stripping and slicing; In the time that Stalk Rot is grown to mycelia, picking colony purifying, cultivation, be inoculated in beef extract-peptone inclined-plane and preserve;
2) Pathogenic qualification
Sow tobacco seed, in the time that cigarette strain has grown to vascular bundle and organizes, destroy tobacco root and also pour the bacteria suspension of cultured Stalk Rot into root, keep hot and humid, after 30d, observe incidence;
3) separation of soil microorganisms
Get the cigarette strain rhizosphere soil around of catching an illness, add sterilized water, after concussion, leave standstill, the soil solution is carried out to concentration gradient dilution, get diluting soln evenly coating on beef-protein medium, Gause I substratum and rose bengal medium respectively, cultivate, picking list bacterium colony purifying is also seeded to corresponding slant medium preservation;
4) Antagonistic Fungi screening
By the cultivation that stands facing each other on NA substratum of the pathogenic bacteria of the different soils microorganism of cultivation and bacterial wilt, judgement has or not antagonistic action;
5) antagonistic action is measured
Wash out Stalk Rot with sterilized water, prepare bacteria suspension, and bacteria suspension is mixed with NA substratum, placement is frozen into flat board, face-off is cultivated to the single and combined inoculation between two of the soil microorganisms with antagonistic action of measuring and on flat board, cultivate, the antibacterial spot of mensuration soil microorganisms to pathogenic bacteria or the size of antibacterial band;
6) Antagonistic Fungi is carried out to morphological observation;
7) Antagonistic Fungi is identified to classification.
The present invention also aims to provide the application of control tobacco bacterial wilt Antagonistic Fungi aspect microbiobacterial agent.
The technical solution adopted in the present invention is also to provide the application of control tobacco bacterial wilt Antagonistic Fungi aspect microbiobacterial agent.
The present invention prevents and treats the production method of tobacco bacterial wilt Antagonistic Fungi microbial inoculum: the Antagonistic Fungi QJ-1 of purifying and Ty-3 are inoculated into respectively on NA substratum, in the incubator of 32 DEG C, cultivate 24h, get 5mL sterilized water contra bevel, vibration gently, then accurately drawing lmL bacteria suspension joins in the 250mL triangular flask that the aseptic NA liquid nutrient medium of 150mL is housed, 1d is cultivated in the shaking bath concussion that is put into 32 DEG C, then cultured bacteria suspension is joined respectively according to the inoculum size of massfraction 10% in the wheat bran of sterilizing, cultivate 3d for 32 DEG C, the air-dry single bacteria agent that is QJ-1 and Ty-3.In single bacteria agent, QJ-1 and Ty-3 reach respectively 10
8cfu/g, then mixes two kinds of single bacteria agents in the ratio of quality 1:1, be Antagonistic Fungi microbial inoculum.
The present invention also aims to provide the application of control tobacco bacterial wilt Antagonistic Fungi aspect microbial organic fertilizer.
The technical solution adopted in the present invention is also to provide the application of control tobacco bacterial wilt Antagonistic Fungi aspect microbial organic fertilizer.
The production method of control tobacco bacterial wilt Antagonistic Fungi microbial organic fertilizer of the present invention: add decomposed manure to mix equably microbial inoculum by the addition of massfraction 8～12%, regulate moisture to 50～60%, pile the stacking of 50～80cm, 5～7d and get final product banks up.
The present invention sets about from improving soil microorganisms aspect, from original position soil screening Antagonistic Fungi, and has studied the application of control tobacco bacterial wilt Antagonistic Fungi in microbiobacterial agent and microbial organic fertilizer.Antagonistic Fungi mixes the microorganism organic fertilizer making with organic fertilizer can significantly reduce tobacco bacterial wilt, and this fertilizer provides effective nutrition and function yeast, and Antagonistic Fungi is surely grown rapidly in soil, regulates soil micro-ecosystem balance to suppress bacterial wilt.Can reach by use this functional fertilizer to vega the ability that improves cigarette strain self and resist bacterial wilt, improve quality of tobacco and reduce the object of chemical fertilizer, pesticide dosage.
Brief description of the drawings
Fig. 1 is the gramstaining that separates the bacterial isolates QJ-1 obtaining from original position soil;
Fig. 2 separates the fungal bacterial strain Ty-3 obtaining from original position soil;
Fig. 3 is that QJ-1 and Ty-3 mix and ralstonia solanacearum face-off growth 1 day;
Fig. 4 is the microscopic morphology figure of Ty-3 spore.
Embodiment
Tobacco bacterial wilt pathogenic bacteria is taken from cigarette district, Shaowu City, Fujian Province and has the cigarette strain of more typical illness, and gets diseased plant rhizosphere soil around.
The preparation of substratum:
Beef-protein medium: NaCl 5g, extractum carnis 3g, peptone 10g, agar 20g, adds water and is settled to 1L, pH7.4～7.6,121 DEG C of sterilizing 20min.
Gause I substratum: Zulkovsky starch 20g, KNO
31g, K
2hPO
40.5g, MgSO
47H
2o 0.5g, NaCl 0.5g, FeSO
47H
2o 0.01g, agar 20g, pH7.4～7.6.When preparation, first use a small amount of cold water, by starch furnishing pasty state, pour in the boiling water that is less than institute's water requirement, on fire, heat, dissolve one by one successively while stirring other compositions, after dissolving, supply moisture to 1000mL, adjust pH, 121 DEG C of sterilizing 20min.
Rose bengal medium: peptone 5g, glucose 10g, KH
2pO
41g, MgSO
47H
2o 0.5g, agar 20g, 1/3000 rose-bengal solution 100mL, paraxin 0.1g.After above-mentioned each composition adds in distilled water and to dissolve, then add rose-bengal solution.Separately, with a small amount of dissolve with ethanol paraxin, add in substratum, after packing, 121 DEG C of sterilizing 20min.
NA substratum: yeast extract 12g, sucrose 20g, K
2hPO
44g, agar 20g, pH7.0～7.2, the constant volume that adds water is to 1L, 121 DEG C of sterilizing 20min.The difference of NA liquid nutrient medium and NA substratum is, NA liquid nutrient medium does not add agar.
Wort agar substratum (MEA): wort 150mL, agar 3g, pH nature (approximately 6.4), 121 DEG C of sterilizing 20min.
The isolation and screening of embodiment 1, tobacco bacterial wilt Antagonistic Fungi
1) pathogenicbacteria separation
Separate before germ, first tobacco rod is rinsed well, with 75% alcohol, tobacco rod and pocket knife are sterilized again, then under aseptic condition, cut the scab on cigarette strain cane open with pocket knife, susceptible position vascular bundle tissue is cut into some fritters, put into the dull and stereotyped culture dish of beef extract-peptone bacterium with the tweezers gripping cigarette piece of sterilizing and cultivate, in every ware, put 5, repeat 3 wares, culture temperature is 32 DEG C, and incubation time is 48h.Treat that bacterial wilt cigarette piece and substratum contact position grow white mobility bacterium colony and outwards disperse tiny thalline, be inoculated in beef extract-peptone and preserve.
Photomicrograph shows that Stalk Rot thalline is shaft-like, the blunt circle in two ends, and amphitrichous, without pod membrane, Gram-negative.
2) pathogenic qualification
Select tobacco K326 for test kind.In basin alms bowl, by tobacco seed after planting, in the time that cigarette strain has grown into vascular bundle and organizes, the root of tobacco destroyed and cultured Stalk Rot weaker concn gradient is made to bacteria suspension, pouring 50mL bacteria suspension in tobacco rhizosphere, and keep hot and humid.After 30d, observe incidence.
According to KochShi rule, the bacterial wilt pathogenic strains separating in cigarette strain of falling ill is inoculated on health tobacco plant, pathogenic to measure it.Isolated Causal Organism of The Bacterial Wilt strains tested is inoculated to 20 strain health tobaccos, after 30d, have 15 strain tobaccos to occur bacterial wilt symptom.
3) separation of soil microorganisms
Obtain bacterial wilt cigarette strain rhizosphere soil 10g around, put into the triangular flask of 90mL sterilized water, after concussion 30min, leave standstill 20min, be then diluted to 10
-4, 10
-5with 10
-6g/mL, draw respectively solution 0.1mL evenly coating on beef-protein medium, Gause I substratum and rose bengal medium of different concns, every kind is repeated 3 wares, is placed in 32 DEG C of incubators and cultivates after 48h, and picking list bacterium colony carries out purifying and move on corresponding slant medium preserving.
Separate through dull and stereotyped dilution spread, from soil, obtain altogether 5 kinds of bacteriums, 4 kinds of fungies and 4 kinds of actinomycetes.Bacterium is numbered respectively to QJ-1 to QJ-5, and fungi is numbered Ty-1 to Ty-4, and actinomycetes are numbered QF-1 to QF-4.In bacterium, QJ-1 is gram-positive microorganism, and all the other 4 strain bacterium are Gram-negative bacteria, and QJ-1 gramstaining photo as shown in Figure 1.The colonial morphology figure of Ty-3 as shown in Figure 2.
4) screening of antagonistic strain
The cultivation that stands facing each other on NA substratum of the soil microorganisms that separation is obtained and Stalk Rot.Substratum coating Stalk Rot, middle inoculation separates the soil microorganisms obtaining, two bacterium are 3mm apart, check cultivation results after 2d, according to having or not inhibition zone etc. to judge that the soil microorganisms of separation has or not antagonistic action to pathogenic bacteria between bacterium colony tempo, bacterium colony.
Cultivate by face-off, from separate the soil microorganisms that obtains, filter out 3 strains tobacco bacterial wilt germ is had the bacterial strain of antagonistic action, be respectively QJ-1, QJ-3 and Ty-3.
5) antagonistic action is measured
Wash out the pathogenic bacteria of the bacterial wilt of cultivating 24h with sterilized water, make bacteria suspension, in the culture dish of the bacteria suspension of absorption 1mL after sterilizing, then pour into 45-50 DEG C NA substratum 15mL, place 2h and solidify rear one-tenth flat board, face-off is cultivated to the single and combined inoculation between two of the soil microorganisms with antagonistic action of measuring cultivates on flat board, every kind is repeated 3 wares, cultivate 2d at 32 DEG C after, observe the growing state of pathogenic bacteria, and measure the antibacterial spot of soil microorganisms to germ or the size of antibacterial band, wherein the antibacterial band of QJ-1 and Ty-3 mixed bacterium is maximum, reach 9mm, there is the effect of good inhibition Stalk Rot, as shown in Figure 3.
6) antagonism mechanism
QJ-1 and the Ty-3 antagonism mechanism to tobacco bacterial wilt germ:
QJ-1 and Ty-3 can produce some meta-bolites, and this substance effectively suppresses the growth of Stalk Rot, and both mix the generation that mutual promotion suppresses pathogenic bacteria material, strengthen bacteriostasis.
Embodiment 2, morphological observation
QJ-1 bacterium initial stage bacterium colony is open and flat, and oyster white is smooth, opaque.
Ty-3 bacterium initial stage white hypha, rear generation dark-brown or chocolate spore, spore is abundant.
The qualification classification of embodiment 3, Antagonistic Fungi
Through institute of microbiology of the Chinese Academy of Sciences, qualification result is:
1, QJ-1 is bacillus cereus (Bacillus cereus).
1) cellular form and physics and chemistry test-results
Gram-positive, cell is shaft-like, and cell dia is greater than 1 μ m, forms gemma, and gemma does not expand; VP test, methyl red test, Starch Hydrolysis, decomposition casein, gelatin hydrolysate, catalase, oxydase, nitrate reduction react, utilize Citrate trianion to be all positive; Acid-producing shows as: the glucose positive, wood sugar, L-arabinose, N.F,USP MANNITOL, lactose feminine gender.
2) 16S rRNA gene sequencing result
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?CGCAGGTGGT?TTCTTAAGTC
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 sequencing result
TGACGGCGGC?GGTTATAAAG?TTTCTGGTGG?TTTGCATGGT?GTTGGGGCAT
CTGTAGTAAA?TGCTCTATCA?ACAGAACTAG?AGGTATTTGT?ACATCGTGAA
GGTAAAATCC?ATTATCAAAA?ATACGAAAGA?GGTATTCCAG?TTGCGGATTT
AAAAGTCATT?GGTGACACAG?ATCAAACAGG?AACGATAACT?CGATTTAAAC
CAGATCCAGA?AATTTTTCAG?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?TAGTATTTTA
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?CGTTAGAAGT?TTCAAGCTTA
CCTGGTAAAT?TAGCAGATTG?CTCTTCAAAA?GATCCAGCAA?TTAGCGAAAT
TTATATTGTA?GAGGGTGATT?CTGCCGGCGG?ATCAGCAAAG?CAAGGTCGTG
ACCGTCACTT?CCAGGCGATT?TTACCGCTAA?AGGGTAAAAT?TATTAACGTT
GAAAAAGCAA?GATTAGATAA?AATTTTATCT?AACGATGAAG?TGCGTACAAT
TATTACTGCA?ATTGGTACGA?ACATTGGCGG?AGATTTTGAT?ATTGAGAAAG
CTCGTTATCA?TAAAGTTATT?ATTATGACGG?ACGCCGACGT?CGACGGCTCG
CACATCCG
4) fermentation condition
Seed culture medium is NA liquid nutrient medium, and the initial pH that ferments is that 7.5,250mL triangular flask bottling amount is 150mL, 32 DEG C of shaking table revolutions are 150r/min, fermentation time is 24h, is inoculated in wheat bran and cultivates after 72h with the inoculum size of massfraction 10%, naturally dries to moisture content and is less than 30%.
2, Ty-3 is aspergillus niger (Aspergillus niger)
1) colonial morphology and microscopic features
The upper colony growth of wort agar substratum (MEA) is fast, lower 7 days colony diameter 50～63mm of 25 DEG C of dark conditions, and quality is velvet-like; Conidial fructification forms in a large number, and conidial head Vandyke brown is to chocolate, and the initial stage is spherical, and the later stage splits for several columnar structures; The bacterium colony back side is light brown, without water colo(u)r.
Conidiophore is tall and big, wide 6～15 μ m, and wall is smooth, straight or bending; Top capsule is spherical, diameter 22～30 μ m, and all surfaces can be educated; Conidial fructification bilayer; Conidium is subsphaeroidal, shallow to brown, the coarse or tool spinule of wall, 3.0～4.8 μ m.There are no spermatium as shown in Figure 4.
2) rRNA gene order sequencing result:
(comprising 18S rRNA fragment, ITS1,5.8S rRNA, ITS2 district complete sequence and 28S rRNA sequence fragment)
GATCCGAGGTCACCTGGAAAGAATGGTTGGAAAACGTCGGCAGGCGCCGGCCAATCCTACAGAGCATGTGACAAAGCCCCATACGCTCGAGGATCGGACGCGGTGCCGCCGCTGCCTTTCGGGCCCGTCCCCCCGGAGAGGGGGACGGCGACCCAACACACAAGCCGGGCTTGAGGGCAGCAATGACGCTCGGACAGGCATGCCCCCCGGAATACCAGGGGGCGCAATGTGCGTTCAAAGACTCGATGATTCACTGAATTCTGCAATTCACATTAGTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAACCAAGAGATCCATTGTTGAAAGTTTTAACTGATTGCATTCAATCAACTCAGACTGCACGCTTTCAGACAGTGTTCGTGTTGGGGTCTCCGGCGGGCACGGGCCCGGGGGGCAGAGGCGCCCCCCCGGCGGCCCACAAGCGGCGGGCCCGCCGAAGCAACAGGGTACAATAGACACGGATGGGAGGTTGGGCCCAAAGGACCCGCACTCGGTAATGATCCTTCCGCAGGTTCACCTACGGAAACCTTGTTAC
4) fermentation condition
Seed culture medium is NA liquid nutrient medium, and the initial pH that ferments is that 7.5,250mL triangular flask bottling amount is 150mL, 32 DEG C of shaking table revolutions are 150r/min, fermentation time is 24h, is inoculated in wheat bran and cultivates after 72h with the inoculum size of massfraction 10%, naturally dries to moisture content and is less than 30%.
The production of embodiment 4, Antagonistic Fungi microbial inoculum
The Antagonistic Fungi QJ-1 of purifying and Ty-3 are inoculated into respectively on NA slant medium, in the incubator of 32 DEG C, cultivate 24h, get 5mL sterilized water contra bevel, vibration gently, then accurately drawing lmL bacteria suspension joins in the 250mL triangular flask that the aseptic NA liquid nutrient medium of 150mL is housed, 1d is cultivated in the shaking bath concussion that is put into 32 DEG C, then cultured bacteria suspension is joined respectively according to the inoculum size of massfraction 10% in the wheat bran of sterilizing, cultivate 3d, the air-dry single bacteria agent that is QJ-1 and Ty-3 for 32 DEG C.In single bacteria agent, QJ-1 and Ty-3 reach respectively 10
8cfu/g, then mixes two kinds of single bacteria agents in the ratio of quality 1:1, be Antagonistic Fungi microbial inoculum.
The production of embodiment 5, microbial organic fertilizer
By the inoculum size of massfraction 10%, Antagonistic Fungi microbial inoculum is added in decomposed manure equably and mixed, regulate moisture to 50-60%, pile the stacking of 80cm, after the 5d that banks up and get final product.
The preparation method of decomposed manure: feces of livestock and poultry regulates moisture to 50-60%, piles 1.1m high, turning every day, compost temperature raises gradually, and when temperature drops to while approaching room temperature, fermentation ends, is the fertilizer becoming thoroughly decomposed.
Experimental example 1, the effect analysis of control tobacco bacterial wilt
1, within 2012, carry out field experiment in cigarette district, Shaowu City, Fujian Province
1) fertilizing time:
The present invention reaches the object that suppresses bacterial wilt by administered twice microbial organic fertilizer.For the first time, microbial organic fertilizer is applied with base manure form, probiotics in soil just can be rolled up in the early stage of cigarette strain growth, thereby suppress the growth of pathogenic bacteria in soil, its quantity is greatly reduced.For the second time, be about to apply with the form of topdress (high density bacterial manure: water=1:10) while generation in local bacterial wilt, utilize the amount reproduction of probiotics to resist the Growth and reproduction of ralstonia solanacearum.
2) for examination soil and block design:
Soil is sand loam, and physical features is smooth, and irrigation and drainage are convenient, and basic fertility is: organic 40.6g/kg, alkali-hydrolyzable nitrogen 198.5mg/kg, rapid available phosphorus (P
2o
5) 20.52mg/kg, available potassium (K
2o) 80.43mg/kg, pH is 5.52.Be K326 for examination flue-cured tobacco cultivars.Adopt floating seedlings technology to grow seedlings, transplant with seedling transplantation technique under film, after transplanting, manage by the requirement of High Quality Tobacco production specifications.Three repetitions of each processing, the cultivation technique measure of each community, bookkeeping require to be substantially consistent.
Test arranges four processing altogether:
Process A: contrast, local conventional fertilizer application and management;
Treatments B: microbial organic fertilizer applies with base manure (250g/ strain) form;
Process C: microbial organic fertilizer applies with base manure (200g/ strain)+(the filling with root 50g/ strain) form of topdressing;
Process D: microbial organic fertilizer applies with (the filling with root 50g/ strain) form of topdressing.
3) researching determining project
Between local bacterial wilt period of disease, date, sickness rate and the onset speed of just morbidity of observed and recorded different treatment bacterial wilt also calculates disease index, and from morbidity, record once weekly; Severity Scaling standard is undertaken by national tobacco industry tobacco diseases investigation grade scale Yc/T39-1996, bacterial wilt investigation method: 0 grade, complete stool is anosis; 1 grade, the even an IOU issued by a post office spot that moves back of stem, or (with) slightly wilting with lower blade or top, or there is scab in bottom minority blade; 2 grades, there is obvious black streak in stem but does not reach cigarette strain top, or (with) sick side is more than half or part waist leaf is wilting with blade; 3 grades, stem's black streak arrives cigarette strain top, or (with) sick lateral lobe sheet is all wilting, or the most of blade of complete stool is wilting; 4 grades, diseased plant is withered.Disease index and prevention effect are pressed tobacco diseases test of pesticide effectiveness method Yc/T40-1996 and are calculated:
Cigarette district, Shaowu City, Fujian Province field experiment result is as shown in table 1.As can be seen from Table 1, use microbial organic fertilizer of the present invention, the sickness rate of tobacco bacterial wilt has reduced more than 98%, and disease index obviously declines, and illustrates that microbial organic fertilizer of the present invention has the effect of good control tobacco bacterial wilt.
Table 1 Shaowu, Fujian Province field experiment in 2012 is prevented and treated bacterial wilt effect
| Process | A | B | C | D |
| Sickness rate | 71.95% | 3.11% | 1.21% | 4.34% |
| Disease index | 33.83% | 0.98% | 0.67% | 1.34% |
The yield of tobacco of experimental example 2, tobacco and mass analysis
The tobacco of Shaowu, Fujian Province field experiment in 2012 in experimental example 1 is carried out to yield of tobacco and mass analysis, and result is as table 2.
Tobacco smoke leaf-making quantity and the mass analysis of table 2 Shaowu, Fujian Province field experiment in 2012
As can be seen from Table 2, use after microbial organic fertilizer of the present invention, the Quality and yield of tobacco tobacco leaf is all greatly improved.
Claims (3)
1. control tobacco bacterial wilt Antagonistic Fungi, is characterized in that, described Antagonistic Fungi be bacillus cereus (
bacillus cereus) QJ-1 and aspergillus niger (
aspergillus niger) Ty-3; Wherein, bacillus cereus (
bacillus cereus) QJ-1, depositary institution: Chinese Typical Representative culture collection center, preservation address: Wuhan, China Wuhan University, preservation date: on July 5th, 2012, preserving number: CCTCC NO:M2012271; Described aspergillus niger (
aspergillus niger) Ty-3, depositary institution: Chinese Typical Representative culture collection center, preservation address: Wuhan, China Wuhan University, preservation date: on July 8th, 2011, preserving number: CCTCC NO:M2011241.
2. the application of control tobacco bacterial wilt Antagonistic Fungi as claimed in claim 1 aspect microbiobacterial agent.
3. the application of control tobacco bacterial wilt Antagonistic Fungi as claimed in claim 1 aspect microbial organic fertilizer.
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