CN105112316A - Application of Bacillus cereus D13 and volatile substances thereof in prevention and treatment of bacterial diseases of paddy rice - Google Patents
Application of Bacillus cereus D13 and volatile substances thereof in prevention and treatment of bacterial diseases of paddy rice Download PDFInfo
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- CN105112316A CN105112316A CN201510334696.6A CN201510334696A CN105112316A CN 105112316 A CN105112316 A CN 105112316A CN 201510334696 A CN201510334696 A CN 201510334696A CN 105112316 A CN105112316 A CN 105112316A
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Abstract
The invention discloses application of Bacillus cereus D13 and volatile substances thereof in prevention and treatment of bacterial diseases of paddy rice. The Bacillus cereus D13 having inhibitory effect on Xanthomonas oryzae pv. oryzae of paddy rice is preserved in China General Microbiological Culture Collection Center on October 25, 2014, with an accession number of CGMCC No. 10243. The invention also discloses application of volatile substances produced by the Bacillus cereus D13 in prevention and treatment of bacterial diseases of paddy rice. Bacillus subtilis OKB105 and the Bacillus cereus D13 producing volatile substances capable of inhibiting xanthomonas are screened out in the invention. The volatile substances produced by D13 exert inhibitory effect on Xoo; under the condition of low concentration, the volatile substances produced by D13 can inhibit swarming and pathogenic ability of xanthomonas of paddy rice; and under the condition of high concentration, the volatile substances can improve membrane permeability and cleavage of intracellular substance, which lead to death of cells.
Description
Technical field
The invention belongs to microorganism field, relate to bacillus cereus D13 and the application of volatile matter in control paddy rice Micobial Disease thereof.
Background technology
Rice Xanthomonas (Xanthomonasoryzaepv.oryzae, Xoo) is the pathogenic bacteria of bacterial blight of rice, is grown on xylem, causes the scab of yellow-white along vein.Paddy rice bacterial leaf spot is the Major Diseases of Asian countries paddy rice, causes the underproduction of 50-60% time serious.Comparatively thorough to the research of rice Xanthomonas, the product that the virulence factor reported comprises anaphylaxis, II and III secretes system secretion, exocellular polysaccharide (EPS), movability, diffustivity signal factors (DSF) and extracellular enzyme etc.
Long ago people just recognize that bacterium can produce special smell, and are developed as the fragrance ingredient of cheese, Yoghourt and red wine.Recent research shows that bacterium can be done mutually by generation volatile matter and plant, fungi, nematode and other bacterium.2003, the bacteriogenic volatile organic compounds (volatileorganiccompounds, VOCs) of the Late Cambrian such as Ryu can affect plant-growth.In the past in 10 years, 2,3-butanediol, 3-hydroxy-2-butanone, hexadecyldimethyl benzyl ammonium amine, 2-amyl furan has been proved has promoter action to plant-growth.The 2,3-butanediol that BacillussubtilisGB03 produces promotes the growth of plant by mechanism such as the synthesis transhipment of acidifying rhizosphere, the absorption improving iron, regulating plant tumor growth element and the expression of suppression ABA synthesis responsive genes thereof.In addition, the VOCs that rhizosphere growth-promoting endophytic bacteria (plantgrowth-promotingrhizobacteria, PGPR) produces can also cause plant to the resistance of the abiotic stress such as drying, salt, nutritive deficiency.Such as bacterium VOCs can improve the balance of plant to sodium in the absorption of iron and regulating plant body.2004, the volatile matter that Ryu reports the secretion of rhizosphere growth-promoting endophytic bacteria first can induce the generation of Arabidopis thaliana system resistant.The 2,3-butanediol that B.amyloliquefaciensIN937a and B.subtilisGB03 produces can significantly improve the resistance of Arabidopis thaliana to carrot soft rot Pectinatus.In addition, the tridecane of PaenibacilluspolymyxaE681 secretion also can produce system resistant (Inducedsystemicresistance, ISR) by inducing plant.
Just be in the news early than bacteriogenic volatile matter in 1966 and can affect the g and D of fungi.Research shows that volatile matter that streptomyces griseus (Streptomycesgriseus) produces obviously can reduce the sporulation of Gleosporiumaridum, promotes the formation of white rot pyrenomycetes (Sclerotiumcepivorum) and dry thread Pyrenomycetes (Rhizoctoniasolani) sclerotium.Report that at present the volatile matter with fungicidal activities comprises dimethyl disulfide, hendecene, phenyl aldehyde, hexalin, capraldehyde, 2-ethyl-1-hexanol, benzothiazole, NSC 97324 and the alkanes of long-chain, alkene class, aldehydes and alcohols etc. in some.
In the recent period, there are some researches show that the volatile matter of producing bacillus subtilis life can reduce colibacillary movability and improve it to antibiotic adaptability, however then relatively less to the research of pathogenetic bacteria about PGPR volatile matter.
Summary of the invention
The object of the invention is the above-mentioned deficiency for prior art, a kind of bacillus cereus D13 inhibited to rice leaf spot bacteria is provided.
Another object of the present invention is the application of same work bacillus cereus D13 in water prevention rice bacterial leaf spot pathogenic bacteria.
Another object of the present invention is to provide the application of volatile matter in water prevention rice bacterial leaf spot pathogenic bacteria of bacillus cereus D13.
Object of the present invention realizes by following technical scheme:
A bacillus cereus D13 inhibited to rice leaf spot bacteria, is preserved in China General Microbiological culture presevation administrative center, and preserving number is CGMCCNo.10243, and preservation date is on December 25th, 2014.
The application of bacillus cereus D13 of the present invention in the biological pesticide of preparation control paddy rice Micobial Disease, the application preferably in the biological pesticide preparing the water prevention bacterial blight of rice.
The application of the volatile matter that bacillus cereus D13 of the present invention produces in control paddy rice Micobial Disease, the application preferably in the water prevention bacterial blight of rice.
The application of the volatile matter that bacillus cereus D13 of the present invention produces in the biological pesticide of preparation control paddy rice Micobial Disease, the application preferably in the biological pesticide preparing the water prevention bacterial blight of rice.
Beneficial effect:
The present invention filters out volatile matter has inhibition bacterial strain bacillus cereus D13 to Xanthomonas campestris.The volatile matter that D13 produces has inhibition to Xoo, during lower concentration, the volatile matter that D13 produces can suppress swarming and the pathogenecity of rice Xanthomonas, then can strengthen the cracking of permeability of cell membrane and intracellular organic matter, thus cause necrocytosis during high density.The present invention utilizes solid-phase microextraction to identify the volatile matter that D13 produces in conjunction with GC-MS, and the volatile matter of generation mainly comprises acids, alcohols, benzene class, ketone and sulfur-bearing nitrogenous compound thereof.Wherein, decyl alcohol (DA) and 3,5,5-trimethyl hexanol (TMH) can suppress the growth of Xoo effectively, in addition 3,5,5-trimethyl hexanols also have same inhibition to xanthomonas oryzae pv. oryzicola, bacterial spot of tomato bacterium and bacterial wilt of tomato bacterium.These results show that the volatile matter that D13 produces has broad spectrum toxicity, have good application prospect.
Accompanying drawing explanation
Fig. 1 genus bacillus volatile compound is to the fungistatic effect of paddy rice bacterial leaf spot
Fig. 2 is based on the BacilluscereusD13 phylogenetic tree of gyrB sequence construct
Fig. 3 genus bacillus volatile matter is to Xooswarming capacity
Fig. 4 genus bacillus volatile matter is on the impact of Xoo colony diameter
Fig. 5 genus bacillus volatile matter is on the impact of Xoo vigor
Fig. 6 genus bacillus volatile matter is on the impact of Xoo pathogenic related gene expression level
Fig. 7 genus bacillus volatile matter affects Xoo virulence
Xoo thalli morphology under Fig. 8 scanning electron microscope after the process of genus bacillus volatile matter
A:control；B:D13
Xoo thalli morphology under Fig. 9 transmission electron microscope after the process of genus bacillus volatile matter
A:control；B:D13。
Figure 10 genus bacillus presses down the screening of Xoo volatile matter
Wherein, LA (dodecylic acid), TA (tridecanoic acid), MA (TETRADECONIC ACID), PA (palmitic acid), MCP (3-methyl isophthalic acid, 2-ring pentanediol), TMH (3,5,5-trimethyl hexanol), DA (decyl alcohol) and PCO (2-pentacosane ketone).
The impact that DA and TMH of Figure 11 different concns grows Xoo
The impact that Figure 12 DA and TMH grows Xoc, Pst and Rst
Wherein xanthomonas oryzae pv. oryzicola (Xanthomonasoryzaepv.oryzicola, Xoc), bacterial spot of tomato bacterium (Pseudomonassyringaepv.tomatoDC3000, and bacterial wilt of tomato bacterium (Ralstoniasolanacearumpv.tomato, Rst) Pst).
Biomaterial preservation information
D13, Classification And Nomenclature is Bacillus cereus Bacilluscereus, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date is on December 25th, 2014, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, and preserving number is CGMCCNo.10243.
Embodiment
Embodiment 1 volatile matter has the Bacillus strain screening pressing down bacterial activity
The method screening volatile matter separating culture dish is adopted to have the Bacillus strain of bacteriostatic activity.Bacillus strain comprises some genus bacillus of bacterial strain and the laboratory preservation be separated from Malaysia.Separate culture dish while pour LB solid medium into, and add 30 μ l10
8cfumL
-1genus bacillus bacterium liquid, and pour NA solid medium and 10 μ l10 at the other side
8cfumL
-1xoo bacterium liquid, with only have Xoo bacterium liquid containing genus bacillus bacterium liquid process for contrast.Xoo colony diameter is measured after 28 DEG C of Dual culture 6d.We filter out the good bacterial strain D13 (Fig. 1) of fungistatic effect from the type strain that wild mushroom and the laboratory of the separation of 50 strain Malaysia are preserved, and its volatile matter produced has obvious inhibition to Xoo, and inhibiting rate is 37.5%.Wherein D13 is the wild mushroom that Malaysia is separated.
16SrRNA and gyrB amplification and order-checking are carried out to D13, and order-checking gained sequence and ncbi database are carried out blast comparison, by MEGA4.0 software, Phylogenetic Analysis is carried out to D13 and genus bacillus type strain, determine that D13 bacterial strain belongs to bacillus cereus (Bacilluscereus) (Fig. 2); D13 is delivered CGMCC preservation, deposit number is CGMCCNo.10243.。
Embodiment 2Xooswarming ability measures
The method separating culture dish is adopted to detect B.cereusD13 volatile matter to the impact of Xooswarming ability.Xoo bacterium liquid is adjusted to OD
600=1.0, and get 2 μ l and drop in the separation culture dish of the NA substratum containing 0.7% agar, and add 30 μ l10 at the other side separating culture dish
8cfumL
-1b.cereusD13 bacterium liquid.Xoo colony diameter is measured after 28 DEG C of Dual culture 3d.Collect the Xoo thalline after D13 process respectively, extract bacteria RNA, use PrimeScriptfirstStrandcDNASynthesisKit (TaKaRa, Dalian) to carry out RNA reverse transcription, method reference reagent box specification sheets.Real-timePCR adopts SYBRPremixExTaq
tMtest kit (TaKaRa, Dalian), AppliedBiosystems7500RealTimePCRSystem (ABI, USA) completes.Detect the expression level of genes involved respectively, the primer of use is in table 1.
The primer that table 1 is used
Xooswarming ability after D13 volatile matter process 3d is weaker than than contrast (Fig. 3), and after measuring show to only have process 4d to the colony diameter of Xoo and viable count, these parameters just can obviously reduction (Figure 4 and 5).Meanwhile, relevant to swarming motA and motC all lowers expression (Fig. 6).When these results show process 3d, D13 volatile matter suppresses the swarming ability of Xoo instead of directly lethal.
Embodiment 3Xoo Pathogenicity
Collect B.cereusD13 volatile matter according to the Xoo thalline after embodiment 1 method process 3d, be resuspended in aqua sterilisa to final concentration be 10
8cfuml
-1, with leaf-cutting method inoculation paddy rice (IR24).Inoculation 14d " Invest, Then Investigate " scab length.
Xoo virulence after D13 volatile matter process 3d is obviously weaker than than contrast (Fig. 7), rpf (regulationofpathogenicityfactor) gene cluster is Xoo biofilm formation and causes a disease necessary, and rpfF, rpfC and rpfG are the genes of its key, rpfC also is correspondingly suppressed to express by D13 volatile matter as shown in Figure 6.
Embodiment 4 scanning electron microscope and transmission electron microscope detect Xoo outside form and internal structure
Adopt and separate culture dish method, after D13 and Xoo28 DEG C of Dual culture 6d, aqua sterilisa cleans 3 times, and fixes by 2.5% glutaraldehyde, carries out scanning electron microscope and transmission electron microscope observing.With the Xoo of LB nutrient solution process for contrast.Contrast Xoo cell walls is smooth, becomes shaft-like, and the Xoo cell walls of D13 volatile matter process depression, cell becomes deformity, does not become shaft-like (Fig. 8).And transmission electron microscope observing shows that normal Xoo cell within a cell matter is evenly distributed, and the Xoo cytoplasm condenses after the process of genus bacillus volatile matter, the cell walls of part cell is destroyed, and causes permeability of cell membrane to strengthen and intracellular organic matter cracking (Fig. 9).
The collection of embodiment 5 volatile matter and qualification
Adopt Solid-phase Microextraction
[1]collect volatile matter, get in the 100ml bottle that 20 μ L genus bacillus bacterium drops add to containing 30mlLB substratum and cultivate 6d in 28 DEG C.SPME extracting head is inserted into the overhead of 100ml bottle, and hatches 30min in 50 DEG C of water-baths, carry out GC-MS analysis.Gas chromatography mass spectrometry program: SPME extracting head is placed in 220 DEG C of 5min that dissociate; Column temperature: starting temperature is after 35 DEG C of maintenance 3min, is warming up to 180 DEG C with 10 DEG C/min, then is warming up to 240 DEG C of maintenance 5min with 4 DEG C/min; Carrier gas is helium, and column flow rate is 1ml/min.Scanning of the mass spectrum scope: 50-500m/z.Scan-data and NIST/EPA/NIH database are compared.The volatile matter that result display produces mainly comprises acids, alcohols, benzene class, ketone and sulfur-bearing nitrogenous compound (table 2) thereof.
The volatile matter that table 2D13 produces
Embodiment 6VOCs reagent presses down bacterial activity screening to Xoo
By 100 μ lVOCs reagent liquid sterlings and 100 μ lVOCs reagent solid pure product, (dissolve with DMSO, concentration is 1gml respectively
-1) drop to containing LB substratum separation culture dish on, the substratum that the other side contains NA adds 10 μ l10
8cfuml
-1xoo bacterium liquid, with the DMSO of respective volume for contrast.Xoo colony diameter is measured after 28 DEG C of Dual culture 6d.
After preliminary screening, select have 3,5, the 5-trimethyl hexanols (TMH) of bacteriostatic activity and decyl alcohol (DA) further to screen.With DMSO, TMH and DA is diluted to 0.8mgml
-1to 40mgml
-1, the diluent detecting 100 μ l different concns, to the bacteriostatic activity of Xoo, determines the minimum inhibitory concentration of TMH and DA.Detect TMH and DA to the fungistatic effect of Xanthomonasoryzaepv.oryzicola (Xoc), Pseudomonassyringaepv.tomatoDC3000 (Pst) and Ralstoniasolanacearumpv.tomato (Rst) simultaneously.
That has bought sterling in more than the ten kind of volatile matter that D13 produces only has 8 kinds, detects the impact that this 8 kinds of volatile matter sterlings grow Xoo.As shown in Figure 10, only have decyl alcohol (DA) and 3,5,5-trimethyl hexanol (TMH) obviously can suppress the growth of Xoo, inhibiting rate is respectively 53.6% and 60.7%.In addition, as interpolation 100 μ l4.8mgml
-1decyl alcohol (DA) and 24mgml
-13,5,5-trimethyl hexanols (TMH) time just can well suppress the growth (Figure 11) of Xoo.TMH can suppress xanthomonas oryzae pv. oryzicola (Xanthomonasoryzaepv.oryzicola equally, Xoc), bacterial spot of tomato bacterium (Pseudomonassyringaepv.tomatoDC3000, and bacterial wilt of tomato bacterium (Ralstoniasolanacearumpv.tomato Pst), Rst) growth, and DA only has inhibition (Figure 12) to Xoc.These results show that TMH has broad spectrum toxicity to pathogenetic bacteria.
Reference
[1]YuanJ,RazaW,ShenQ,HuangQ.AntifungalActivityofBacillusamyloliquefaciensNJN-6VolatileCompoundsagainstFusariumoxysporumf.sp.cubense.Appliedandenvironmentalmicrobiology.2012；78:5942-4.
Claims (7)
1. to the bacillus cereus D13 that rice leaf spot bacteria is inhibited, it is characterized in that being preserved in China General Microbiological culture presevation administrative center, preserving number is CGMCCNo.10243, and preservation date is on December 25th, 2014.
2. the application of bacillus cereus D13 according to claim 1 in the medicine of preparation control paddy rice Micobial Disease.
3. application according to claim 2, is characterized in that the application of bacillus cereus D13 according to claim 1 in the biological pesticide preparing the water prevention bacterial blight of rice.
4. the volatile matter that bacillus cereus D13 according to claim 1 produces is preventing and treating the application in paddy rice Micobial Disease.
5. application according to claim 4, is characterized in that the application of volatile matter in the water prevention bacterial blight of rice that bacillus cereus D13 according to claim 1 produces.
6. the volatile matter that bacillus cereus D13 according to claim 1 produces prevents and treats the application in the biological pesticide of paddy rice Micobial Disease in preparation.
7. application according to claim 6, is characterized in that the application of volatile matter in the biological pesticide preparing the water prevention bacterial blight of rice that bacillus cereus D13 according to claim 1 produces.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108531415A (en) * | 2017-08-14 | 2018-09-14 | 浙江师范大学 | The pale air bacillus Jh-7 of one plant of antagonism rice leaf spot bacteria |
| CN115024338A (en) * | 2022-06-30 | 2022-09-09 | 金正大生态工程集团股份有限公司 | Application of resuscitation promotion factor protein in preventing and treating crop continuous cropping diseases and promoting crop growth and application method |
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Non-Patent Citations (5)
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| CHITHRASHREE ET AL.: "Plant growth-promoting rhizobacteria mediate induced systemic resistance in rice against bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae", 《BIOLOGICAL CONTROL》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108531415A (en) * | 2017-08-14 | 2018-09-14 | 浙江师范大学 | The pale air bacillus Jh-7 of one plant of antagonism rice leaf spot bacteria |
| CN108531415B (en) * | 2017-08-14 | 2021-05-25 | 浙江师范大学 | Bacillus air pallidus Jh-7 for antagonizing rice bacterial blight |
| CN115024338A (en) * | 2022-06-30 | 2022-09-09 | 金正大生态工程集团股份有限公司 | Application of resuscitation promotion factor protein in preventing and treating crop continuous cropping diseases and promoting crop growth and application method |
| CN115024338B (en) * | 2022-06-30 | 2023-05-12 | 金正大生态工程集团股份有限公司 | Application of resuscitating promoting factor protein in preventing and treating crop continuous cropping diseases and promoting crop growth and application method |
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