CN105907655A - Typhonium giganteum Engl endogenous Bacillus licheniformis with antagonism effects on a plurality of plant pathogenic fungi and separation method thereof - Google Patents
Typhonium giganteum Engl endogenous Bacillus licheniformis with antagonism effects on a plurality of plant pathogenic fungi and separation method thereof Download PDFInfo
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- CN105907655A CN105907655A CN201610117215.0A CN201610117215A CN105907655A CN 105907655 A CN105907655 A CN 105907655A CN 201610117215 A CN201610117215 A CN 201610117215A CN 105907655 A CN105907655 A CN 105907655A
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Abstract
The invention provides a Bacillus licheniformis strain. The Bacillus licheniformis strain has a preservation number of CGMCC No. 9048 and is marked as TG119. The strain is fungal special biocontrol bacterium, can form a flat round-shaped bacterial colony with the irregular edge and the smooth surface on an NA medium, does not produce a pigment, is milky and has a Gram staining positive result. A scanning electron microscope observation result shows that the strain is in a shape of a rod and has the size of (0.7-0.9) microns * (3-6) microns. A morphology-physiological and biochemical index-16S rRNA sequence comprehensive analysis result shows that the TG119 strain is preliminarily identified as Bacillus licheniformis. A test shows that the TG119 strain has good inhibition effects on seven phytopathogens, is a wide spectrum inhibition bacterium and is suitable for development and use of agricultural biocontrol bacteria.
Description
Technical field
The present invention relates to a bacillus licheniformis bacterial strain, particularly relate to one and various plants pathogenic fungi is had antagonism
Raw lichem bacillus strain in the Herba Typhonii gigantei of effect；Raw lichem bacillus strain in present invention simultaneously relates to this Herba Typhonii gigantei
Isolation and purification method.
Background technology
Herba Typhonii gigantei ( Typhonium giganteum Engl) it is China's endemic plant, belongs to Araeceae
(Araceae), Typhonium Schott (Tyhonium), its dry tuber is used as medical material, is frequently referred to " Rhizoma Typhonium Gigantei (Rhizoma Typhonii) "；Its chemical composition
Enrich and pharmacological action is extensive, there is antimicrobial antiphlogistic, antitumor, tetanus toxin, immunomodulating, convulsion, calmness, dispel the wind
The effects such as expectorant；And Herba Typhonii gigantei infects pest and disease damage in growth course hardly, can produce and host according to medicinal plants endophyte
The most identical similar active substance, and some active substance may participate in plant antibacterial during, thus it is speculated that with only angle
It is unique that the endophyte of lotus coevolution also has it.
From Herba Typhonii gigantei tuber, isolated one strain all has the endophyte of good inhibitory action to various plants pathogenic fungiB. licheniformis, and named TG119.Endophyte of plant and host plant are formed during long-term coevolution
Very close relationship, endophyte also can be by producing hydrolytic enzyme while obtain stable living environment from host plant
The metabolites such as class, antibiotics and alkaloids, compete nutrient substance with pathogen, and induction host plant produces system resistant
And the approach such as resistance strengthening plant plays Biological control effect.
Summary of the invention
It is an object of the invention to provide a strain and various plants pathogenic fungi is had Radix Rehmanniae clothing in the Herba Typhonii gigantei of antagonism
Bacillus cereus；
It is a further object of the present invention to provide the separation method of raw Bacillus licheniformis in a kind of above-mentioned Herba Typhonii gigantei.
One, raw Bacillus licheniformis in Herba Typhonii gigantei
Raw Bacillus licheniformis in the Herba Typhonii gigantei of the present inventionB.licheniformis, bacterial strain is attributed to bacillus, is designated as
TG119.Deposit number is CGMCC No.9048, and preservation date is on April 14th, 2014, depositary institution: Chinese microorganism strain
Preservation administration committee common micro-organisms center, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode
100101。
Raw Bacillus licheniformis TG119 in the Herba Typhonii gigantei of the present invention, for fungus specificity biocontrol microorganisms, bacterial strain is in NA culture medium
Upper bacterium colony is flattened round, and edge is irregular, and surface is more smooth, not chromogenesis, and Herb is milky, and Gram’s staining is sun
Property；Scanning electron microscopic observation is club-like, and size is (0.7 ~ 0.9) μm × (3 ~ 6) μm (see figure 1).
Two, the separation of raw Bacillus licheniformis and purification in Herba Typhonii gigantei
The separation method of raw Bacillus licheniformis in Herba Typhonii gigantei: after Herba Typhonii gigantei tuber is carried out surface detoxicating process, at aseptic bar
It is cut into the piece of tissue of size about 0.5cm × 0.5cm under part, is placed on isolation medium, make cut sides contact with culture medium；It is placed in
Cultivate 2 ~ 5 days for 37 DEG C；After growing bacterium colony around piece of tissue, picking is transferred in new culture medium, and method of scoring purification obtains pure
Fungal bacterial strain, save backup.
The culture medium that the cultivation of raw Bacillus licheniformis in Herba Typhonii gigantei, separation, purification use is nutrient agar
(NA), its constituent is: beef extract 3g, peptone 10g, sodium chloride 5g, and agar 18g adds water and is settled to 1000mL,
pH7.0~7.2。
Three, in Herba Typhonii gigantei, the Physiology and biochemistry of raw Bacillus licheniformis TG119 is identified
1, Physiology and biochemistry is identified
TG119 being carried out Physiology and biochemistry qualification with reference to " common bacteria system identification handbook ", experimental result is shown in Table 1:
2,16S rRNA sequencing and Phylogenetic Analysis
Using bacterial universal primers PCR to expand 16S rRNA, amplified fragments is cloned after purification and checks order, by the 16S of TG119 bacterial strain
RRNA sequence is submitted to GenBank, and its sequence accession number is KJ145796.Sequencing result homology comparison shows, TG119 bacterial strain
16S rRNA sequence is the most similar to bacillus 16S rRNA.Gene order is shown in sequence table.Based on 16S rRNA sequence structure
The phylogenetic tree of the TG116 bacterial strain built is as shown in Figure 2.
Growth curve (Fig. 3) display of raw Bacillus licheniformis TG119 in the Herba Typhonii gigantei of above-mentioned separation, 0 ~ 8 h growth is slow
Slowly, it is in lag phase；8 ~ 20h viable count rapid development, is in exponential phase；After 20h, curve is steady, grows into stable
Phase, 20 ~ 48h is in stable trophophase；Phase of decline is entered after inoculation 48h.
In the Herba Typhonii gigantei of above-mentioned separation, raw Bacillus licheniformis can grow in PDA culture medium, the temperature growth to TG119 bacterial strain
Impact is relatively big, and it all can grow in the range of 20 ~ 60 DEG C, growth temperature range relatively extensively, optimum growth temperature be 45 DEG C (see figure
4).
In conjunction with above-mentioned morphology, physiological and biochemical index and 16S rRNA sequence synthesis analysis result, by preliminary for TG116 bacterial strain
It is accredited as Bacillus licheniformis (Bacillus licheniformis).During culture presevation, Chinese microorganism strain
Preservation administration committee common micro-organisms center has also carried out further confirmation to strain, confirms as Bacillus licheniformis.
Four, the raw Bacillus licheniformis TG119 inhibitory action to plant pathogenic fungi in Herba Typhonii gigantei
Use the rejection ability of asynchronous culture method, flat board face-off method research TG119 strains on plant pathogenic fungi: will activation and expansion
Test plant pathogenic fungi after big cultivation breaks into the bacterium cake of a diameter of 6mm, and moves it in PDA plate culture medium respectively,
Oxford cup is placed on when growing to 2cm Deng it two symmetrical positions, and 0.2ml Bacteria suspension is accessed in the inside, cultivates 7d for 28 DEG C.Press
The suppression ratio of following formula calculating TG119 strains on plant pathogenic fungi:
Colony diameter × 100% of bacteriostasis rate (%)=(colony diameter of the colony diameter of comparison-generation inhibition zone)/comparison
Fig. 5 be raw Bacillus licheniformis in asynchronous culture method Herba Typhonii gigantei of the present invention (Bacillus lichenifomis) TG119
Condition diagram cultivated by the flat board of bacterial strain fermentation liquor process group test plant pathogen and matched group；Fig. 6 be flat board face-off method with this
Raw Bacillus licheniformis in bright Herba Typhonii gigantei (Bacillus lichenifomis) TG119 bacterial strain bacterium solution process test plant cause of disease
Condition diagram cultivated by the flat board of bacterium.Fig. 5,6 display, asynchronous culture method process group test plant pathogen compared with matched group the most almost
Do not grow, show in TG119 bacterial strain fermentation liquor containing the material that test plant pathogen is had high inhibition effect.By flat board pair
The rejection ability of bacterial strain has been done quantitative assessment by method of standing erect (Fig. 7), and result is as shown in table 2.Display TG119 bacterial strain is to seven kinds for trying
Phytopathogen all has good inhibitory action, suppresses bacterium for broad spectrum type, it is adaptable to agricultural biocontrol microorganisms is developed.
Five, the application of TG119 bacterial strain
The TG119 bacterial strain that this experiment is separated in Herba Typhonii gigantei tuber all has good suppression to 7 kinds of test plant pathogenic fungi
Effect, for broad spectrum type plant pathogenic fungi Antagonistic Fungi, it is possible to make the distortion of pathogen mycelia, deformation etc..TG119 is carried out form
, physiological and biochemical property and molecular biology identification, its morphology and biochemical reactions phase basic with bacillus feature
Seemingly, the comparison of 16S rRNA sequence is analyzed and is shown that TG119 bacterial strain belongs to bacillus, with Bacillus licheniformis (Bacillus
Licheniformis) homology is up to 99%, consistent with morphology and biochemical reactions result, thus determines TG119 bacterial strain
For Bacillus licheniformis (Bacillus licheniformis).Bacillus licheniformis metabolism can produce antibiotic and antibacterial egg
Wait many kinds of substance in vain, be widely used in industry and pharmaceutical sector, the preventing and treating of plant disease also has value, be a kind of tool
There is the microorganism of high using value.TG119 enriches the resource of interior raw Bacillus licheniformis, carries for Biological control crop pest
Having supplied new microbial resources, and the study show that, the breeding of TG119 strain growth is fast, and acid-base condition and temperature environment adapt to model
Enclosing extensively, antimicrobial spectrum is wide, and these all show this bacterium potential using value in terms of biocontrol of plant disease, its anti-pathogenesis
Reason, growth-promoting functions, practical fermentation condition and Field information effect etc. also need to make further research.
Accompanying drawing explanation
Fig. 1 be raw Bacillus licheniformis in Herba Typhonii gigantei of the present invention (Bacillus lichenifomis) TG119 bacterial strain is at electricity
Picture under mirror；
Fig. 2 be raw Bacillus licheniformis in Herba Typhonii gigantei of the present invention (Bacillus lichenifomis) TG119 bacterial strain and close bacterium
The 16S rRNA sequence of strain carries out the phylogeny tree graph of sequence analysis structure；
Fig. 3 be raw Bacillus licheniformis in Herba Typhonii gigantei of the present invention (Bacillus lichenifomis) TG119 strain growth curve
Figure；
Fig. 4 be raw Bacillus licheniformis in Herba Typhonii gigantei of the present invention (Bacillus lichenifomis) TG119 bacterial strain optimum temperature
Figure；
Fig. 5 be raw Bacillus licheniformis in asynchronous culture method Herba Typhonii gigantei of the present invention (Bacillus lichenifomis) TG119
Condition diagram cultivated by the flat board of bacterial strain fermentation liquor process group test plant pathogen and matched group；
Fig. 6 be flat board face-off method Herba Typhonii gigantei of the present invention in raw Bacillus licheniformis (Bacillus lichenifomis) TG119
Bacterial strain bacterium solution processes the flat board of test plant pathogen and cultivates condition diagram；
Fig. 7 be raw Bacillus licheniformis in Herba Typhonii gigantei of the present invention (Bacillus lichenifomis) bacteriostasis rate of TG119 bacterial strain
Figure.
Detailed description of the invention
Below by specific embodiment, the isolation identification of Bacillus licheniformis raw in Herba Typhonii gigantei of the present invention is made furtherly
Bright.The method is conventional method if no special instructions.
1, material
Healthy Herba Typhonii gigantei (T. giganteun Engl.) plant, picks up from Longnan of Gansu Province Wen County.Test plant pathogenic fungi is peppery
Green pepper phytophthora pathogenic bacteria (Phytophthora capsici), Colletotrichum capsici (Colletotrichum capsici), Rhizoma Solani tuber osi
Rhizoctonia solani Kuhn (Rhizoctonia solani), fusarium graminearum (Fusarium graminearum), cucumber fusarium axysporum
Bacterium (Fusarium oxysporum), botrytis cinerea pers (Botrytis cinerea), Fructus Caricae cotton disease bacterium (Pythium
Aphanidermatum), given by Inst. of Plant Protection, Gansu Prov. Academy of Agricultural Sciences.
The separation of Herba Typhonii gigantei endogenetic bacteria, purification and cultivation use nutrient agar (NA), plant pathogenic fungi
Cultivate and Antagonism experiment uses potato dextrose agar (PDA).Nutrient agar (NA): beef extract 3
G, peptone 10g, sodium chloride 5g, agar 18g, add water and be settled to 1000mL, pH7.0 ~ 7.2；Potato dextrose agar is cultivated
Base (PDA): Rhizoma Solani tuber osi 200g, glucose 20g, agar 18g, add water and be settled to 1000mL, pH is natural.
2, the isolation and purification of bacillus subtilis TG119
Herba Typhonii gigantei tuber is carried out surface detoxicating, takes the Herba Typhonii gigantei tuber after surface sterilization processes, under aseptic condition, be cut into size
The piece of tissue of about 0.5cm × 0.5cm, is placed on isolation medium, makes cut sides contact with culture medium, be placed in 37 DEG C cultivate 2 ~
5d, after growing bacterium colony around piece of tissue, picking is transferred in new culture medium, and method of scoring makees short-term at cryogenic refrigerator after purification
Preserve or preserve for a long time.Rinsing liquid method of inspection and Tissue blot-ELISA is utilized to set two kinds of blanks.TG119 is in isolated strains
One strain.
3, the morphologic observation of bacillus subtilis TG119 and physiological and biochemical analysis
Fig. 1 is the Electronic Speculum electron microscopic picture of the TG119 bacterial strain that the present invention separates.Scanning electron microscope shows, TG119 bacterial strain is club-like,
Size is (0.7 ~ 0.9) μm × (3 ~ 6) μm.
TG119 bacterial strain bacterium colony in NA culture medium is flattened round, and edge is irregular, and surface is more smooth, not chromogenesis,
Herb is milky, and Gram’s staining is positive, for fungus specificity biocontrol microorganisms.
With reference to " common bacteria system identification handbook ", TG119 is carried out Physiology and biochemistry qualification, the results are shown in Table 1.
4, the 16S rRNA sequencing of bacillus subtilis TG119 and Phylogenetic Analysis
Use the 16S rRNA fragment of the method amplification TG119 of bacterium colony PCR.30 μ L sterilized water are added in 1.5mL centrifuge tubes also
The a small amount of thalline of picking makes suspension in water, first boils 10min, then puts into rapidly 5min in ice, with 12000rmin-1Centrifugal
Supernatant after 10min, as pcr template, uses the 16S rRNA of bacterial universal primers 27F and 1492R amplification bacterial strain TG119.
Gained sequence carries out BLAST comparison analysis with the existing sequence in GenBank data base after check and correction, adopts with MEGA6.0 software
With adjacent (Neighbor Joining) cluster analysis, and phylogenetic tree construction.
Pcr amplification primer thing sequence is as follows:
27F (5 ' AGAGTTTGATCCTGGCTCAG 3 ')
1492R(5 ' GGTTAC CTTGTTACGACTT 3 ')
PCR amplification condition is: 95 DEG C of denaturations 5min, 95 DEG C of degeneration 30s, and 55 DEG C of annealing 30s, 72 DEG C extend 90s, and 30 are followed
Ring；72 DEG C extend 10min eventually.
Bacillus licheniformis (Bacillus lichenifomis) the 16SrRNA sequence of TG119 is shown in sequence table.
Combining form, physiological and biochemical index and 16S rRNA sequence synthesis analysis result, by TG116 bacterial strain Preliminary Identification
For Bacillus licheniformis (Bacillus licheniformis).
＜ 110 ＞ Northwest Normal University
＜ 120 ＞ mono-strain various plants pathogenic fungi is had the Herba Typhonii gigantei of antagonism in raw Bacillus licheniformis
〈160〉1
〈210〉1
〈211〉1390
〈212〉16SrRNA
＜ 213 ＞ bacillus (Bacillus)
＜ 223 ＞ Bacillus licheniformis (Bacillus lichenifomis) the 16SrRNA sequence of TG119
gcagtcgagc ggacagatgg gagcttgctc cctgatgtta gcggcggacg ggtgagtaac 60
acgtgggtaa cctgcctgta agactgggat aactccggga aaccggggct aataccggat 120
ggttgtttga accgcatggt tcaaacataa aaggtggctt cggctaccac ttacagatgg 180
acccgcggcg cattagctag ttggtgaggt aacggctcac caaggcgacg atgcgtagcc 240
gacctgagag ggtgatcggc cacactggga ctgagacacg gcccagactc ctacgggagg 300
cagcagtagg gaatcttccg caatggacga aagtctgacg gagcaacgcc gcgtgagtga 360
tgaaggtttt cggatcgtaa agctctgttg ttagggaaga acaagtaccg ttcgaatagg 420
gcggtacctt gacggtacct aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg 480
taatacgtag gtggcaagcg ttgtccggaa ttattgggcg taaagggctc gcaggcggtt 540
tcttaagtct gatgtgaaag cccccggctc aaccggggag ggtcattgga aactggggaa 600
cttgagtgca gaagaggaga gtggaattcc acgtgtagcg gtgaaatgcg tagagatgtg 660
gaggaacacc agtggcgaag gcgactctct ggtctgtaac tgacgctgag gagcgaaagc 720
gtggggagcg aacaggatta gataccctgg tagtccacgc cgtaaacgat gagtgctaag 780
tgttaggggg tttccgcccc ttagtgctgc agctaacgca ttaagcactc cgcctgggga 840
gtacggtcgc aagactgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca 900
tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc tctgacaatc 960
ctagagatag gacgtcccct tcgggggcag agtgacaggt ggtgcatggt tgttgtcagc 1020
tcgtgtcgtg agatgtttgg ttaagtcccg caacgagcgc aaccgttgat cttagttgcc 1080
agcattcagt tgggcacttt aaggtgactg ccggtgacaa accggaggaa ggtggggatg 1140
acgtcaaatc atcatgcccc ttatgacctg ggctacacac gtgttacaat ggacagaaca 1200
aagggcagcg aaaccgcgag gttaagccaa tcccacaaat ctgttctcag ttcggatcgc 1260
agtctgcaac tcgattgcgt gaagccggaa tcgctagtaa tcgcggatca gcatgccgcg 1320
gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccacgagagt ttgtaacacc 1380
cgaagtcggt gagg 1390
Claims (4)
1. a strain raw Bacillus licheniformis in various plants pathogenic fungi is had the Herba Typhonii gigantei of antagonism, deposit number is
CGMCC No.9048, is labeled as TG119.
2. raw Bacillus licheniformis in as claimed in claim 1 various plants pathogenic fungi being had the Herba Typhonii gigantei of antagonism,
It is characterized in that: bacterial strain is fungus specificity biocontrol microorganisms, and in NA culture medium, bacterium colony is flattened round, edge is irregular, surface
More smooth, not chromogenesis, Herb is milky, and Gram’s staining is positive；Scanning electron microscopic observation is club-like, and size is
(0.7 ~ 0.9) μm × (3 ~ 6) μm.
3. raw Bacillus licheniformis in as claimed in claim 1 various plants pathogenic fungi being had the Herba Typhonii gigantei of antagonism
Separation method, after Herba Typhonii gigantei tuber carries out surface detoxicating process, is aseptically cut into size about 0.5cm × 0.5cm's
Piece of tissue, is placed on isolation medium, makes cut sides contact with culture medium；It is placed in 37 DEG C to cultivate 2 ~ 5 days, treats around piece of tissue long
After going out bacterium colony, picking is transferred in new culture medium, and method of scoring preserves after purification.
4. raw Bacillus licheniformis in as claimed in claim 1 various plants pathogenic fungi being had the Herba Typhonii gigantei of antagonism
Separation method, it is characterised in that: described culture medium is nutrient agar, and its constituent is: beef extract 3g, peptone
10g, sodium chloride 5g, agar 18g, add water and be settled to 1000mL, pH7.0 ~ 7.2.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108690817A (en) * | 2018-04-08 | 2018-10-23 | 西北师范大学 | A kind of multifunctional broad-spectrum complex micro organism fungicide and its preparation and application |
| CN109777758A (en) * | 2019-03-04 | 2019-05-21 | 青岛红樱桃生物技术有限公司 | One kind having the active bacillus licheniformis HYT-9 of broad-spectrum antibacterial and its bacterial preparation process and application |
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| CN1793331A (en) * | 2005-11-14 | 2006-06-28 | 浙江大学 | Lichen spore bacillus strain and application of bacillocin produced by it |
| CN104805017A (en) * | 2014-11-27 | 2015-07-29 | 中华全国供销合作总社南京野生植物综合利用研究所 | Plant endophytic fungus for generation of beta-glucosidase and application thereof |
| CN104928211A (en) * | 2015-05-27 | 2015-09-23 | 广东省农业科学院作物研究所 | Sugarcane endophyte with antagonistic activity and application of sugarcane endophyte |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1793331A (en) * | 2005-11-14 | 2006-06-28 | 浙江大学 | Lichen spore bacillus strain and application of bacillocin produced by it |
| CN100352916C (en) * | 2005-11-14 | 2007-12-05 | 浙江大学 | Lichen spore bacillus strain and application of bacillocin produced by it |
| CN104805017A (en) * | 2014-11-27 | 2015-07-29 | 中华全国供销合作总社南京野生植物综合利用研究所 | Plant endophytic fungus for generation of beta-glucosidase and application thereof |
| CN104928211A (en) * | 2015-05-27 | 2015-09-23 | 广东省农业科学院作物研究所 | Sugarcane endophyte with antagonistic activity and application of sugarcane endophyte |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108690817A (en) * | 2018-04-08 | 2018-10-23 | 西北师范大学 | A kind of multifunctional broad-spectrum complex micro organism fungicide and its preparation and application |
| CN109777758A (en) * | 2019-03-04 | 2019-05-21 | 青岛红樱桃生物技术有限公司 | One kind having the active bacillus licheniformis HYT-9 of broad-spectrum antibacterial and its bacterial preparation process and application |
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| CN105907655B (en) | 2019-05-07 |
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