CN106172507B - Applications of the bacillus subtilis HMB19198 in prevention powdery mildew of cucumber - Google Patents
Applications of the bacillus subtilis HMB19198 in prevention powdery mildew of cucumber Download PDFInfo
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- CN106172507B CN106172507B CN201610879296.8A CN201610879296A CN106172507B CN 106172507 B CN106172507 B CN 106172507B CN 201610879296 A CN201610879296 A CN 201610879296A CN 106172507 B CN106172507 B CN 106172507B
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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
Abstract
The invention discloses applications of bacillus subtilis (Bacillus subtilis) the bacterial strain HMB19198 in prevention powdery mildew of cucumber；Also disclose application of the microbial bacterial agent containing bacillus subtilis HMB19198 in prevention powdery mildew of cucumber.Bacillus subtilis HMB19198 of the present invention is high to the drug effect of powdery mildew of cucumber, and average preventive effect is 90.0% or more；Secondly, strong using HMB19198 preventions powdery mildew of cucumber specialization, and it is not likely to produce drug resistance, persistence is strong；Microbial bacterial agent in the method for the present invention, without environmental pollution, belongs to environmentally friendly to people, animal safety.
Description
Technical field
The invention belongs to field of biological control, and in particular to bacillus subtilis HMB19198 is in prevention powdery mildew of cucumber
Application.
Background technology
Powdery mildew of cucumber is commonly called as " white hair disease ", is caused by monofilament shell powdery mildew (Sphaerotheca fuliginea)
Fungal disease, which mainly infects cucumber leaves, next infects petiole and stem, seriously affects the photosynthetic of cucumber leaves
Effect, causes the underproduction and influences cucumber quality.The disease can fall ill from Seedling Stage to Adult plant, have popular strong, incidence
It high, short incubation period and infects the features such as frequent again.The cucumber yield caused by powdery mildew loses the general time 10% or so, and
The popular time is up to 30% or so.
Mainly have to the control method of powdery mildew of cucumber：(1) disease-resistant variety is cultivated：It is most economical effective to cultivate disease-resistant variety
Method, but since the resource of mildew-resistance is less, resistant variety phase is cultivated in the continuous evolution of pathogen biological strain in addition
To relatively difficult.(2) agricultural measures are prevented：Planting density is such as reduced, ventilation and penetrating light is paid attention to, avoids excessive nitrogen fertilizer application, it is this
Method control effect is extremely limited.(3) chemical prevention：It is the main method of current prevention powdery mildew of cucumber, mainly by spraying
Thiophanate, triadimefon, auspicious malicious copper, carbendazim, Flusilazole, celestial life or generation advanced chemical fungicide prevent powdery mildew of cucumber.So
And it is long-term largely using chemical pesticide, pathogen easy tos produce drug resistance, control effect is caused to reduce；It is easy to cause agriculture simultaneously
Product and environmental pollution cause new expression or the big generation of secondary disease.(4) biological control：Biological control has specialization by force, no
It is also easy to produce drug resistance, advantages of environment protection nontoxic to people and animals, thus is increasingly subject to the favor of people.It is known that there are many micro- lifes
Object such as bacillus licheniformis (CN102399712A), bacillus subtilis (CN1934241A, CN104403970A,
CN1911043A), trichoderma harzianum (CN102388922A, CN101575574A), Trichoderma viride (CN101724572A), trichoderma
Bacterium (CN1911043A) and Strepiomyces lavendulae (CN101481669A) etc. have good control effect to powdery mildew of cucumber, but
It is due to diversity of organism, for different pathogens, different microorganisms is different to its resistance, and due to common evolutionary
Characteristic, pathogen constantly generate new biological strain, therefore, new microorganism should constantly be selected to cope with the physiology of powdery mildew of cucumber
The variation of microspecies.
Bacillus subtilis strain HMB19198 is mainly used in cucumber downy mildew (CN102586142A) and ginseng stand it is withered
The prevention of sick (CN105746579A).
Through retrieval, reports of the bacillus subtilis HMB19198 for preventing powdery mildew of cucumber is not found.
Invention content
Present invention aims at provide applications of the bacillus subtilis strain HMB19198 in prevention powdery mildew of cucumber.
Another object of the present invention is to provide a kind of microbial bacterial agents containing bacillus subtilis strain HMB19198
Application in prevention powdery mildew of cucumber.
Third of the present invention is designed to provide the method for preventing powdery mildew of cucumber using mentioned microorganism microbial inoculum.
The invention is realized by the following technical scheme：
The present invention provides bacillus subtilis (Bacillus subtilis) bacterial strain HMB19198 in prevention cucumber white powder
Application in disease.
Bacillus subtilis (Bacillus subtilis) bacterial strain HMB19198 described in above application was in 2011 12
The moon is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 20th, and deposit number is：CGMCC
No.5613。
The present invention also provides the microbial bacterial agents containing bacillus subtilis HMB19198 in prevention powdery mildew of cucumber
Application.
The microbial bacterial agent, active constituent are bacillus subtilis HMB19198 thalline.
The microbial bacterial agent be can be liquid preparation.
The liquid preparation of the microbial bacterial agent, is prepared as follows：
(1) actication of culture：The HMB19198 bacterial strains of Cord blood are activated on LB plating mediums, picking single bacterium colony exists
On LB slant mediums, is cultivated 16~24 hours at 30~32 DEG C, obtain the bacterial strain of activation；
(2) prepared by seed liquor：The bacterial strain of an oese step (1) activation is scraped with sterile oese and is inoculated into
In 100mL LB liquid mediums, in 28~34 DEG C, shaking speed to be cultivated 20~24 hours under the conditions of 170~210rpm, obtain
Seed liquor；
(3) fermented and cultured：The seed liquor of step (2) is linked into sucrose beans by the ratio for being 0.5~3.0% according to volume ratio
In cake powder culture medium (pH value 5.8-6.2), ferment under the conditions of temperature is 28~34 DEG C, shaking speed is 170~210rpm
36~54h is cultivated, zymotic fluid is obtained；The constituent and its weight percent of the sucrose beancake powder culture medium be：Sucrose 2.0
~3.0%, beancake powder 1.6~2.4%, NaCl 0.1~0.2%, CaCO30.1~0.3%, KH2PO40.01~0.03%
And MgSO4·7H2O 0.01~0.03%, remaining is water；
(4) thalline and Number of spores in zymotic fluid are detected, waits for that grown spore in zymotic fluid accounts for gemma and thalline sum
Stop fermented and cultured when 90%；Gained is the liquid preparation of HMB19198 bacterial strains.
Aforesaid liquid preparation, the temperature described in step (2) or (3) are preferably 30~32 DEG C；The shaking speed
Preferably 210rpm；The incubation time is preferably 48h.
LB plating mediums, LB slant mediums and the LB liquid medium is conventionally prepared.
The preparation method of the sucrose soya-bean cake culture medium, according to weight percent by sucrose, beancake powder, NaCl,
CaCO3、KH2PO4And MgSO4·7H2O is mixed, and is added water, is stirred evenly.
The viable count of mentioned microorganism microbial inoculum, bacillus subtilis HMB19198 is more than 1.0 × 109cfu/mL。
The method for preventing powdery mildew of cucumber using mentioned microorganism microbial inoculum includes with water by the liquid of mentioned microorganism microbial inoculum
Preparation diluent to viable bacteria body number is 107Cfu/mL is sprayed before the onset of powdery mildew of cucumber on cucumber blade face.
The present invention has the advantage that and advantageous effect：(1) present invention provides one efficiently for prevention powdery mildew of cucumber
Microorganism opens an effective controlling way；(2) drug effects of the bacillus subtilis HMB19198 of the present invention to powdery mildew of cucumber
Height, average preventive effect is 90.0% or more；(3) it utilizes the method for the present invention prevention powdery mildew of cucumber specialization strong, and is not likely to produce anti-
Pharmacological property, persistence are strong；(4) microbial bacterial agent in the method for the present invention is to people, animal safety, without environmental pollution；(5) present invention makes
Microbial bacterial agent preparation method is simple, application method is simple, at low cost, application easy to spread.
Specific implementation mode
The present invention is further clearly explained with specific embodiment below, but is not constituted in any way to the present invention
Limitation.Experimental method in following embodiments is unless otherwise instructed conventional method；Involved by following embodiments
LB plating mediums, LB slant mediums, LB liquid medium etc. are conventionally prepared.
The preparation of 1 HMB19198 microbial bacterial agents of embodiment
It carries out in accordance with the following steps：
(1) actication of culture：- 80 DEG C bacillus subtilis (Baci11us subtilis) bacterial strain will be stored in
HMB19198 (deposit number is CGMCC No.5613) is activated (30 DEG C) on LB plating mediums, and picking single bacterium colony exists
It is cultivated 20 hours at 32 DEG C on LB slant mediums, obtains the bacterial strain of activation；
(2) preparation of seed liquor：LB liquid medium 100mL is packed into 250mL triangular flasks, high pressure moist heat sterilization waits for
Activated bacterial strain in an oese step (1) is accessed after temperature drops to room temperature, in every bottle, in 30 DEG C, shaking speed 190rpm
Under conditions of shaken cultivation hour for 24 hours, obtain seed liquor；
(3) preparation of sucrose soya-bean cake culture medium：According to weight percent by sucrose 3.0%, beancake powder 2%, NaCl
0.1%, CaCO30.3%, KH2PO40.02% and MgSO4·7H2O 0.03% is added to the water, and is uniformly mixed to get sugarcane
Sugared soya-bean cake culture medium；It is sub-packed in triangular flask, per bottled 200mL；30 points of sterilizing is carried out to sucrose soya-bean cake culture medium at 121 DEG C
Clock, then cool to 30 DEG C it is spare；
(4) fermented and cultured：The seed obtained by inoculation step (2) into every bottle of sucrose soya-bean cake culture medium 200mL obtained by step (3)
Liquid 2mL；Fermented and cultured 36h is carried out under the conditions of 30 DEG C, shaking speed 190rpm, is sampled from triangular flask every 30 minutes later
Carry out microscopy, in the visual field gemma and total thalline number count, and calculate gemma rate (gemma rate (%)=grown spore
Number/(grown spore number+thalline number) × 100)；Gemma rate stops fermented and cultured when reaching 90%；Common fermentation culture about 48h, obtains
The liquid preparation of bacillus subtilis HMB19198.
2 HMB19198 microbial bacterial agents of embodiment are to powdery mildew of cucumber control effect contrast test
(1) test material：
(1), cucumber variety：Excellent No. 1 of Tianjin.
(2), powdery mildew of cucumber pathogen conidial suspension：Initial strains come from Agricultural University Of Hebei, and inoculation is yellow
Melon powdery mildew pathogenic bacteria conidial suspension plants diease occurrence by initial strains in Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie
Numerous get is expanded in anti-laboratory.
(2) test process：
(1), 100 times of water diluents of HMB19198 liquid preparations prepared by embodiment 1；
(2), blank control：Clear water.
(3) test method：
This experiment carries out in Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie's disease flocking biocontrol laboratory.In diameter 6cm
Nutritive cube in cultivate excellent No. 1 cucumber seedling in Tianjin, seedling medium is vermiculite, and each nutritive cube sows two seeds, is left and taken after emergence
One strong sprout.Carry out experiment after cucumber seedling cotyledon completely flattening.Experiment sets 2 processing, and processing 1 is to be sprayed in cucumber cotyledons
100 times of water diluents of HMB19198 liquid preparations prepared by embodiment 1, processing 2 are blank control, spray equivalent clear water.Often
A processing is repeated 4 times, and each repeats 20 plants of cucumber seedlings.At 25 DEG C moisturizing for 24 hours, then spray inoculation powdery mildew of cucumber pathogen
Conidial suspension (1 × 105A/mL), in 25 DEG C of constant temperature incubations (illumination 14 hours, 10 hours dark).Wait for blank control
After the onset of cotyledon is abundant, investigates the cucumber cotyledons powdery mildew state of an illness and calculate disease index and preventive effect.Investigation method, grade scale and
Control effect computational methods are with reference to national standard pesticide field efficacy medicine test criterion (one) bactericidal agent for preventing and treating powdery mildew of cucumber (GB/
T17980.30-2000)。
As a result the disease index for (being shown in Table 1) and being handled through HMB19198 liquid preparations is 0.46, substantially less than blank control
Disease index (31.83), HMB19198 liquid preparations are to the preventive effect of powdery mildew of cucumber up to 98.56%.The above results illustrate withered grass
Bacillus strain HMB19198 and its HMB19198 liquid preparation have control effect outstanding to powdery mildew of cucumber.
1 HMB19198 microbial bacterial agents of table are to powdery mildew of cucumber control effect comparative test result
| Processing | Disease index | Preventive effect (%) |
| HMB19198 liquid preparations | 0.46b | 98.56 |
| Blank control | 31.83a | / |
3 HMB19198 microbial bacterial agents of embodiment prevent powdery mildew of cucumber Piglet s colibacillosis
(1) test material：
(1), cucumber variety：Excellent No. 1 of Tianjin.
(2), powdery mildew of cucumber pathogen conidial suspension, initial strains come from Agricultural University Of Hebei, and inoculation is yellow
Melon powdery mildew pathogenic bacteria conidial suspension plants diease occurrence by initial strains in Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie
Numerous get is expanded in anti-laboratory.
(2) test process：
(1), 100 times of water diluents of HMB19198 liquid preparations prepared by embodiment 1；
(2), blank control：Clear water.
(3) test method：
This experiment carries out in Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie's disease flocking biocontrol laboratory.In diameter 6cm
Nutritive cube in cultivate excellent No. 1 cucumber seedling in Tianjin, seedling medium is vermiculite, and each nutritive cube sows two seeds, is left and taken after emergence
One strong sprout.Be transplanted to when cucumber seedling grows two panels true leaf in the plastic flowerpot of diameter 15cm (in flowerpot load vermiculite with
Rural area soil is according to 1:The matrix that 1 ratio is made into), in 25 DEG C of constant temperature incubations (illumination 14 hours, 10 hours dark).Cucumber seedling is used
Bamboo pole support is fixed, and carries out experiment after growing 5-7 piece true leaves.Processing 1 sprays the HMB19198 liquid preparations of the preparation of embodiment 1
100 times of water diluents；Processing 2 is blank control, sprays equivalent clear water.Each processing is repeated 4 times, each to repeat 5 basin cucumber
Seedling.Moisturizing is for 24 hours under the conditions of 25 DEG C.Spray inoculation powdery mildew of cucumber pathogen conidial suspension (1 × 10 after for 24 hours5A/
ML), 25 DEG C of constant temperature incubations (illumination 14 hours, 10 hours dark) are continued at.After the onset of waiting for that blank control true leaf is abundant, investigation is yellow
The melon true leaf powdery mildew state of an illness simultaneously calculates disease index and preventive effect.Investigation method, grade scale and control effect computational methods reference
GB/T17980.30-2000。
As a result the cucumber leaves of the HMB19198 liquid preparations that (are shown in Table 2) processing are not fallen ill, and disease index 0.00 is aobvious
The disease index (87.05) less than blank control is write, HMB19198 liquid preparations are to the anti-of the powdery mildew of cucumber on cucumber leaves
Effect is controlled up to 100%.The above results illustrate bacillus subtilis strain HMB19198 and its HMB19198 liquid preparation to cucumber
Powdery mildew has control effect outstanding.
2 HMB19198 microbial bacterial agents of table prevent powdery mildew of cucumber Piglet s colibacillosis result
| Processing | Disease index | Preventive effect (%) |
| HMB19198 liquid preparations | 0.00b | 100.00 |
| Blank control | 87.05a | / |
4 HMB19198 microbial bacterial agents of embodiment are to powdery mildew of cucumber control effect contrast test
(1) test material：Excellent No. 1 of cucumber variety Tianjin.
(2) test process：
(1) 100 times of water diluents of HMB19198 liquid preparations prepared by embodiment 1.
(2) 10% difenoconazole water dispersible granules (Swiss Syngenta Crop Protection Co., Ltd.).
(3) blank control：Clear water.
(3) test method：
This experiment was satisfied in Hebei province Baoding Xushui County in cities and towns north of the city village village poplar Golconda cucumber booth in spring in 2015
Carry out.It ploughs, apply fertilizer, sowing, watering with local farming operations.3 processing of experiment setting, processing 1 is microbial bacterial agent, is sprayed
100 times of water diluents of HMB19198 liquid preparations prepared by embodiment 1；Processing 2 is chemical agent, sprays 10% difenoconazole
800 times of water diluents of water dispersible granules (Swiss Syngenta Crop Protection Co., Ltd.)；Processing 3 is blank control, sprays equivalent
Clear water.Each processing is repeated 4 times.3 rows are each repeated, often about 25 cucumber seedlings of row.Start to try before powdery mildew of cucumber morbidity
Test, it is primary to be spaced processing in 7-10 days, until blank control is fully fallen ill, investigate the powdery mildew of cucumber state of an illness and calculate disease index with
Preventive effect.Investigation method, grade scale and control effect computational methods are with reference to GB/T17980.30-2000.
As a result the disease index of the HMB19198 liquid preparations that (are shown in Table 3) processing is 4.53, substantially less than the blank control state of an illness
The disease index (8.36) of index (76.25) and chemical agent is 94.06% to powdery mildew of cucumber preventive effect, is better than chemical agent
The preventive effect (89.04%) of control.The above results illustrate bacillus subtilis HMB19198 and its microbial bacterial agent to cucumber white powder
Disease has control effect outstanding.
3 HMB19198 liquid preparations of table are to powdery mildew of cucumber preventive effect comparative test result
| Processing | Disease index | Preventive effect (%) |
| HMB19198 liquid preparations | 4.53c | 94.06 |
| Chemical agent | 8.36b | 89.04 |
| Blank control | 76.25a | / |
Claims (8)
1. applications of bacillus subtilis (Bacillus subtilis) the bacterial strain HMB19198 in prevention powdery mildew of cucumber；Its
Described in bacillus subtilis (Bacillus subtilis) bacterial strain HMB19198 be preserved in China on December 20th, 2011
Microbiological Culture Collection administration committee common micro-organisms center, deposit number are：CGMCC No.5613.
2. application of the microbial bacterial agent containing bacillus subtilis HMB19198 in prevention powdery mildew of cucumber.
3. application according to claim 2, it is characterised in that the active constituent of the microbial bacterial agent is withered grass gemma
Bacillus HMB19198 thalline.
4. application according to claim 2 or 3, it is characterised in that the microbial bacterial agent is liquid preparation.
5. application according to claim 4, it is characterised in that the liquid preparation of the microbial bacterial agent, according to such as lower section
It is prepared by method：
(1) the HMB19198 bacterial strains of Cord blood are activated on LB plating mediums, picking single bacterium colony is in LB slant mediums
On, it is cultivated 16~24 hours at 30~32 DEG C, obtains the bacterial strain of activation；
(2) it scrapes the bacterial strain of an oese step (1) activation with sterile oese and is inoculated into 100mLLB fluid nutrient mediums
In, in 28~34 DEG C, shaking speed to be cultivated 20~24 hours under the conditions of 170~210rpm, obtain seed liquor；
(3) seed liquor of step (2) is linked into the sucrose that pH value is 5.8-6.2 by the ratio for being 0.5~3.0% according to volume ratio
In beancake powder culture medium, 36~54h of fermented and cultured under the conditions of temperature is 28~34 DEG C, shaking speed is 170~210rpm is obtained
Zymotic fluid；The constituent and its weight percent of the sucrose beancake powder culture medium be：Sucrose 2.0~3.0%, beancake powder
1.6~2.4%, NaCl 0.1~0.2%, CaCO30.1~0.3%, KH2PO40.01~0.03% and MgSO4·7H2O
0.01~0.03%, remaining is water；
(4) thalline and Number of spores in zymotic fluid are detected, waits for that grown spore accounts for the 90% of gemma and thalline sum in zymotic fluid
When stop fermented and cultured；Gained is the liquid preparation of HMB19198 bacterial strains.
6. application according to claim 5, it is characterised in that the temperature described in its step (3) is 30~32 DEG C；It is described
Shaking speed be 210rpm；The incubation time is 48h.
7. application according to claim 4, it is characterised in that bacillus subtilis in the microbial bacterial agent
The viable count of HMB19198 is more than 1.0 × 109cfu/mL。
8. utilizing the method for the microbial bacterial agent prevention powdery mildew of cucumber containing bacillus subtilis HMB19198, it is characterised in that
It is 10 that the liquid preparation of the microbial bacterial agent obtained by claim 5, which is diluted to viable bacteria body number, with water7Cfu/mL, in cucumber white powder
It is sprayed on cucumber blade face before the onset of disease.
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106942276A (en) * | 2017-03-16 | 2017-07-14 | 河北省农林科学院植物保护研究所 | Bacillus subtilis BAB 1 prevents and treats the application of cucurbits powdery mildew |
| CN109423461A (en) * | 2017-08-31 | 2019-03-05 | 上海多利农业发展有限公司 | A kind of method and its application for the microorganism formulation preparing bacillus subtilis |
| CN107593802A (en) * | 2017-09-27 | 2018-01-19 | 贵州蓝天生态农业科技发展有限公司 | A kind of composition for treating Momordica grosvenori powdery mildew and preparation method thereof |
| CN109691467A (en) * | 2017-10-23 | 2019-04-30 | 上海崇明低碳农业科技有限公司 | A kind of application of bacillus subtilis microbial preparation in prevention and treatment crop powdery mildew |
| CN109234172A (en) * | 2018-10-18 | 2019-01-18 | 天津大学 | A kind of complex micro organism fungicide for preventing and treating Powdery Mildew of Euonymus japonicus L |
| CN112400870A (en) * | 2020-11-27 | 2021-02-26 | 云南省微生物发酵工程研究中心有限公司 | Bacillus licheniformis water dispersible granule and application thereof |
| CN113265480B (en) * | 2021-06-08 | 2022-03-18 | 河北省农林科学院植物保护研究所 | Specific primer of bacillus subtilis HMB19198 strain, Taqman probe and application |
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| CN102586142A (en) * | 2012-02-09 | 2012-07-18 | 河北省农林科学院植物保护研究所 | Bacillus subtilis for preventing and curing cucumber downy mildew and microbial inoculants thereof |
| CN105746579A (en) * | 2016-02-02 | 2016-07-13 | 安建慧 | Application of bacillus subtilis HMB19198 preparation in control of rhizoctonia silani |
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