CN106190920B - Bacillus subtilis YN145 and its application - Google Patents

Bacillus subtilis YN145 and its application Download PDF

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CN106190920B
CN106190920B CN201610643690.1A CN201610643690A CN106190920B CN 106190920 B CN106190920 B CN 106190920B CN 201610643690 A CN201610643690 A CN 201610643690A CN 106190920 B CN106190920 B CN 106190920B
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刘二明
邹秋霞
任佐华
刘敏捷
周鑫钰
刘双清
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Hunan Agricultural University
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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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Abstract

The present invention relates to technical field of biological control, specifically disclose a bacillus subtilis YN145 and its application in prevention and treatment rice blast, and the deposit number of the bacillus subtilis YN145 is CCTCC No:M2016339.Rice blast is prevented and treated using bacterial strain YN145, avoids the susceptibleization risk that chemical prevention utilizes the potential adverse effect of environment and rice safety and disease-resistant variety.Bacterial strain YN145 living body bacterium and its metabolite have preferable preventive effect to rice blast, and protection and therapeutic effect have both, can develop as the formulation of living microbial inoculum and metabolite.In addition, bacterial strain YN145 has inhibitory effect to a variety of cause of disease indicator bacterias.

Description

Bacillus subtilis YN145 and its application
Technical field
The present invention relates to technical field of biological control, specifically, being related to a kind of bacillus subtilis YN145 and its anti- Control the application in rice blast.
Background technique
Rice (Oryza sativa) is one of most important cereal crops, and the whole world has the populations more than half to be with rice Food.Rice blast (germ is Magnaporthe oryzae, and asexual generation is Pyicularia oryzae) is rice most serious One of fungal disease wanted.Pyricularia oryzae (M.oryzae) is that a kind of half living body is parasitic, is had in the infection initial period very bright The aobvious biotroph stage.Rice blast can be divided into seedling pest, leaf pest, section pest, fringe according to its site of pathological change and occurrent time difference Pest, grain pest, branch stalk pest etc..Production at present is upper mainly to control rice blast using disease-resistant variety and using chemical bactericide.Mainly The fungicide used has tricyclazole, Isoprothiolane, Prochloraz, fenoxanil, different rice blast net, kitazine, edifenphos (Hinosan), third Ring azoles, Fluoxastrobin, Tebuconazole, probenazole, chlorobromide isocyanuric acid, sym-closene, Rabcide, oxime bacterium Tebuconazole (taking enemy steady), sulphur tricyclazole, first sulphur tricyclazole (rice saliva), different rice Isoprothiolane, propiconazole prochloraz etc.；Biological production Product chemistry effective component or living microorganism such as kasugarnycin, S-Ethyl ethylthio sulfonate, amino-oligosaccharide, bacillus subtilis, solution starch bud Spore bacillus etc..Since the genetic background of Pyricularia oryzae is complicated, Yi Bianyi, in addition anti-pest single variety, the heredity of main breed Homogeney is high, and disease-resistant variety is hard to keep resistance to rice blsat, frequently results in a new anti-pest kind and plants several years in large area After lose resistance, and susceptibleization.In the case where disease-resistant variety is not able to satisfy production needs, chemical prevention is prevention and treatment rice blast Effective means, but since medication is unreasonable, potential adverse effect is brought to ecological safety, is also worth people's concern.
Biological control be utilize beneficial microbe living body or metabolite kill or reduce the quantity of pathogenic microorganism with Plant disease is controlled to occur and endanger.Consider from the sustainable control of plant disease, biological control, which is that one kind is green safe, to be had The control measure of effect.
However, can often be encountered in field of biological control antagonistic microbe colonazition under the natural conditions of field it is poor, to disease The problems such as control efficiency is unstable.A large amount of research also indicates that the antagonistic microbe that lab screening goes out is in the greenhouse to pathogen There is stronger inhibiting effect, and effect is undesirable in control in field, this may be since one side bacterial strain is not suitable with crop field life State environment and the rhizosphere that plant cannot be colonized well, on the other hand with microbe species and antagonism complicated in field soil It interacts between bacterial strain, leads to the reduction of antagonistic microbe preventive effect, therefore, the field that biological bacterial strain is screened in selection adapts to energy Power is strong or efficient metabolite to be applied to crop field disease control be the important channel to solve the above problems.
Summary of the invention
The object of the present invention is to provide one plant of biocontrol microorganisms-bacillus subtilises newly separated (Bacillus subtilis) YN145。
It is a further object of the present invention to provide application of the bacillus subtilis YN145 in prevention and treatment rice blast.
In order to achieve the object of the present invention, bacillus subtilis of the invention (B.subtilis) YN145 is from Hunan Province peach The disease in morning Xian 24, rice blast rice kind Hunan of Jiang Shiniu Jiang Zhenhua Tian Cun (N28 ° 42 ' 55.56 ", E112 ° 15 ' 74.24 ") sense Choose what healthy rice strain separation obtained in clump, using with Pyricularia oryzae tablet face-off method filter out to Pyricularia oryzae Mycelial growth Antagonistic strain of the rate up to 84.31%, while the bacterial strain has good antagonism to a variety of fungal pathogens, such as to the red star of tobacco Germ (Alternaria alternata), grape botrytis cinerea (Botrytis cinerea), capsicum colletotrichum gloeosporioides Penz The percentage mycelial inhibition of (Colletotrichum gloeosporioides), Causal Organism of Cucumber Blight (Phytophthora melonis) Respectively reach 74.60%, 53.50%, 70.00% and 63.49%；Using inhibition zone method discovery bacterial strain YN145 to bacteria pathogeny There is also certain effects for bacterium, such as (Xanthomonas oryzae pv.oryzae, is abbreviated as rice leaf spot bacteria Xoo), the inhibiting rate of avenae subsp.citrull (Pseudomonas syringae pv.lachrymans) respectively reaches 33.09% With 30.15%.And there is certain inhibiting effect to the ability of the production melanin of Pyricularia oryzae.It has been preserved in Chinese Typical Representative culture Object collection, address: Wuhan, China, Wuhan University, postcode 430072, deposit number CCTCC NO:M2016339, preservation day June 22 2016 phase.
Bacterial strain YN145 is cultivated on NA plate to carry out observation to its colonial morphology and sees to the scanning electron microscope (SEM) photograph of its thallus It examines, colonial morphology is irregular, and rough surface is opaque, marginal swell, there is fold, yellowish.YN145 thallus is in the shape of a rod, there is whip Hair, size are (0.6~0.8) μ m (2.0~4.0) μm, and gemma ellipse to column is 0.5~1.0 μm long, form such as Fig. 1 institute Show.
Bacterial strain YN145 can make Starch Hydrolysis, gelatin liquefaction, and catalase test, VP test, methyl red test, nitrate are also Original test is positive, and so that hydrogen sulfide production test, indole test and phenylalanine deaminase test is negative, can utilize D-Fructose, Portugal Grape sugar, sucrose, xylose, corn flour, PEARLITOL 25C, D-arabinose.
The 16S rDNA sequence of bacterial strain YN145 is as shown in SEQ ID NO.1.
The present invention also provides the microbial inoculums prepared by the bacillus subtilis YN145.
The present invention also provides the bacillus subtilis YN145 or microbial inoculum prepared therefrom in prevention and treatment rice blast Using.
Application above-mentioned includes that the bacillus subtilis YN145 or microbial inoculum prepared therefrom are applied to rice crop. Wherein, in the microbial inoculum bacillus subtilis YN145 viable bacteria concentration >=1 × 107cfu/mL。
The present invention further provides a kind of methods for preventing and treating rice blast comprising by the bacillus subtilis The culture solution of YN145 is centrifuged, and living body bacterium precipitating and supernatant are obtained, and living body bacterium preparation bacteria suspension is applied to rice and is made Object, or supernatant (containing metabolite) is applied to rice crop.
Optionally, the preparation method of the supernatant (metabolite) includes: by bacterial strain YN145 in NA solid medium It is activated on (peptone 5g/L, beef extract 3g/L, glucose 20g/L, agar 17g/L, pH 7.0, distilled water are prepared) plate Afterwards, it is beaten with punch and 2 bacteria cakes of diameter 5mm is taken to access equipped with 150mL NB fluid nutrient medium (peptone 5g/L, beef extract 3g/L, glucose 20g/L, pH 7.0, distilled water are prepared) 250mL triangular flask in, 28 DEG C, under the conditions of 180r/min, shaking table vibration Culture 4d is swung, is filtered with biofilter, bacillus subtilis YN145 metabolite is obtained.
Further, the method for administration is preferably foliage-spray.
I.e. by bacterial strain YN145 metabolite or living body bacterium (concentration >=1 × 107Cfu/mL it) before Pyricularia oryzae infects or invades 1~3d after dye is applied on rice crop using foliar spray method, can be played preferable preventive effect effect, be also demonstrated the bacterium Strain has good treatment rice blast effect.
When prevention, it can be applied at Rice Heading initial stage, method of administration is same as above.
Preferably, amount of application is thallus 4.5 × 1012cfu/hm2~4.5 × 1013cfu/hm2；Thallus and metabolite It is used in mixed way 4.5 × 1011cfu/hm2~4.5 × 1012cfu/hm2。
In addition, have certain effect to a variety of fungal pathogen indicator bacterias using plate opposite culture method discovery bacterial strain YN145, Such as tobacco brown spot pathogen (A.alternata), grape botrytis cinerea (B.cinerea), capsicum colletotrichum gloeosporioides Penz (C.gloeosporioides), the bacteriostasis rate of Causal Organism of Cucumber Blight (P.melonis) growth be respectively 74.60%, 53.50%, 70.00% and 63.49%；Using inhibition zone method discovery bacterial strain YN145, to bacterial disease opportunistic pathogen, there is also certain effects, such as water Rice bacterial leaf spot pathogenic bacteria (X.oryzae pv.oryzae, be abbreviated as Xoo), avenae subsp.citrull (P.syringae Pv.lachrymans inhibiting rate) respectively reaches 33.09% and 30.15%.
The beneficial effects of the present invention are:
The present invention obtains a kind of bacillus subtilis YN145 through screening and has carried out preservation to it, and the present invention is utilized to provide Bacillus subtilis YN145 rice blast is prevented and treated, avoid chemical prevention to environment and rice safety it is potential The susceptibleization risk that adverse effect and disease-resistant variety utilize.Bacterial strain YN145 living body bacterium and its metabolite have to rice blast compared with Good preventive effect, protection and therapeutic effect have both, can develop as the formulation of living microbial inoculum and metabolite.
Detailed description of the invention
Fig. 1 is the bacterial strain scanning electron microscope hypothallus form of bacillus subtilis YN145 of the present invention.
Fig. 2 is bacillus subtilis YN145 cultivation results on NA plate in the embodiment of the present invention 1；Wherein, A is bacterium colony Form, B are scribing line cultivation results.
Fig. 3 is bacillus subtilis YN145 in the embodiment of the present invention 1 to the growth inhibitory effect of Different Kinds of Pathogens indicator bacteria； Wherein, A1 is tobacco brown spot pathogen (A.alternata)；A2 is YN145 to tobacco brown spot pathogen mycelia growth inhibitory effect；B1 For grape botrytis cinerea (B.cinerea)；B2 is YN145 to grey mold mycelia growth inhibitory effect.
Fig. 4 is bacillus subtilis YN145 in the embodiment of the present invention 2 and its metabolite to Pyricularia oryzae (M.oryzae) Mycelia growth inhibitory effect；Wherein, A is bacterial strain YN145 to Growth of Magnaporthe Grisea inhibitory effect, and CK is Pyricularia oryzae；B is bacterium For the metabolite of strain YN145 to Pyricularia oryzae mycelia growth inhibitory effect, CK is Pyricularia oryzae.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
The separation of 1 bacillus subtilis YN145 of embodiment obtains
From Hunan Province, Hunan Province, the town Shi Niujiang, Taojiang flower Tian Cun (N28 ° 42 ' 55.56 ", E112 ° 15 ' 74.24 ") sense rice blast Choose healthy rice strain in sick rice varieties Hunan morning Xian 24 sick clumps, isolate 517 plants of bacterial strains, using with Pyricularia oryzae plate Face-off method filters out 1 plant of Antagonistic Fungi YN145 to Pyricularia oryzae (M.oryzae) percentage mycelial inhibition up to 84.31%.
By in NA solid medium (peptone 5g/L, beef extract 3g/L, glucose 20g/L, agar 17g/L, pH 7.0, distilled water is prepared) bacterial strain YN145 (Fig. 2A and Fig. 2 B) is cultivated on plate, its colonial morphology is observed, to its thallus Scanning electron microscope (SEM) photograph observation, and combine the Physiology and biochemistry of the bacterium, microbiologic inhibition tests as a result, and 16S rDNA sequencing result (see SEQ ID NO.1), it is final to determine that bacterial strain YN145 is bacillus subtilis (B.subtilis).
In addition, there is inhibitory effect to a variety of fungal pathogen indicator bacterias using plate opposite culture method discovery bacterial strain YN145 (Fig. 3), such as tobacco brown spot pathogen (A.alternata), grape botrytis cinerea (B.cinerea), capsicum colletotrichum gloeosporioides Penz (C.gloeosporioides), the bacteriostasis rate of Cucumber Blight (P.melonis) growth be respectively 74.60%, 53.50%, 70.00% and 63.49%；Using inhibition zone method discovery bacterial strain YN145, to bacterial disease opportunistic pathogen, there is also certain effects, such as water Rice bacterial leaf spot pathogenic bacteria (Xoo), avenae subsp.citrull (P.syringae pv.lachrymans) inhibiting rate respectively reach 33.09% and 30.15%.
Application of the 2 bacillus subtilis YN145 of embodiment in rice blast prevention and treatment
After bacterial strain YN145 is activated in NA solid medium tablets, is beaten with punch and 2 bacteria cakes of diameter 5mm is taken to access Equipped with 150mL NB fluid nutrient medium (peptone 5g/L, beef extract 3g/L, glucose 20g/L, pH 7.0, distilled water are prepared) 250mL triangular flask in, 28 DEG C, 180r/min condition shaken cultivation 4d in shaking table are filtered with biofilter, obtain withered grass Bacillus YN145 metabolite.
Bacterial strain YN145 metabolite and PDA culture medium are mixed with the amount of volume (mL) 1:8, connect the rice blast that diameter is 7mm Germ bacteria cake is on the culture medium prepared, and replacing antagonism bacteria culture fluid with sterile water is control, and 28 DEG C are cultivated, and measures rice after 10d The size of the bacteria cake of seasonal febrile diseases bacterium, 3 repetitions are as the result is shown 84.31% to the inhibiting rate of Pyricularia oryzae mycelia.
Greenhouse pot culture test includes YN145 bacterial strain various concentration living body bacterium and metabolite to rice height sense rice blast kind The rice blast control efficiency of Hunan evening Xian 12 tests two parts, and test carries out in tissue culture temperature control illumination cultivation room.
YN145 living body bacterium is to rice height sense kind Hunan evening Xian 12 rice blast control efficiency: being inoculated with rice using spray-on process (conidium concentration is 1 × 10 to seasonal febrile diseases bacterium5A/mL) induce rice blast.By YN145 viable bacteria concentration gradient design are as follows: 1 × 1010cfu/mL、1×109cfu/mL、1×108cfu/mL、1×107Cfu/mL totally 4 concentration gradients.Test includes 3 kinds of processing Mode is respectively as follows: A group and first sprays magnaporthe grisea spore liquid, and 3d sprays the YN145 bacterial strain living bacterial liquid of 4 concentration gradients；The inoculation of B group 1d sprays the YN145 bacterial strain living bacterial liquid of 4 concentration gradients respectively before magnaporthe grisea spore suspension；C group is inoculated with magnaporthe grisea spore 3d first sprays the YN145 bacterial strain living bacterial liquid of 4 concentration gradients respectively before liquid；Clear water is set, 20% tricyclazole WP is control and 40% Isoprothiolane EC (× 500 times of liquid) is control, every kind of 3 alms bowl of processing mode, 30 plants of every alms bowl.10d carries out rice blast resistance after inoculation Classification investigation and prevention and treatment rice blast effect assessment.
YN145 Metabolite evaluates rice height sense kind Hunan evening Xian 12 rice blast control efficiency: using water Rice living body inocalation method is inoculated with rice blast pathogen conidiospore suspension, and (concentration is 1 × 105A/mL) induce rice blast.Specific test Including 3 kinds of processing modes, it is respectively as follows: A group and first sprays magnaporthe grisea spore liquid, 3d sprays metabolite；B group first sprays metabolite, Magnaporthe grisea spore liquid is sprayed after 1d；C group first sprays metabolite, sprays magnaporthe grisea spore liquid after 3d.Clear water, fungicide 40% are set Isoprothiolane EC (× 500 times of liquid) and 20% tricyclazole WP (× 400 times of liquid) is control, every kind of 3 alms bowl of processing mode, 30 plants of every alms bowl. 7-10d carries out classification investigation to rice blast resistance after inoculation and control efficiency is evaluated.
Greenhouse pot culture test result: YN145 living body bacterium is to rice height sense kind Hunan evening Xian 12 rice blast control efficiency The rice blast control efficiency that 1, YN145 Metabolite is shown in Table to rice height sense kind Hunan evening Xian 12 is shown in Table 2.
1 YN145 living body bacterium of table is to Hunan evening Xian 12 rice blast therapeutic effects
Note: above data is 3 reproducible results.
2 YN145 Metabolite of table is to Hunan evening Xian 12 rice blast control efficiency
Processing Diseased plant rate (%) Disease index Relative control effect (%)
YN145 (A group) 65.56 17.53 72.59
40% Isoprothiolane (A group) 85.55 29.87 53.28
20% tricyclazole (A group) 85.55 36.42 43.05
Clear water (A group) 97.78 63.95 /
YN145 (B group) 96.67 56.05 24.71
40% Isoprothiolane (B group) 100.00 61.85 15.08
20% tricyclazole (B group) 94.44 41.60 42.88
Clear water (B group) 100.00 74.44 /
YN145 (C group) 95.56 43.20 40.68
40% Isoprothiolane (C group) 100.00 62.22 14.57
20% tricyclazole (C group) 95.56 56.54 22.37
Clear water (C group) 100.00 74.44 /
Note: above data is 3 reproducible results.
Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

Claims (9)

  1. Bacillus subtilis 1. (Bacillus subtilis) YN145, deposit number is CCTCC No:M2016339.
  2. 2. the microbial inoculum of the preparation of the bacillus subtilis YN145 as described in claim 1.
  3. 3. the answering in prevention and treatment rice blast of microbial inoculum described in bacillus subtilis YN145 or claim 2 described in claim 1 With.
  4. 4. application according to claim 3, which is characterized in that it includes by bacillus subtilis described in claim 1 YN145 or microbial inoculum as claimed in claim 2 are applied to rice crop.
  5. 5. application according to claim 4, which is characterized in that the viable bacteria of bacillus subtilis YN145 is dense in the microbial inoculum Degree >=1 × 107cfu/mL。
  6. 6. a kind of method for preventing and treating rice blast, which is characterized in that by bacillus subtilis YN145 described in claim 1 Culture solution be centrifuged, obtain living body bacterium precipitating and supernatant, living body bacterium preparation bacteria suspension be applied to rice crop, or will Supernatant is applied to rice crop.
  7. 7. according to the method described in claim 6, it is characterized in that, method of administration is foliar spray.
  8. 8. the method according to the description of claim 7 is characterized in that imposing on rice blast miaoye pest and leaf pest beginning before and after onset 1~3d, fringe pest prevention and treatment is in heading initial stage application.
  9. 9. according to the method described in claim 8, it is characterized in that, when using the viable bacteria body, amount of application be thallus 4.5 × 1012cfu/hm2~4.5 × 1013cfu/hm2；When the viable bacteria body and the supernatant are used in mixed way, amount of application is thallus 4.5×1011cfu/hm2~4.5 × 1012cfu/hm2。
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CN107502570B (en) * 2017-08-18 2019-07-16 华中农业大学 One plant of Biocontrol Bacillus subtilis BJ-1 and its application
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