CN104342393A - Bacillus methylotrophicus and application thereof on aspect of preventing and controlling fusarium graminearum - Google Patents
Bacillus methylotrophicus and application thereof on aspect of preventing and controlling fusarium graminearum Download PDFInfo
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- CN104342393A CN104342393A CN201410601775.4A CN201410601775A CN104342393A CN 104342393 A CN104342393 A CN 104342393A CN 201410601775 A CN201410601775 A CN 201410601775A CN 104342393 A CN104342393 A CN 104342393A
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- fusarium graminearum
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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
- C12R2001/07—Bacillus
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- 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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
Abstract
The invention discloses Bacillus methylotrophicus and an application of the Bacillus methylotrophicus on the aspect of preventing and controlling fusarium graminearum. The preservation number of the Bacillus methylotrophicus disclosed by the invention is CGMCC (China General Microbiological Culture Collection Center) No. 9500, and the nucleotide sequence of the Bacillus methylotrophicus is shown as SEQ ID No. 1. The bacterial strain is used for the application of biological prevention and control in plant diseases caused by the fusarium, wheat and corns serve as biological prevention and control materials for crop diseases; no matter new biological pesticide or biological prevention and control fungicide is developed, the bacterial strain has an excellent application prospect.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of Methylotrophic genus bacillus and and preventing and treating the application in Fusarium graminearum.
Background technology
Fusarium graminearum (Fusarium graminearum) is one of important pathogen of gramineous crop, Gibberella zeae (Sch.) Petch (Fusarium Head Blight can be caused, FHB), Fusarium graminearum is a principal element (Dubin, 1997) of restriction wheat yield.China's weather condition are extremely conducive to the generation of Gibberella zeae (Sch.) Petch, and the long-term disease in In Middle And Lower Reaches of Changjiang River causes production loss 10%-15%, the time underproduction nearly 50% of being very popular.Due to the impact that global warming and the cropping system such as straw-returning, no-tillage cultivation change, the generation area of China's Gibberella zeae (Sch.) Petch is rapidly to northwest, North China by the Yangtze valley and expands, particularly in 2008,2010 and 2012, whole nation Gibberella zeae (Sch.) Petch occurs very serious, cause serious product grain loss and bad social influence, serious threat China grain security.
The deoxynivalenol (DON, mould-9 alkene-8 ketone of the single-ended spore of 3,7,15 trihydroxy--12,13 epoxy) that Fusarium graminearum produces is one of main natural toxin be present in gibberellic hypha infection wheat.DON has been identified as one of the most dangerous naturally-occurring contaminants in food, is put into the precedence of international research.DON can cause people's immunity degradation, anaemia, headache, stomachache, feel sick; Activate thing food refusal, growth retardation and miscarriage (Rocha et al., 2005; Sob rova et al., 2010; Pestka, 2010).DON extensively exists and a large amount of pollution wheat and goods thereof, forms very large harm, serious threat China food safety to people and animals.Therefore, strengthen Fusarium graminearum prevention and control research and become the active demand ensureing China's grain security and food safety.
For many years mainly chemical pesticide is relied on to Gibberella zeae (Sch.) Petch control, but along with the environmental problem that chemical pesticide brings, in addition medication has for many years caused the appearance (Changjun Chen et al., 2007) of pathogenic bacteria resistance Fusarium graminearum bacterial strain, and difficulty of prevention and cure is strengthened.Although drop into a large amount of energy both at home and abroad to carry out correlative study in scab resistance quality, there is no desirable high resistance wheat breed (Li Zhenghui etc., 2007) so far.
Biological control is reduced environmental impact not only, also not easily develops immunity to drugs, and the research of correlation theory and technology is day by day by people are paid close attention to.The nutrition of Methylotrophic genus bacillus is simple, is widespread in nature, nontoxic to people and animals, free from environmental pollution, can multiple antibiotic and enzyme be produced, there is broad spectrum antibiotic activity and extremely strong anti-adversity ability, have more the potentiality being developed as biological prevention and control microbial inoculum compared with other microorganisms.
Summary of the invention
The object of this invention is to provide a strain Methylotrophic genus bacillus and the application in control Fusarium graminearum thereof.
The present invention is achieved through the following technical solutions:
A kind of Methylotrophic genus bacillus provided by the invention (Bacillus methylotrophic us), this bacterial strain carries out preservation, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center; Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Preservation date: on July 18th, 2014; Hide numbering: CGMCCNo.9500.
Methylotrophic genus bacillus of the present invention is used for application in the microbial Plant diseases of biological control reaping hook, and described sickle-like bacteria is Fusarium graminearum, and described disease is head blight or ear rot or root rot, and described plant is cereal; Described cereal is wheat crops or corn; Described wheat crops is specially wheat.
Methylotrophic genus bacillus of the present invention is for the preparation of the application in the medicine of the microbial Plant diseases of control reaping hook, by solid culture after the liquid of routine, acquisition comprises microorganism culture, produced by the liquid fermenting of routine, add tensio-active agent as one or more in dispersion agent, stablizer, wetting agent, binding agent, defoamer, disintegrating agent, antifreezing agent etc., or after absorption carrier mixes by a certain percentage, be prepared into wettable powder, water dispersible granules, suspension agent, suspended emulsion agent, aqueous emulsion or microemulsion.
The method of the microbial Plant diseases of Methylotrophic genus bacillus control reaping hook of the present invention also belongs to protection scope of the present invention, and the method is in growing process, carry out spraying process; Described spraying is the bacteria suspension of this Methylotrophic genus bacillus or the pesticide preparation of fermented liquid or meta-bolites or preparation.
Beneficial effect of the present invention is that Methylotrophic genus bacillus provided by the invention not only efficiently can suppress Fusarium graminearum when slat chain conveyor, and in greenhouse experiments its prevention effect up to 77.4%.As the biological control material of wheat scab, no matter develop new biological pesticide or biological control microbial inoculum, this bacterium all has good application prospect.
Accompanying drawing explanation
Fig. 1 is that JS20GA tests the opposite culture of Fusarium graminearum, and Figure 1A is the dull and stereotyped control group of PDA only inoculating Fusarium graminearum bacterium dish, and Figure 1B is at the JS20GA apart from PDA plate center 25mm place four long and short dash line inoculation same volume and concentration;
Fig. 2 is the bacteriostatic experiment of the volatile gases that JS20GA produces, and Fig. 2 A is the control group of the PDA flat board of inoculation Fusarium graminearum bacterium dish, and Fig. 2 B is that back-off is in the flat board being connected to Fusarium graminearum at NA plate center inoculation JS20GA.
Embodiment
Below in conjunction with embodiment, technical solution of the present invention is further described, the following stated, only to preferred embodiment of the present invention, not do other forms of restriction to the present invention, any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed or be modified as the Equivalent embodiments of equal change.Everyly do not depart from the present invention program's content, according to technical spirit of the present invention to any simple modification made for any of the above embodiments, equivalent variations and remodeling, all drop in protection scope of the present invention.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
NB liquid nutrient medium: be made up of solvent and solute; Solute is peptone, extractum carnis and NaCl, and solvent is water; The concentration of peptone in NB liquid nutrient medium is 1%, and the concentration of extractum carnis in NB liquid nutrient medium is the concentration of 0.3%, NaCl in NB liquid nutrient medium is 0.5%, and described % is quality volume percent (g/100ml).
NA solid medium: add agar (ratio of agar and liquid nutrient medium is 1.5g:100ml) in NB liquid nutrient medium, obtain NA solid medium.
PDA substratum: potato 200g, adds a small amount of poach 20min, and three layers of filtered through gauze, get filtrate water and be settled to 1L, add 20g glucose, 15g agar, 121 DEG C of autoclaving 20min.
Sweet mung bean soup substratum: mung bean 10g, adds a small amount of poach 20min, and three layers of filtered through gauze, get filtrate water and be settled to 1L, 121 DEG C of autoclaving 20min after packing.
Phosphate buffered saline buffer: be made up of solvent and solute; Solute is potassium primary phosphate and magnesium chloride, and solvent is water; The concentration of potassium primary phosphate is 0.004%, and the concentration of magnesium chloride is 0.019%, and described % is quality volume percent (g/100ml).
Fusarium graminearum (Fusarium graminearum) FG1 document " Yang Huiyong; Li Feifeng; Lu Qiongxian; Xu Jianhong; Shi Jianrong. antagonistic strain AFR0406 is to the biological activity determination of fusarium graminearum and sheath blight fungus. Jiangsu's agriculture science; 2006 (06), 142-144. " in be disclosed, the public can obtain from Institute of Agricultural Product Processing, Chinese Academy of Agricultural Sc.
New 828 kinds of wheat stone document " Sun Zhijun; Yang Xiaoli; Zhang Hui; Zhang Guanghui. new 828 Breeding Process of new variety of wheat stone and high-yield culture technique. modern agriculture science and technology; 2011 (09); 96-97. " in be disclosed, the public can obtain from Institute of Agricultural Product Processing, Chinese Academy of Agricultural Sc.
The Isolation and ldentification of embodiment 1 bacterium
One, the separation of bacterium
In June, (one) 2012, in Bechtop, the sick fringe of wheat gathered in 10 fringe Jiangsu is placed on concussion in 100ml sterile distilled water and prepares bacteria suspension in 15 minutes, then 80 DEG C of water bath processing 10 minutes.
(2) be coated on after bacteria suspension sterile distilled water being carried out concentration gradient dilution on NA culture medium flat plate, cultivate 24 hours under 30 DEG C of conditions, bacterium colony is covered with whole flat board, purify with form on transfering loop picking flat board, size, color, bacterial strain plate streaking that transparency is different, the bacterial strain after point connects purifying is applied to the experiment of Fusarium graminearum opposite culture.By the strain bacterium called after JS20GA strong to Fusarium graminearum antagonistic ability obtained.
Two, identify
(1) according to " the outstanding Bacteria Identification handbook of uncle " (the 8th edition) and " common bacteria system identification handbook " (eastern elegant pearl, Cai Miaoying etc. write, Beijing: Science Press, 2001.2) method described in, carry out the qualification of morphological specificity, cultural characters and physio-biochemical characteristics to bacterial strain JS20GA, concrete outcome is as follows:
The physiology and morphology biochemical characteristic of thalline:
At NA substratum, cultivate 1 day for 28 DEG C, thalline is shaft-like, and diameter is about 0.5-0.7 μm × 1.5-3.0 μm; Bacterium colony creamy white, circular, dimpling, neat in edge.
Physiological and biochemical property:
Oxydase :+; Arginine dihydrolase :-; Gelatin hydrolysis :+; Starch Hydrolysis :+; Catalase :+; Butyrin is hydrolyzed :+; Nitrate reduction :+; Polychrom is hydrolyzed :+; Casein is hydrolyzed :+; Tween80 is hydrolyzed :-; Indoles is tested :-; Urase :-; Citric acid utilizes :+; H2S generates :-; VP tests :+; Anaerobic growth :-; 4%NaCl:-; PH4.0:+; 50 DEG C :-; 20 DEG C :+.
Glycerine can be utilized, L-arabinose, D-ribose, D-wood sugar, L-wood sugar, ribitol, D-semi-lactosi, D-Glucose, D-Fructose, D-MANNOSE, L-rhamnosyl, inositol, N.F,USP MANNITOL, sorbyl alcohol, Alpha-Methyl-D-Glucose glycosides, N-acetyl-glucosamine, arbutin, polychrom, saligenin, D-cellobiose, D-Maltose, D-lactose, D-melibiose, D-sucrose, synanthrin, D-melizitose, D-raffinose, starch, glycogen, D-Toulon sugar, D-lyxose, D-Fucose, L-fucose produces acid.
(2) 16SrDNA test
Extract the STb gene of JS20GA, with it for template, utilize bacterial 16 S rDNA universal primer to carry out pcr amplification, obtain the amplified production that length is about 1.4kb, reclaimed by amplified production and check order, the sequence recorded is as shown in SEQIDNo.1.
Compare according to Gen-Bank sequence homology, bacterial strain JS20GA and Bacillus sp.B1 (GenBank accession number KF922381.1) homology is 99%, with Bacillus amylol iquefaciens subsp.plantarumNAU-B3 (GenBank accession number HG514499.
1) homology is 99%, and this bacterium of preliminary judgement is bacillus (Bacillussp.).Based on above feature, bacterial strain JS20GA is accredited as Methylotrophic genus bacillus (Bacillusmethylotrophicus).This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 18th, 2014 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCCNo.9500.
Embodiment 2 Methylotrophic genus bacillus JS20GA is to the antagonistic action of Fusarium graminearum
Opposite culture method is adopted to detect Methylotrophic genus bacillus JS20GA to the antagonism of Fusarium graminearum.
One, be that the punch tool of 5mm is beaten get bacterium dish at cultured pathogenic fungi Fusarium graminearum (F usarium graminearum) FG1 colony edge with diameter, with the toothpick of sterilizing, bacterium dish chosen into PDA plate center.
Two, at the JS20GA apart from PDA plate center 25mm place four long and short dash line inoculation same volume and concentration, dull and stereotyped for control group with the PDA only inoculating Fusarium graminearum bacterium dish, cultivate 4-5 days for 28 DEG C, observe and formed with or without antibacterial band.Each group do three parallel.Result is as shown in table 1:
Table 1 to cultivate after 5 days JS20GA to the restraining effect of Fusarium graminearum
As Fig. 1 and table 1 is known, show that Methylotrophic genus bacillus JS20GA has very strong inhibition to Fusarium graminearum.
Embodiment 3JS20GA fermented liquid is to the inhibition of Fusarium graminearum
Opposite culture method is adopted to detect Methylotrophic genus bacillus JS20GA to the antagonism of Fusarium graminearum.
One, be that the punch tool of 5mm is beaten get bacterium dish at cultured pathogenic fungi Fusarium graminearum (F usarium graminearum) FG1 colony edge with diameter, with the toothpick of sterilizing, bacterium dish chosen into PDA plate center.
Two, at the JS20GA apart from 4, PDA plate center 25mm place punching inoculation same volume and concentration, dull and stereotyped for control group with the PDA only inoculating Fusarium graminearum bacterium dish, cultivate 4-5 days for 28 DEG C, observe and formed with or without antibacterial band.Each group do three parallel.Result is as shown in table 2.Show that JS20GA has very strong inhibition to Fusarium graminearum.
Table 2 to cultivate after 5 days JS20GA to the restraining effect of Fusarium graminearum
Embodiment 4JS20GA volatile gases is to the inhibition of Fusarium graminearum
Opposite culture method is adopted to detect the volatile gases of Methylotrophic genus bacillus JS20GA generation to the antagonism of Fusarium graminearum.
One, be that the punch tool of 5mm is beaten get bacterium dish at cultured pathogenic fungi Fusarium graminearum (F usarium graminearum) FG1 colony edge with diameter, with the toothpick of sterilizing, bacterium dish chosen into PDA plate center.
Two, at NA plate center inoculation JS20GA, back-off is in the flat board being connected to Fusarium graminearum, dull and stereotyped for control group with the PDA only inoculating Fusarium graminearum bacterium dish, cultivates 4-5 days, observes with or without fungistatic effect for 28 DEG C.Each group do three parallel.Result as shown in Figure 2.Show that JS20GA can produce the volatile gases suppressing Fusarium graminearum.
Embodiment 5JS20GA is to the greenhouse experiments of Fusarium graminearum inhibition
One, JS20GA 28 DEG C of shaken overnight in NB substratum cultivated, adopting phosphate buffered saline buffer to be diluted to concentration is 2 × 108cfu/ml.
Two, by Fusarium graminearum FG1 with sweet mung bean soup substratum shaking culture (28 DEG C, 5 days), obtain bacteria suspension, adopt phosphate buffered saline buffer dilution bacterial concentration be 2 × 105cfu/ml.
Three, mixed according to the ratio of 1:1 with Fusarium graminearum FG1 by the JS20GA after dilution, final concentration is respectively 108cfu/ml and 105cfu/ml.
Four, carry out in early days inoculating in the middle part of the wheat head in the flowering of new 828 kinds of stone, connecing that thalline amasss is 10 μ l, as experimental group.Simultaneously using the phosphate buffered saline buffer only containing Fusarium graminearum as positive controls, using only inoculate phosphoric acid buffer as negative control.Often group establishes 10 repetitions, experiment repetition 3 times, results averaged.
Fusarium graminearum bacterial suspension inoculation is after 16 days, observes the morbidity of the wheat head, has that the wheat head is withered to turn white or rubescent, and the wheat head of inner hollow kernels, weight saving performance is the morbidity wheat head.Wherein head blight index=sickness rate × disease index/100.
Table 3 JS20GA is to the field control effect of Fusarium graminearum
Result is as shown in table 3, and Methylotrophic genus bacillus JS20GA can prevent and treat the wheat scab that Fusarium graminearum causes very well under greenhouse experiment.
Claims (6)
1. a Methylotrophic genus bacillus, is characterized in that: this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 18th, 2014, and deposit number is CGMCC No.9500.
2. a kind of Methylotrophic genus bacillus according to claim 1, is characterized in that: the nucleotides sequence of this Methylotrophic genus bacillus is classified as shown in SEQ ID NO.1.
3. Methylotrophic genus bacillus according to claim 1 is used for the application in the microbial Plant diseases of biological control reaping hook.
4. Methylotrophic genus bacillus according to claim 1 is for the preparation of the application in the medicine of the microbial Plant diseases of control reaping hook.
5. the application according to claim 3 or 4, is characterized in that:
Described sickle-like bacteria is Fusarium graminearum;
Described disease is the microbial head blight of reaping hook or ear rot or root rot;
Described plant is cereal.
6. application according to claim 5, is characterized in that: described cereal is wheat or corn.
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Cited By (5)
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| CN105543132A (en) * | 2015-12-31 | 2016-05-04 | 河南省农业科学院植物保护研究所 | Bacillus methylotrophicus YB-F7 and application thereof in preventing plant diseases |
| CN106747804A (en) * | 2016-12-15 | 2017-05-31 | 中国热带农业科学院环境与植物保护研究所 | A kind of Methylotrophic gemma bacillus agent and preparation method thereof |
| CN108641989A (en) * | 2018-06-28 | 2018-10-12 | 山东农业大学 | One plant of Methylotrophic bacillus and its application |
| CN108753666A (en) * | 2018-07-02 | 2018-11-06 | 山东农业大学 | The preparation and application of one plant of salt tolerant Antagonistic Fungi M4-1 and its microbial inoculum |
| CN109825455A (en) * | 2019-03-11 | 2019-05-31 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | A kind of Methylotrophic bacillus and application |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543132A (en) * | 2015-12-31 | 2016-05-04 | 河南省农业科学院植物保护研究所 | Bacillus methylotrophicus YB-F7 and application thereof in preventing plant diseases |
| CN105543132B (en) * | 2015-12-31 | 2019-01-18 | 河南省农业科学院植物保护研究所 | A kind of Methylotrophic bacillus YB-F7 and its application in controlling plant diseases |
| CN106747804A (en) * | 2016-12-15 | 2017-05-31 | 中国热带农业科学院环境与植物保护研究所 | A kind of Methylotrophic gemma bacillus agent and preparation method thereof |
| CN108641989A (en) * | 2018-06-28 | 2018-10-12 | 山东农业大学 | One plant of Methylotrophic bacillus and its application |
| CN108641989B (en) * | 2018-06-28 | 2021-06-15 | 山东农业大学 | Bacillus methylotrophicus and application thereof |
| CN108753666A (en) * | 2018-07-02 | 2018-11-06 | 山东农业大学 | The preparation and application of one plant of salt tolerant Antagonistic Fungi M4-1 and its microbial inoculum |
| CN108753666B (en) * | 2018-07-02 | 2021-05-14 | 山东农业大学 | Salt-tolerant antagonistic bacterium M4-1 and preparation and application of microbial inoculum thereof |
| CN109825455A (en) * | 2019-03-11 | 2019-05-31 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | A kind of Methylotrophic bacillus and application |
| CN109825455B (en) * | 2019-03-11 | 2022-06-14 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | Bacillus methylotrophicus and application thereof |
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