CN105969686A - Bacterial-wilt-resistant Streptomyces rochei strain and bio-organic fertilizer thereof - Google Patents

Bacterial-wilt-resistant Streptomyces rochei strain and bio-organic fertilizer thereof Download PDF

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CN105969686A
CN105969686A CN201610329907.1A CN201610329907A CN105969686A CN 105969686 A CN105969686 A CN 105969686A CN 201610329907 A CN201610329907 A CN 201610329907A CN 105969686 A CN105969686 A CN 105969686A
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strain
bacterial
wilt
bio
fertilizer
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石俊雄
刘艳霞
李想
孟琳
蔡刘体
张恒
邹焱
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GUIZHOU JILONG ECOLOGY TECHNOLOGY CO LTD
Guizhou Institute of Tobacco Science
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F3/00—Fertilisers from human or animal excrements, e.g. manure
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00—Technologies for solid waste management
    • Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse

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  • Organic Chemistry (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
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  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
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  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Fertilizers (AREA)

Abstract

The invention discloses a bacterial-wilt-resistant antagonistic bacterium strain. The strain is named Streptomyces rochei L-9, and was collected by China General Microbiological Culture Collection Center on September 25th, 2013; and the collection number is CGMCC No.8264. The strain L-9 has high bacterial wilt resistance, and the antagonistic ring radius reaches 15mm. The invention provides a new microbial resource for biological control on bacterial wilt, and overcomes the defects in the prior art.

Description

The Lou Che Shi streptomycete bacterial strain of a kind of resistance to bacterial wilt and biological organic fertilizer thereof
Technical field
The invention belongs to biological technical field, particularly to the Antagonistic Fungi of a kind of resistance to bacterial wilt, be specifically related to a kind of Lou thorough Family name streptomycete (Streptomyces rochei) L-9 bacterial strain, relates to the application in terms of resistance to bacterial wilt of this bacterial strain simultaneously.
Background technology
Tobacco bacterial wilt is one of Major Diseases of tropical and subtropical region Nicotiana tabacum L., at present in Yangtze River in China basin and On the south cigarette district generally occur, wherein with Guangdong, Fujian, Taiwan, Hunan, Jiangxi, east Guangxi, Southern Anhui Province, Sichuan and Guizhou Locally cigarette district causes harm seriously, usually outbreak of epidemic of indivedual times, causes crushing loss.In recent years, the morbidity of this disease and be Evil scope has the trend of north cigarette district extension, and in Shandong, Henan, Shaanxi and Liaoning etc. save has generation, some areas to cause harm Heavier.The loss that this disease causes at present has occupied the 4th of all kinds of disease.The conventional strategy preventing and treating bacterial wilt include chemical pesticide, Soil improvement, disease-resistant variety, grafting and crop rotation etc., but effect is the most unstable.Additionally, excess uses the environment that chemical pesticide brings Pollute and food-safety problem, cause people's extensive concern and worry.Therefore, how development environment friendly prophylactic measures, Realize reducing pesticide dosage, suppression disease develops, recovery soil health is the problem needing solution at present badly.
Summary of the invention
The technical problem to be solved is to provide Lou Che Shi streptomycete bacterial strain and the biology thereof of a kind of resistance to bacterial wilt Fertilizer, for the microbial resources that the Biological control offer of bacterial wilt is new.
The Antagonistic Fungi of resistance to bacterial wilt of the present invention, this Strain Designation is Lou Che Shi streptomycete (Streptomyces Rochei) L-9, was deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 09 25th, 2013 (Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number is CGMCC No.8264.
The bacterial strain L-9 of the present invention has the property that
1, L-9 is on Gause I solid medium, and mycelia is flourishing, and milky bit brownish, center is black, and gas silk is rich Cyclopentadienyl, velvet-like, Lycoperdon polymorphum Vitt.Base silk is colourless, and after switching, slightly blush is adjusted sometimes.
2, L-9 shows the performance of stronger antagonism Ralstonia solanacearum, and antagonism circle radius reaches 15mm.
3, bacterial genomes DNA small volume of reagent box (AxyPrep, the U.S.) high salt precipitation method is used to extract DNA.PCR expands Universal primer is 27F/1541R.I.e. forward primer: 5 '-GAGAGTTTGATCCTGGCTCAG-3 '；Reverse primer: 5 '- AAGGAGGTGATCCA GCCGCA-3′.16S rRNA gene order sequencing result shows, the 16S rRNA gene order of L-9 is 1488bp, the sequence Genbank in NCBI website online sequence alignment result shows, L-9 Yu Streptomyces rochei's Homology, more than 99%, is accredited as Lou Che Shi streptomycete according to morphological characteristic and 16S rDNA.
The bacterial strain of the present invention is accessed the rapeseed cake fertilizer of rapeseed cake enzymolysis with cow dung compost with weight with the inoculation weight of 5% Ratio fermentation culture in the fertilizer that 1:1 mixes, fermentation culture conditions is: temperature 30～45 DEG C, water content 30～40%, time Between 4～5 days, rear water content of having fermented reaches 34.9%, is biological organic fertilizer.Bacterial wilt is had substantially by this biological organic fertilizer Prevention effect.
Accompanying drawing explanation
Accompanying drawing 1 is the flat board antagonistic effect of Antagonistic Fungi L-9；
Accompanying drawing 2 is that the system that 16S rRNA gene order based on isolated strains and the close bacterial strain of its sibship builds is sent out Educate tree (adjacent method)；
Accompanying drawing 3 is the quantity of Ralstonia solanacearum in potting media；
Accompanying drawing 4 is the quantity of Antagonistic Fungi L-9 in potting media.
Detailed description of the invention
The separation of embodiment 1 Antagonistic Fungi of the present invention L-9 and qualification
The separation of 1.1 Antagonistic Fungi L-9 and screening
Gather, from many places, Guizhou Province tobacco bacterial wilt morbidity field, the cigarette strain and Rhizosphere Soil, cryopreservation do not fallen ill, be used for Separate Antagonistic Fungi.Process to cigarette strain sample: take rhizome tissue, first with 2% sodium hypochlorite surface sterilization 2min, then use Deionization washing rinse 3～5 times after sterilizing.To pedotheque or or process after cigarette strain sample, according to dilution spread flat board The soil liquid or Plant samples solution are diluted to 10 by method-1～10-6Stand-by.
The soil liquid or Plant samples solution are coated in Gause I culture medium uniformly, at 30 DEG C of dark conditions Under, after cultivating 24h, after bacterium colony grows, Stalk Rot Ralstonia solanacearum bacteria suspension is uniformly sprayed on flat On plate, carry out cultivating and observe.Separating from soil and cigarette strain, screen 219 strain antagonistic strains, its antagonism circle of comprehensive assessment is big The indexs of correlation such as growing state under little, the speed of growth, lean nutrition, whether salt tolerant, finally choose a strain Antagonistic Fungi L-9.Often Rule qualification result shows, L-9 is on Gause I solid medium, and mycelia is flourishing, milky bit brownish, and center is black, Gas silk is luxuriant, velvet-like, Lycoperdon polymorphum Vitt.Base silk is colourless, and after switching, slightly blush is adjusted sometimes.L-9 shows stronger antagonism Ralstonia solanacearum Performance, antagonism circle radius reaches 15mm.
The 16S rRNA gene identification of 1.2 Antagonistic Fungis and gene sequencing
Bacterial genomes DNA small volume of reagent box (AxyPrep, the U.S.) high salt precipitation method is used to extract DNA.PCR amplification is logical It is 27F/1541R with primer.I.e. forward primer: 5 '-GAGAGTTTGATCCTGGCTCAG-3 '；Reverse primer: 5 '- AAGGAGGTGATCCA GCCGCA-3′。
16S rRNA gene order sequencing result shows, the 16S rRNA gene order of L-9 is 1488bp, and sequence is at NCBI The Genbank online sequence alignment result of website shows, the homology of L-9 Yu Streptomyces rochei more than 99%, It is accredited as Lou Che Shi streptomycete (referring to Fig. 2) according to morphological characteristic and 16S rDNA.
1.3 bacterial strain L-9 production methods
1.3.1 prepared by bacterial strain primary seed solution
L-9 is in Gause I fluid medium, and in 30 DEG C of shaking tables, 170rpm cultivates 48h, makes primary seed solution.
1.3.2 function strain L-9 bacterial strain first order seed amplification culture
Test through repeatedly first order seed lab scale amplification culture, excellent to L-9 bacterial strain first order seed amplification culture technical parameter Change, preliminarily form following scale up test technical parameter.
Culture medium prescription: PDA improved culture medium 1；
Fermentation pH: before disappearing 7.2, after disappearing 7.0；
Inoculation weight: 1%；
Cultivation temperature: 37 degree；
First order seed rotating speed: 160～170 revs/min；
Incubation time: 16～18h；
Bacteria containing amount: 2～3 × 108cfu/mL。
1.3.3 function strain L-9 bacterial strain secondary seed amplification culture
Test through repeatedly secondary seed lab scale amplification culture, L-9 bacterial strain secondary seed amplification culture technical parameter optimized, Preliminarily form following scale up test technical parameter.
Culture medium prescription: No. 2+0.1% defoamer of PDA improved culture medium；
Cultivate pH: before disappearing 7.2, after disappearing 7.0；
Inoculation weight: 5%；
Cultivation temperature: 35 DEG C；
Two grades of cultivation rotating speeds: 45～50Hz；
Ventilation: 35～40m3/h；
Pressure: 0.045～0.05MPa；
Dissolved oxygen: 80～85%；
Incubation time: 17～18h；
Bacteria containing amount: 1～2 × 109cfu/mL。
1.3.4 function strain L-9 bacterial strain 500L amplification culture
Test through repeatedly amplification culture lab scale, L-9 bacterial strain amplification culture technical parameter is optimized, preliminarily forms following pilot scale Technological production norm.
Culture medium prescription: No. 3+0.15% defoamer of PDA improved culture medium；
Fermentation pH: before disappearing 7.2, after disappearing 7.0；
Inoculation weight: 10%；
Cultivation temperature: 32 DEG C；
Amplification culture rotating speed: 75～80Hz；
Ventilation: 60～80m3/h；
Pressure: 0.045～0.05MPa；
Dissolved oxygen: early stage 60～75%, mid-term 90～100%, the later stage 75～100%；
Incubation time: 24～27h；
Bacteria containing amount: 4～5 × 109Cfu/mL (spore rate >=90%).
The preparation of embodiment 2 biological organic fertilizer of the present invention
Cultivating Antagonistic Fungi L-9 in Gause I fluid medium, in 30 DEG C of shaking tables, 170rpm cultivates 48h.Organic The fertile rapeseed cake fertilizer for rapeseed cake enzymolysis mixes with weight ratio 1:1 with cow dung compost, after measured containing organic matter 33.8%, ammonia Base acid 4.3%, N4.20%, P2O52.26%, K2O1.08%.Antagonistic Fungi L-9 culture fluid is accessed with the inoculation weight of 5% Stating in fertilizer, regulation temperature is 30～45 DEG C, and water content is 30～40%, ferments 4～5 days, and period constantly turns over throwing.Ferment After one-tenth, water content reaches 34.9%, is biological organic fertilizer (hereinafter referred to as " BIO ").
Embodiment 3 BIO effect of resistance to bacterial wilt in seedling growth test
Experimental cultivar: the big gold dollar of K326 Flos Carthami.
Earthworm excrement seedling medium: preparation method is shown in patent ZL200710202167.6.
3.1 EXPERIMENTAL DESIGN
3.1.1 the BIO impact on Nicotiana tabacum L. germination percentage
Every 100 K326 seeds of flat board or Flos Carthami big gold dollar seed, substrate is added different proportion by conventional seedbed system substrate Earthworm excrement seedling medium and BIO form, and process and are shown in Table 1.1, and about paving 1cm is thick, and each process repeats for 5 times, in illumination box 25 DEG C, 75% moisture content is cultivated, results of regular determination bud ratio.
Tobacco seed is sprouted and is affected EXPERIMENTAL DESIGN by table 1.1 BIO
3.1.2 the impact that tobacco in seedling stage is grown by BIO
The result affected Nicotiana tabacum L. germination percentage according to BIO, using to sprout Nicotiana tabacum L. does not has ratio (the i.e. earthworm excrement of inhibitory action Seedling medium 68%, BIO 2%) apply Nicotiana tabacum L. conventional seedbed system substrate.Being respectively adopted two kinds of method for culturing seedlings, a kind of is conventional drift Floating nursery, another kind of in view of the growth and breeding problem of Antagonistic Fungi in BIO, use black plastic seedlings nursing plate educating relative to floating Nursery under conditions of Seedling is more arid, is i.e. directly placed in black plastic seedlings nursing plate in nursery groove, and about 1cm is only saved in bottom Water.Test processes and is shown in Table 1.2.
Table 1.2 BIO nursery processes
3.1.3 the BIO impact on tobacco seedling growth in pot experiment
Process by table 1.3 transplants in new seedling medium by tobacco seedlings, every basin 10Kg seedling medium, often processes 12 weights Multiple.In seedling medium, add a certain proportion of Ralstonia solanacearum bacteria suspension, BIO or rapeseed cake fertilizer fermented, make Rs Concentration reaches 107cfu g-1Substrate, and BIO and rapeseed cake fertilizer add with the ratio of every basin 50g respectively.Respectively with nursery stage pair Compare substrate-pathogen substrate two according to substrate-transplant health substrate and nursery stage to process as comparison.Transplant and gathered the same day Different substrates sample, gathered a rhizosphere substrate every 10 days and adds up sickness rate, measures base with fluorescence quantifying PCR method later Pathogen and the quantity of Antagonistic Fungi in quality sample.
The potted plant process of table 1.3 BIO nursery
Note: be process during nursery before "-".Being the process after transplanting after "-", wherein, CK represents that blank processes, n Representing that substrate seedling processes, p represents potted plant process；+ Rs represents addition Ralstonia solanacearum in soil；OF refers to not add antagonism The fertilizer of bacterium L-9.
3.2 results:
3.2.1 the BIO impact on Nicotiana tabacum L. germination percentage
Bud ratio testing result is shown in Table 2.1.Compared with the control, when BIO concentration is within 2%, the germination percentage of Nicotiana tabacum L. with Comparison is compared, without significant difference；When BIO concentration is higher than 2%, germination percentage is substantially reduced.
Table 2.1 BIO impact on Tobacco Seed Germination rate
Process CK T1 T2 T3 T4 T5
Germination percentage (%) 97.3 93.3 95.6 68.3 53.7 53.3
3.2.2 BIO nursery passes the impact of bacterial wilt sickness rate to Nicotiana tabacum L. germination percentage and soil
Tobacco seedlings is transplanted the sickness rate of latter 30 days statistics pot experiments and is referred to table 2.2.From table 2.2 it can be seen that K326 green grass or young crops is withered Sick sickness rate is the most relatively low, the most only investigates in the substrate of the big gold dollar of Flos Carthami pathogen and Antagonistic Fungi in the change of different times Change.
Table 2.2 different disposal passes the impact of bacterial wilt sickness rate to Nicotiana tabacum L. soil
30 days sampling results from Fig. 3 it can be seen that BIO2-BIOp+Rs HD (i.e. drought cultivates Seedling, nursery and potted plant disease Soil all applies BIO) process relative to other, pathogen quantity can be preferably minimized.This result is likely due to nursery Period Antagonistic Fungi is grown at tobacco seedlings rhizosphere the most surely, and therefore, in the presence of having pathogen in pot experiment substrate, Antagonistic Fungi is in the same old way Can play a role, the quantity of pathogen can be reduced from the beginning, play the effect of protection cigarette strain.
From fig. 4, it can be seen that Antagonistic Fungi also can present a kind of trend first dropping and rising afterwards in substrate.And relative to traditional Floating seedlings, the Antagonistic Fungi of drought training is more easy to existence, and therefore after tobacco seedlings is transplanted, the quantity of its Antagonistic Fungi is also most.During nursery Just adding Antagonistic Fungi, add the process of Antagonistic Fungi than after tobacco seedlings is transplanted, Antagonistic Fungi can enter build phase quickly, by This ensure that Antagonistic Fungi quantity in substrate, so provides for sound assurance for antagonism pathogen.

Claims (6)

1. an Antagonistic Fungi for resistance to bacterial wilt, this Strain Designation is Lou Che Shi streptomycete (Streptomyces rochei) L-9, Being deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 09 25th, 2013, deposit number is CGMCC No.8264。
The bacterial strain the most according to claim 1 application in resistance to bacterial wilt.
3. the preparation method of a biological organic fertilizer, it is characterised in that: by the bacterial strain described in claim 1 with 5% inoculation weight Amount accesses fermentation culture in fertilizer.
Preparation method the most according to claim 3, it is characterised in that: described fertilizer is the rapeseed cake fertilizer of rapeseed cake enzymolysis Mix with weight ratio 1:1 with cow dung compost.
Preparation method the most according to claim 3, it is characterised in that described fermentation culture conditions is: temperature 30～45 DEG C, Water content 30～40%, time 4～5 days, rear water content of having fermented reaches 34.9%.
6. the biological organic fertilizer prepared according to method described in claim 3～5 any claim application in resistance to bacterial wilt.
CN201610329907.1A 2016-05-18 2016-05-18 Bacterial-wilt-resistant Streptomyces rochei strain and bio-organic fertilizer thereof Withdrawn CN105969686A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108300681A (en) * 2018-04-20 2018-07-20 海南大学 One plant of Lou Che Shi streptomycete and its application
CN109874808A (en) * 2019-01-25 2019-06-14 青岛农业大学 The application of arbuscular mycorrhizal fungi and/or plant symbiosis actinomyces in the biological prevention and control agent that preparation is used for capsicum and eggplant
CN114456973A (en) * 2022-01-11 2022-05-10 湖北省生物农药工程研究中心 Streptomyces rochei strain in tobacco and application thereof in prevention and control of tobacco diseases

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CN105309082A (en) * 2015-10-21 2016-02-10 贵州省烟草科学研究院 Comprehensive preventing and controlling method for tobacco bacterial wilt

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CN105309082A (en) * 2015-10-21 2016-02-10 贵州省烟草科学研究院 Comprehensive preventing and controlling method for tobacco bacterial wilt

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YANXIA LIU等: "Tobacco bacterial wilt can be biologically controlled by the application of antagonistic strains in combination with organic fertilizer", 《BIOL. FERTIL. SOILS.》 *
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108300681A (en) * 2018-04-20 2018-07-20 海南大学 One plant of Lou Che Shi streptomycete and its application
CN108300681B (en) * 2018-04-20 2021-07-23 海南大学 Streptomyces rochei and application thereof
CN109874808A (en) * 2019-01-25 2019-06-14 青岛农业大学 The application of arbuscular mycorrhizal fungi and/or plant symbiosis actinomyces in the biological prevention and control agent that preparation is used for capsicum and eggplant
CN109874808B (en) * 2019-01-25 2021-02-02 青岛农业大学 Application of arbuscular mycorrhizal fungi and/or plant symbiotic actinomycetes in preparation of biocontrol preparation for hot pepper and solanaceae vegetables
CN114456973A (en) * 2022-01-11 2022-05-10 湖北省生物农药工程研究中心 Streptomyces rochei strain in tobacco and application thereof in prevention and control of tobacco diseases
CN114456973B (en) * 2022-01-11 2022-08-05 湖北省生物农药工程研究中心 Streptomyces rochei in tobacco and application thereof in prevention and control of tobacco diseases

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Application publication date: 20160928