CN109554317A - Bei Laisi bacillus and its application in wheat sharp eyespot disease prevention growth-promoting - Google Patents

Bei Laisi bacillus and its application in wheat sharp eyespot disease prevention growth-promoting Download PDF

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CN109554317A
CN109554317A CN201811635012.6A CN201811635012A CN109554317A CN 109554317 A CN109554317 A CN 109554317A CN 201811635012 A CN201811635012 A CN 201811635012A CN 109554317 A CN109554317 A CN 109554317A
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bei laisi
laisi bacillus
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wheat
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CN109554317B (en
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安超
薛文娇
马赛箭
刘晨
丁浩
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SHAANXI PROVINCE INSTITUTE OF MICROBIOLOGY
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Abstract

The present invention relates to Bei Laisi bacillus and its application in wheat sharp eyespot disease prevention growth-promoting, the strain be Bei Laisi bacillus (Bacillus velezensis) M6, it is preserved in China General Microbiological culture presevation administrative center, its deposit number is CGMCC No.15628, it can generate including growth-promotings substances such as heteroauxin (IAA), thermophilic iron elements, simultaneously, the bacterial strain also has significant dissolved organic phosphorus effect, in addition, the bacterium to by Rhizoctonia solani Kuhn (Rhizoctoniasolani) caused by wheat sharp eyespot have preferable control efficiency.

Description

Bei Laisi bacillus and its application in wheat sharp eyespot disease prevention growth-promoting
Technical field
The present invention relates to a kind of Bei Laisi bacillus, and in particular to a kind of Bei Laisi bacillus and its withered in wheat line Application in sick disease prevention growth-promoting.
Background technique
Fungal diseases of plants is prevalent in grain and vegetable crop, has been caused by fungus-caused cereal crops disease Global Food Security, if can control propagation of the fungal disease in staple food crop, in the world every year 1.25 hundred million tons are reduced, 60,000,000,000 or more economic loss is reduced.Wheat is a kind of grass family grain work planted extensively all over the world Object, total output occupy the second in the world, there is very important status, wheat in Food Security system and agricultural production Fungal disease have very much, including banded sclerotial blight, root rot, full rot, head blight, powdery mildew and stripe rust etc. are according to statistics, small Wheat disease can generally cause wheat yield 10%~15%, serious to cause 30% or more wheat yield.
Currently, lack effective disease-resistant variety for wheat sharp eyespot, therefore, the prevention and treatment for wheat sharp eyespot be still with Based on chemical prevention, however, a large amount of use of chemical pesticide causes environmental pollution, Practice for Pesticide Residue in Agricultural Products and germ drug resistance It generates.As people are to the further attention of ecological environment and the needs of sustainable agriculture development, life efficiently, less toxic is found Object pesticide has become a kind of trend.Meanwhile as the large area of chemical fertilizer is largely applied, although the yield of crops obtains It significantly improves, but exceedingly equally also brings some environmental problems using chemical fertilizer, it is especially raw to soil and water source State system causes eutrophication etc., therefore, promotes plant growth, improves to be also not to the utilization rate of fertilizer using microbial manure The development trend come.
Bei Laisi bacillus (Bacillus velezensis) after 2008 are confirmed as bacillus amyloliquefaciens Phase obform body is a novel species resource.It is more for the research of Bei Laisi bacillus at present, it is concentrated mainly on using the bacterium Kind carries out the research of biological control, but does not report that the strain can generate heteroauxin (IAA), thermophilic iron element growth-promoting substance, Also being not directed to Bei Laisi bacillus has effects that dissolved organic phosphorus.
Summary of the invention
The object of the present invention is to provide a kind of with Bei Laisi bacillus and its in wheat sharp eyespot disease prevention growth-promoting Using, which can generate including growth-promotings substances such as heteroauxin (IAA), thermophilic iron elements, meanwhile, which also has significantly Dissolved organic phosphorus effect, in addition, the bacterium to by Rhizoctonia solani Kuhn (Rhizoctoniasolani) caused by wheat sharp eyespot have Preferable control efficiency.
The technical scheme adopted by the invention is as follows:
Bei Laisi bacillus, it is characterised in that:
The strain be Bei Laisi bacillus (Bacillus velezensis) M6, it is preserved in China General Microbiological bacterium Kind preservation administrative center, deposit number are CGMCC No.15628.
The Bei Laisi bacillus can dissolve the phosphorus in soil, be used to prepare soil conditioner.
The Bei Laisi bacillus is used to prepare bio-fertilizer, promotes the effective use of P elements in soil.
The Bei Laisi bacillus can generate plant growth hormone, be used to prepare growth-promoting agent.
The Bei Laisi bacillus can generate thermophilic iron element, be used to prepare growth-promoting agent.
The Bei Laisi bacillus can significantly inhibit by Rhizoctonia solani Kuhn (Rhizoctoniasolani) caused by it is small Wheat banded sclerotial blight, as wheat sharp eyespot resistive connection bacterium.
The Bei Laisi bacillus is used as wheat sharp eyespot resistive connection bacterium, is used to prepare wheat planting biological organic Fertilizer.
The invention has the following advantages that
Bei Laisi bacillus M6 of the invention is to separate to obtain from facility fruits and vegetables Orchard Soil, and no eco-toxicity is highly-safe； The bacterium can be generated during the growth process including growth-promotings substances such as somatotropin (IAA), thermophilic iron elements, while having dissolution phosphorus and suppression The effect of wheat sharp eyespot processed.Therefore, the effective use of P elements in soil can be promoted by preparing bio-fertilizer using the bacterial strain, simultaneously The IAA for promoting plant growth and thermophilic iron element can be generated, in addition, the microorganism is to by Rhizoctonia solani Kuhn (Rhizoctoniasolani) caused by wheat sharp eyespot have preferable inhibitory effect, therefore, the bacterium is in wheat biological organic Having a extensive future in fertilizer and biological pesticide.
Detailed description of the invention
Fig. 1 be Bei Laisi bacillus (Bacillus velezensisi) M6 CGMCC No.15268 bacterium colony photo And microphoto.Wherein, A is bacterium colony photo, and B is microphoto.
Fig. 2 is Bei Laisi bacillus systematic evolution tree.
Fig. 3 is that Bei Laisi bacillus generates thermophilic iron plain (periphery of bacterial colonies has transparent circle generation), IAA, dissolved organic phosphorus Experiment (compared to the control group, experimental group culture medium color disappears).Wherein, A: protease；B: thermophilic iron element；C: IAA is produced；D: it is dissolved with Machine phosphorus.
Fig. 4 is Inhibition test of the Bei Laisi bacillus to wheat sharp eyespot.Wherein, A: plate experiment；B: fermentation liquid Organize in vitro efficiency test.
Fig. 5 is experiment of the Bei Laisi bacillus to wheat growth-promoting.Wherein, A: growth-promoting effect picture；B: growth-promoting data.
Specific embodiment
The present invention will be described in detail With reference to embodiment.
The present invention relates to a kind of Bei Laisi bacillus, the strain be Bei Laisi bacillus (Bacillus velezensis) M6, it is located away from the facility orchard soil of Shaanxi Province Baqiao District, is preserved on April 19th, 2018 Chinese common Microbiological Culture Collection administrative center, deposit number are CGMCC No.15628.The bacterium has effects that promotion plant growth, And can be applied in wheat sharp eyespot prevention and treatment, specifically:
1, the strain can dissolve organic phosphorus in soil, be used to prepare soil conditioner；It is used to prepare bio-fertilizer, is promoted in soil The effective use of P elements.
2, the strain can generate plant growth hormone (IAA) and thermophilic iron element, be used to prepare growth-promoting agent.
3, the strain can significantly inhibit by Rhizoctonia solani Kuhn (Rhizoctoniasolani) caused by wheat sharp eyespot, make For wheat sharp eyespot resistive connection bacterium, it is used to prepare wheat planting biological organic fertilizer.
Therefore, the bacterium is without eco-toxicity, and highly-safe, the application prospect in wheat biological organic fertilizer and biological pesticide is wide It is wealthy.In addition, the bacterium can generate the strong gemma of resistance, can compound to form stable commercial preparation product with organic fertilizer etc..
1: Bei Laisi bacillus of embodiment (Bacillus velezensisi) M6 separation, screening and identification:
(1) separation and screening: from the facility plastic greenhouse of Shaanxi Province Baqiao District, soil is acquired, is taken back with freshness protection package；
It weighs soil sample 10g, is added in the triangular flask equipped with 90mL sterile water, be equipped with bead 30 in triangular flask, 37 DEG C, 180rpm shakes 30min, obtains soil supension after standing；
Soil supension is diluted to 10-5、10-6、10-7, the 100 μ L of soil supension for drawing different dilutions is coated on NA culture medium On, 37 DEG C of 24 h of culture are placed in, the different bacterium single colonie purifying such as picking color, form saves；
Using the tablet face-off method bacterium that is separated to of measurement to the bacteriostatic activity of plant pathogenic fungi.By Botrytis cinerea germ, kind Solanum cinerea bacterium and botrytis cinerea pers activate 3d on PDA plate respectively, are beaten with punch in colony edge and take fungus block (diameter 5mm), it is inoculated into PDA plate center.After cultivating 1d, it is inoculated at 2 symmetric points at gray mold fungus block 2cm to be measured thin Bacterium, 25 DEG C of cultures, observes antibacterial result at any time.It is calculated using following equation:
Bacteriostasis rate (%)=(respectively compare colony radius-and respectively handle colony radius)/respectively compares colony radius × 100%.
Test result is as shown in Figure 2 and Table 1, it can be seen that: M6 bacterial strain has preferable antagonism to various ash arrhizus bacterias Effect.
(2) it identifies: the preferable M6 bacterial strain of the fungistatic effect screened is subjected to morphology and 16S rDNA Sequence Identification.
Morphological observation: by the streak inoculation on NA culture medium flat plate of M6 bacterial strain, 37 DEG C of culture 18h, observation bacterium colony is special Sign.Bacterium colony is white, translucent, slightly protuberance, round flat, smooth wet toughness (see figure 1).
16S rDNA sequencing: TaKaRa MiniBEST Universal Genomic DNA Extraction is used Kit extracts the genomic DNA of M6 bacterial strain, according to bacterial universal primers 27F(5 '-AGAGTTTGATCCTGGCTCAG-3 ') and 1492R(5 '-GGTTACCTTGTTACGACTT-3 ') carry out PCR amplification.
Pcr amplification reaction program are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 54 DEG C of annealing 30s, 72 DEG C extend Lmin30s, totally 30 recycle；Last 72 DEG C of extensions 1Omin.
Pcr amplification product is sent to Hua Da gene Co., Ltd and is sequenced.Through being sequenced, the 16S rDNA sequence of M6 bacterial strain Length is 1458bp, and sequence is SEQ ID NO:1.
Sequencing result is subjected to sequence alignment on NCBI GenBank, finds itself and Bei Laisi bacillus (Bacillus velezersis FZB42 16S rDNA sequence similarity) is up to 99%, primarily determines that sw17 bacterial strain is shellfish Lay this bacillus (Bacillus velezersi).Clustering is carried out using MEGA4.1 software, constructs chadogram.Such as Fig. 2 It is shown, the results showed that, M6 withBacillus velezersiFZB42 is in a subbranch.Combining form feature as a result, into One step illustrates that M6 bacterial strain is Bei Laisi bacillus.
2: Bei Laisi bacillus of embodiment (Bacillus velezensisi) M6 fermentation liquid is to rhizoctonia cerealis Preventive effect evaluation:
Embodiment 1 is obtained Bei Laisi bacillus (Bacillus velezensisi) M6 be inoculated in nutrient agar panel training Base is supported, 37 DEG C of incubator cultures are for 24 hours；
The bacterial strain M6 of activation is inoculated in LB liquid medium, 37 DEG C, 230rpm cultivates 18h；
The bacterial strain M6 fermentation liquid of acquisition is removed into thallus under conditions of centrifugal rotational speed 10000rpm, centrifugation 6min, is fermented Liquid supernatant；
Rhizoctonia cerealis activates on PDA plate culture medium, 28 DEG C of condition of culture, cultivates 5d；
The wheat leaf blade for taking growth 10d or so with hypochlorite disinfectant 1min, then uses sterile purified water with after rinsing with ruinning water It rinses 3 times, wraps root with wet absorbent cotton and be placed in plate, smear blade surface with M6 fermentation liquid；
28 DEG C of Wheat Pathogenic Fungi fungus block cultures are inoculated in central vane, observe bacterium colony growing state at any time；
As a result it as can be seen that the processed wheat leaf blade of bacterial strain M6 fermented liquid supernatant can effectively resist infecting for pathogen, reaches To certain protection effect.
3: Bei Laisi bacillus of embodiment (Bacillus velezensisi) M6 growth-promoting Potential Evaluation:
Embodiment 1 is obtained Bei Laisi bacillus (Bacillus velezensisi) M6 be inoculated in nutrient agar panel training Base is supported, 37 DEG C of incubator cultures are for 24 hours；
Produce IAA(heteroauxin) detection: using the colorimetric determination of the descriptions such as Patten, i.e., activated strains are inoculated in and are contained Have in the LB liquid medium of tryptophan, fermented and cultured, takes 1mL supernatant that 4mL Salkowski ' s is added after culture Reagent (the 150mL concentrated sulfuric acid, 250mL water, 7.5mL 0.5M FeCl3 .6H2O), it is placed at room temperature for 20min, observes color Variation, when producing IAA, supernatant reddens color.
Phosphate solubilization detection: Antagonistic Endophytic is inoculated in phosphorus decomposing detection culture medium Pikovskaya`s(PVK), it is placed in 28 DEG C of 2 d of culture.Observation periphery of bacterial colonies transparent circle size can determine whether the phosphorus solvability of bacterial strain.
Produce the detection of thermophilic iron element ability: the generation of the thermophilic iron element of Antagonistic Endophytic detects flat band method, life in activity using CAS Bacterium observes the size of bacterium colony periphery yellow halo after 28 DEG C of 3 d of culture.Due to the EDTA in thermophilic iron element competition culture medium The iron ion of chelating makes culture medium become yellow, therefore the generation for representing thermophilic iron element of periphery of bacterial colonies yellow halo from blue.
It produces protease detection: Antagonistic Endophytic strain being inoculated in non fat dry milk protein enzyme detection culture medium, is placed in 28 DEG C of trainings Support 2 d.Observation periphery of bacterial colonies transparent circle size can determine whether the protein hydrolysis ability of bacterial strain.
4: Bei Laisi bacillus of embodiment (Bacillus velezensisi) M6 is to the pot experiment of wheat growth-promoting:
Growing way 10 days wheat seedlings of consistent germination are selected, is divided into two groups, every group 40 plants, carries out plant height statistics respectively, then move It plants in flowerpot；
By Bei Laisi bacillus (Bacillus velezensisi) M6 is inoculated in nutrient agar plate medium, 37 DEG C of cultures Case culture is for 24 hours；
The bacterial strain M6 of activation is inoculated in LB liquid medium, 37 DEG C, 230rpm cultivates 18h；
The M6 fermentation liquid that the application fermentation of processing group obtains, the fermentation liquid without M6 that control group applies equivalent pours, respectively at 5 days When with 12 days, 20 plants of wheat seedlings is respectively taken to carry out the statistics of plant height；
Compared to control group, the seedling of microbial inoculum M6 fermentation liquid is applied, under ground portion is grown also superior to control group, and its aerial part Branch is more, and growth is very fast, is counted by the growing state to aerial part, when growing 5d, the growth of processing group stem 6.36 ± 0.72cm, and control group has a net increase of long 5.95 ± 0.76cm；When growing 12d, having a net increase of for processing group stem be long by 10.48 ± 1.01cm, and control group has a net increase of a length of 9.87 ± 0.98cm, therefore, bacterial strain M6 has significant growth-promoting functions to wheat seedling.
The contents of the present invention are not limited to cited by embodiment, and those of ordinary skill in the art are by reading description of the invention And to any equivalent transformation that technical solution of the present invention is taken, all are covered by the claims of the invention.
SEQUENCE LISTING
<110>Shaanxi Institute of Microbiology
<120>Bei Laisi bacillus and its application in wheat sharp eyespot disease prevention growth-promoting
<130> 2018
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1442
<212> DNA
<213>Bei Laisi bacillus (Bacillus velezensisi) M6
<400> 1
tgctaataca tgcaagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga 60
cgggtgagta acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg 120
ctaataccgg atggttgttt gaaccgcatg gttcagacat aaaaggtggc ttcggctacc 180
acttacagat ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcga 240
cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac 300
tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg 360
ccgcgtgagt gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtgc 420
cgttcaaata gggcggcacc ttgacggtac ctaaccagaa agccacggct aactacgtgc 480
cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc 540
tcgcaggcgg tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg 600
gaaactgggg aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg 660
cgtagagatg tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg 720
aggagcgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg 780
atgagtgcta agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac 840
tccgcctggg gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca 900
agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat 960
cctctgacaa tcctagagat aggacgtccc cttcgggggc agagtgacag gtggtgcatg 1020
gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg 1080
atcttagttg ccagcattca gttgggcact ctaaggtgac tgccggtgac aaaccggagg 1140
aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca 1200
atggacagaa caaagggcag cgaaaccgcg aggttaagcc aatcccacaa atctgttctc 1260
agttcggatc gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat 1320
cagcatgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccacgaga 1380
gtttgtaaca cccgaagtcg gtgaggtaac cttttaggag ccagccgccg aaggtgacag 1440
ga 1442

Claims (7)

1. Bei Laisi bacillus, it is characterised in that:
The strain be Bei Laisi bacillus (Bacillus velezensis) M6, it is preserved in China General Microbiological bacterium Kind preservation administrative center, deposit number are CGMCC No.15628.
2. the application of Bei Laisi bacillus according to claim 1, it is characterised in that:
The Bei Laisi bacillus can dissolve the phosphorus in soil, be used to prepare soil conditioner.
3. the application of Bei Laisi bacillus according to claim 2, it is characterised in that:
The Bei Laisi bacillus is used to prepare bio-fertilizer, promotes the effective use of P elements in soil.
4. the application of Bei Laisi bacillus according to claim 1, it is characterised in that:
The Bei Laisi bacillus can generate plant growth hormone, be used to prepare growth-promoting agent.
5. the application of Bei Laisi bacillus according to claim 1, it is characterised in that:
The Bei Laisi bacillus can generate thermophilic iron element, be used to prepare growth-promoting agent.
6. the application of Bei Laisi bacillus according to claim 1, it is characterised in that:
The Bei Laisi bacillus can significantly inhibit by Rhizoctonia solani Kuhn (Rhizoctoniasolani) caused by wheat line Blight, as wheat sharp eyespot resistive connection bacterium.
7. the application of Bei Laisi bacillus according to claim 6, it is characterised in that:
The Bei Laisi bacillus is used as wheat sharp eyespot resistive connection bacterium, is used to prepare wheat planting biological organic fertilizer.
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Cited By (7)

* Cited by examiner, † Cited by third party
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CN109868250A (en) * 2019-04-15 2019-06-11 沈阳农业大学 One plant inhibits virus, the Bei Laisi bacillus for promoting plant growth and its application
CN110029080A (en) * 2019-04-08 2019-07-19 陕西省微生物研究所 A kind of nif gene engineering bacteria producing Methyl Parathion Hydrolase and construction method and application
CN111269851A (en) * 2020-01-20 2020-06-12 安徽农业大学 Bacillus belgii and application thereof in wheat take-all prevention and treatment
CN113980836A (en) * 2021-09-23 2022-01-28 中化化肥有限公司临沂农业研发中心 Bacillus belgii and application thereof in prevention and treatment of strawberry root rot
CN114058541A (en) * 2021-11-17 2022-02-18 河北农业大学 Pesticide-resistant and bolas-resistant biocontrol bacillus beiLeisi for wheat sheath blight and application
CN114806931A (en) * 2022-03-31 2022-07-29 河北科技大学 Bacillus belgii YQ-1-8 and application thereof
CN114854650A (en) * 2022-07-05 2022-08-05 烟台泓源生物肥料有限公司 Microbial agent and preparation method thereof

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