CN108719331A - A kind of combination microbial inoculum and preparation method thereof of control Ralstonia solanacearum Population, application - Google Patents
A kind of combination microbial inoculum and preparation method thereof of control Ralstonia solanacearum Population, application Download PDFInfo
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- CN108719331A CN108719331A CN201810247473.XA CN201810247473A CN108719331A CN 108719331 A CN108719331 A CN 108719331A CN 201810247473 A CN201810247473 A CN 201810247473A CN 108719331 A CN108719331 A CN 108719331A
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- ralstonia solanacearum
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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
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/45—Tobacco
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
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Abstract
The invention discloses a kind of combination microbial inoculum of control Ralstonia solanacearum Population and preparation method thereof, applications, microbial inoculum is combined by being formed with to bacillus solution mixed preparing of the Ralstonia solanacearum with antagonistic ability with the phage solution for infecting cracking ability to Ralstonia solanacearum, preparation method is：(1) bacteriophage strain for having to Ralstonia solanacearum and infecting cracking ability is obtained using the method culture co-cultured, and phage solution is obtained after membrane filtration；(2) it uses NB fluid nutrient medium cultures to obtain the Bacillus species strains that there is antagonistic ability to Ralstonia solanacearum, is centrifuged after shaken cultivation and obtain bacillus solution；(3) phage solution and bacillus solution are formulated in the ratio to get combination microbial inoculum；During tobacco growing, combination microbial inoculum is applied in the vega soil of Ralstonia solanacearum；Compared with prior art, the present invention can preferably control the Population of tobacco Ralstonia solanacearum, to reduce the generation of tobacco bacterial wilt.
Description
Technical field
The present invention relates to biological Control Technology fields more particularly to a kind of use to infect the bacteriophage for cracking tobacco Ralstonia solanacearum
And the preparation of the combination microbial inoculum of the Antagonistic Fungi of antagonism tobacco Ralstonia solanacearum, and its answering in terms of controlling tobacco Ralstonia solanacearum Population
With.
Background technology
Bacterial wilt is systemic disease caused by being infected by Ralstonia solanacearum (abbreviation Ralstonia solanacearum), it is
A kind of typical vascular bundle diseases, most significant symptom are withered, can cause whole strain dead when falling ill serious, cause to have no harvest, give
Leaf tobacco production causes heavy economic losses.Currently, tobacco bacterial wilt mainly produces cigarette provinces and regions in China generation, cause China's cigarette
The Ralstonia solanacearum of careless bacterial wilt is mainly Evolution Type I, No. 1 biological strain.Tobacco bacterial wilt is tobacco, pathogen, environment
It is coefficient as a result, in environment temperature, humidity, soil pH and soil Population of Ralstonia solanacearum etc. all with tobacco bacterial wilt
There is close relationship.
The method of Traditional control Ralstonia solanacearum Population mainly has：In time cleaning vega in tobacco rod tobacco leaf residue and its
The host plant of its bacterial wilt adds Ralstonia solanacearum using chemicals control Ralstonia solanacearum quantity, by using biological organic fertilizer
The methods of Antagonistic Fungi, soil disinfection, rational farming and cultivation step control the Population of Ralstonia solanacearum in vega.
With antibiotic be widely used and the appearance of drug resistance or drug resistance, green prevention and control and biological prevention and control conduct
The replacement of traditional drug therapies attracts attention again.It utilizes bacteriophage to prevent bacterial diseases of plants in recent years, utilizes Ralstonia solanacearum
Antagonistic microbe carry out biological prevention and control and cause to pay close attention to and inquire into.
Invention content
The technical problem to be solved by the present invention is to：There is provided it is a kind of control Ralstonia solanacearum Population combination microbial inoculum and its preparation
Method, application, with to tobacco Ralstonia solanacearum with the bacteriophage for infecting crack characteristic and to tobacco Ralstonia solanacearum with strong antagonistic ability
Bacillus combine microbial inoculum, the Population of Collaborative Control tobacco Ralstonia solanacearum infects cigarette strain to reduce tobacco Ralstonia solanacearum, drops
The generation of low tobacco bacterial wilt, with overcome the deficiencies in the prior art.
The technical scheme is that：A kind of combination microbial inoculum of control Ralstonia solanacearum Population, the combination microbial inoculum is by right
Ralstonia solanacearum is matched with being mixed to bacillus solution of the Ralstonia solanacearum with antagonistic ability with infecting the phage solution of cracking ability
It makes.
A concentration of the 1 × 10 of the phage solution7-1×109PFU/ml, the bacillus solution a concentration of 1 ×
1010-1×1012CFU/ml, phage solution are 1 by volume proportion with bacillus solution：The ratio of 10-15 is formulated.
The present invention also provides a kind of preparation methods of the combination microbial inoculum of control Ralstonia solanacearum Population, include the following steps：
(1) bacteriophage strain for having to Ralstonia solanacearum and infecting cracking ability is obtained using the method culture co-cultured, through filter
Phage solution is obtained after membrane filtration；
(2) NB fluid nutrient medium cultures is used to obtain the Bacillus species strains that there is antagonistic ability to Ralstonia solanacearum, oscillation training
Centrifugation obtains bacillus solution after supporting；
(3) phage solution and bacillus solution are formulated in the ratio to get combination microbial inoculum.
The present invention also provides the applications of the combination microbial inoculum of above-mentioned control Ralstonia solanacearum Population, during tobacco growing,
The combination microbial inoculum is applied in the vega soil of Ralstonia solanacearum.
The beneficial effects of the invention are as follows：The present invention use have to Ralstonia solanacearum infect the bacteriophage of splitting action with it is withered to blueness
Bacterium has the Population of the bacillus combination co- controlling tobacco Ralstonia solanacearum of antagonistic effect, to reduce tobacco bacterial wilt
Occur.Compared with prior art, the present invention has the advantage that：
1. safety and environmental protection, environmental-friendly, withered with the bacteriophage for infecting crack characteristic and to tobacco blueness to tobacco Ralstonia solanacearum
There is bacterium the bacillus of antagonistic properties to be all derived from vega soil.
2. pair tobacco Ralstonia solanacearum, with specificity, it is withered only to infect cracking tobacco blueness with the bacteriophage for infecting crack characteristic
Bacterium, it is harmless to other floras in soil.
3. there is pair tobacco Ralstonia solanacearum the bacillus of antagonistic properties can compete nutrition, secondary metabolite with pathogen
It is inhibited to pathogen.
Specific implementation mode
The present invention is described further with reference to specific embodiment：
Embodiment 1：Bacteriophage combines microbial inoculum with bacillus
By to Ralstonia solanacearum with infect the phage solution of cracking ability with to gemma bar of the Ralstonia solanacearum with antagonistic ability
Bacterium solution mixed preparing obtains.
Embodiment 2：Bacteriophage combines microbial inoculum with bacillus
By to Ralstonia solanacearum with infect the phage solution of cracking ability with to gemma bar of the Ralstonia solanacearum with antagonistic ability
Bacterium solution mixed preparing obtains, wherein a concentration of the 1 × 10 of phage solution7PFU/ml, a concentration of the 1 of bacillus solution
×1012CFU/ml, the two volume proportion are 1：10.
Embodiment 3：Bacteriophage combines microbial inoculum with bacillus
By to Ralstonia solanacearum with infect the phage solution of cracking ability with to gemma bar of the Ralstonia solanacearum with antagonistic ability
Bacterium solution mixed preparing obtains, wherein a concentration of the 1 × 10 of phage solution9PFU/ml, the concentration of the bacillus solution
It is 1 × 1010CFU/ml, the two volume proportion are 1：15.
Embodiment 4：Bacteriophage combines microbial inoculum with bacillus
By to Ralstonia solanacearum with infect the phage solution of cracking ability with to gemma bar of the Ralstonia solanacearum with antagonistic ability
Bacterium solution mixed preparing obtains, wherein a concentration of the 1 × 10 of phage solution8PFU/ml, a concentration of the 1 of bacillus solution
×1011CFU/ml, the two volume proportion are 1：12.
Embodiment 5：Combine the preparation method of microbial inoculum
Material：Tobacco Ralstonia solanacearum strain has tobacco Ralstonia solanacearum and infects the bacterium bacteriophage strain of cracking ability, to tobacco
Ralstonia solanacearum has the Bacillus species strains of antagonistic ability, CPG culture mediums, NB fluid nutrient mediums, SMSA solid mediums, sterile
Water, pipettor, autoclave, superclean bench etc..
CPG fluid nutrient mediums：Caseinhydrolysate 1g, peptone 10g, glucose 5g, distilled water 1000mL；pH7.0.
NB fluid nutrient mediums：Beef extract 3g, peptone 10g, distilled water 1000mL, pH 6.8~7.2.
1, the preparation of phage solution
The bacteriophage strain for having to tobacco Ralstonia solanacearum and infecting cracking ability is obtained using the method culture of co-cultivation, such as
φ PB2, φ PB12, φ PB16 (host's tobacco Ralstonia solanacearum be RsZP) in 500ml triangular flasks after sterilization, are added
300mlCPG fluid nutrient mediums and 300 sterile waters of the μ l containing Ralstonia solanacearum, shake culture 2-4h, light absorption value OD at 28 DEG C600About
Between 0.2-0.3,300 sterile waters of the μ l containing bacteriophage are added, continue shake culture 2-3h at 28 DEG C；Later with 8000r/
The rotating speed centrifugation medium 10min of min, takes supernatant, with 0.22 μm of membrane filtration, can get a concentration of 1 × 107-1×
109The phage solution of PFU/ml, 4 DEG C of Cord bloods.
2, the preparation of bacillus solution
The Bacillus species strains that there is antagonistic ability to Ralstonia solanacearum are obtained using NB fluid nutrient medium cultures, such as solve starch
Bacillus X60, bacillus amyloliquefaciens LBX-13, bacillus amyloliquefaciens LBX-34,30 DEG C of 180rpm shaken cultivations 36-
48h, then 10000rpm centrifugations 10min, obtains bacillus thalline, obtains a concentration of 1 × 1010-1×1012The bud of CFU/ml
Spore bacillus solution.
3, mixed preparing
By phage solution and bacillus solution mixed preparing, it is preferred that according to phage solution and gemma
Bacillus liquor capacity proportioning is 1：The ratio of 10-15 is configured to the combination microbial inoculum liquid of bacteriophage and bacillus, 4-10 DEG C of refrigeration
It is spare.
Embodiment 6：Combine application of microbial inoculum during tobacco growing
During tobacco growing, the combination microbial inoculum is applied in the vega soil of Ralstonia solanacearum, it is green withered with prevention
Influence of the disease to tobacco growing quality.
Test results and analysis：
Dripped using liquid/inocalation method is filled, space management group, control treatment group and processing group solution are directly dripped to containing cigarette
In the CPG culture solutions of careless Ralstonia solanacearum or it is poured into the soil of potting tobacco seedlings of tobacco Ralstonia solanacearum.
Using the culture of SMSA semi-selectives and colony counting method, group's number in different time sections tobacco Ralstonia solanacearum is detected
Amount.
SMSA solid mediums：Bacto peptone 10g, glycerine 5mL, casamino acids/casamino acid
1.0g, bacterial agar 15g, distilled water 1000mL, pH7.0.After sterilizing, when being cooled to 50 DEG C, 1.0% polymyxins is added
2.5mL, 1.0% 125 μ L of crystal violet, 1.0% triphenyltetrazolium chloride 1.25mL, 1.0% 625 μ L of bacitracin,
0.1% 125 μ L of penicillin, 1.0% 125 μ L of chloramphenicol.
1, the Experimental Comparison in CPG fluid nutrient mediums
Distilled water 40ul is added in space management group 1 in 200mlCPG fluid nutrient mediums；
Control treatment group 2 is inoculated with 20ul a concentration of 1 × 10 in 200mlCPG fluid nutrient mediums10The cigarette of CFU/ml concentration
Distilled water 20ul is added in careless Ralstonia solanacearum strain RsZP；
Processing group 3 is inoculated with 20ul a concentration of 1 × 10 in 200mlCPG fluid nutrient mediums10The tobacco of CFU/ml concentration is green
Dry strain system RsZP, be added above-mentioned phage phi PB2 and bacillus amyloliquefaciens X60 combination microbial inoculum 20ul (by a concentration of 1 ×
109The phage phi PB2 solution of PFU/ml and a concentration of 1 × 1012The bacillus amyloliquefaciens X60 solution of CFU/ml is according to volume
Than being 1：10 light and slow are uniformly mixed obtain)；
Processing group 4 is inoculated with 20ul a concentration of 1 × 10 in 200mlCPG fluid nutrient mediums10The tobacco of CFU/ml concentration is green
Withered bacterium RsZP, be added above-mentioned phage phi PB2 and bacillus amyloliquefaciens X60 combination microbial inoculum 20ul (by a concentration of 1 ×
108The phage phi PB2 solution of PFU/ml and a concentration of 1 × 1010The bacillus amyloliquefaciens X60 solution of CFU/ml is according to volume
Than being 1：15 light and slow are uniformly mixed obtain).
Processing group 5 is inoculated with 20ul a concentration of 1 × 10 in 200mlCPG fluid nutrient mediums10The tobacco of CFU/ml concentration is green
Withered bacterium RsZP is added above-mentioned a concentration of 1 × 107PFU/ml phage phi PB2 solution 1ul, distilled water 19ul.
Processing group 6 is inoculated with 20ul a concentration of 1 × 10 in 200mlCPG fluid nutrient mediums10The tobacco of CFU/ml concentration is green
Withered bacterium RsZP, is added above-mentioned concentration 1 × 1012CFU/ml bacillus amyloliquefaciens X60 solution 19ul, distilled water 1ul.
3 repetitions are often handled, shake culture at 28 DEG C is placed in, is sampled when cultivating 12,24,48 and 72h, using semi-selection
Culture medium SMSA cultures combine the Population of colony counting method detection tobacco Ralstonia solanacearum RsZP.
Control tobacco Ralstonia solanacearum Population in culture solution experiments have shown that, phage phi PB2 and bacillus amyloliquefaciens X60
The tobacco Ralstonia solanacearum Population that can control well of combination microbial inoculum, it is obviously preferable when effect is administered alone compared with the two.
1 bacteriophage of table combines the effect of the Population of tobacco Ralstonia solanacearum in microbial inoculum control culture medium with bacillus
2, the Experimental Comparison in potting soil
Distilled water 15ml is added in space management group 1 in potting soil (2.0kg)；
Control treatment group 2 is added a concentration of 1 × 10 in potting soil (2.0kg)10The tobacco Ralstonia solanacearum strain of CFU/ml
Distilled water 5ml is added in RsZP10ml；
Processing group 3 is added a concentration of 1 × 10 in potting soil (2.0kg)10The tobacco Ralstonia solanacearum strain of CFU/ml
RsZP10ml, be added above-mentioned phage phi PB2 and bacillus amyloliquefaciens X60 combination microbial inoculum 5ml (by a concentration of 1 ×
109The phage phi PB2 solution of PFU/ml and a concentration of 1 × 1012The bacillus amyloliquefaciens X60 solution of CFU/ml is according to volume
Than being 1：10 light and slow are uniformly mixed obtain)；
Processing group 4 is added a concentration of 1 × 10 in potting soil (2.0kg)10The tobacco Ralstonia solanacearum strain of CFU/ml
RsZP10ml, be added above-mentioned phage phi PB2 and bacillus amyloliquefaciens X60 combination microbial inoculum 5ml (by a concentration of 1 ×
108The phage phi PB2 solution of PFU/ml and a concentration of 1 × 1010The bacillus amyloliquefaciens X60 solution of CFU/ml is according to volume
Than being 1：15 light and slow are uniformly mixed obtain).
Processing group 5 is added a concentration of 1 × 10 in potting soil (2.0kg)10The tobacco Ralstonia solanacearum strain of CFU/ml
RsZP10ml is added above-mentioned a concentration of 1 × 107PFU/ml phage phi PB2 solution 0.25ml, distilled water 4.75ml.
Processing group 6 is added a concentration of 1 × 10 in potting soil (2.0kg)10The tobacco Ralstonia solanacearum strain of CFU/ml
Above-mentioned concentration 1 × 10 is added in RsZP10ml12CFU/ml bacillus amyloliquefaciens X60 solution 4.75ul, distilled water 0.25ml.
3 repetitions are often handled, is placed in room temperature and cultivates, sampled when cultivating 3,5,7 and 9d, using semi-selection culture medium
SMSA cultures combine the Population of colony counting method detection tobacco Ralstonia solanacearum RsZP.
In potting soil control tobacco Ralstonia solanacearum Population experiments have shown that, phage phi PB2 and bacillus amyloliquefaciens
The tobacco Ralstonia solanacearum Population that the combination microbial inoculum of X60 can control well, it is obviously preferable when effect is administered alone compared with the two.
2 bacteriophage of table combines the effect of the Population of tobacco Ralstonia solanacearum in microbial inoculum control potting soil with bacillus
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that
The specific implementation of the present invention is confined to these explanations.For those of ordinary skill in the art to which the present invention belongs, exist
Under the premise of not departing from present inventive concept, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to the present invention's
Protection domain.
Claims (4)
1. a kind of combination microbial inoculum of control Ralstonia solanacearum Population, it is characterised in that：The combination microbial inoculum is by having Ralstonia solanacearum
The phage solution for infecting cracking ability with to Ralstonia solanacearum there is the bacillus solution mixed preparing of antagonistic ability to form.
2. the combination microbial inoculum of control Ralstonia solanacearum Population according to claim 1, it is characterised in that：The bacteriophage is molten
A concentration of the 1 × 10 of liquid7-1×109PFU/ml, a concentration of the 1 × 10 of the bacillus solution10-1×1012CFU/ml bites
Phage solution is 1 by volume proportion with bacillus solution：The ratio of 10-15 is formulated.
3. the preparation method of the combination microbial inoculum of control Ralstonia solanacearum Population as claimed in claim 1 or 2, it is characterised in that：
Include the following steps：
(1) bacteriophage strain for having to Ralstonia solanacearum and infecting cracking ability is obtained using the method culture co-cultured, through filter membrane mistake
Phage solution is obtained after filter；
(2) NB fluid nutrient medium cultures are used to obtain the Bacillus species strains that there is antagonistic ability to Ralstonia solanacearum, after shaken cultivation
Centrifugation obtains bacillus solution；
(3) phage solution and bacillus solution are formulated in the ratio to get combination microbial inoculum.
4. the application of the combination microbial inoculum of control Ralstonia solanacearum Population as claimed in claim 1 or 2, it is characterised in that：In cigarette
In careless growth course, the combination microbial inoculum is applied in the vega soil of Ralstonia solanacearum.
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Cited By (4)
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| CN110592029A (en) * | 2019-10-18 | 2019-12-20 | 贵州省烟草科学研究院 | Method for expanding propagation of ralstonia solanacearum bacteriophage by utilizing ralstonia solanacearum complex properties |
| CN111789132A (en) * | 2020-07-04 | 2020-10-20 | 菲吉乐科(南京)生物科技有限公司 | Novel composite preparation and application thereof in bacterial diseases |
| CN111925995A (en) * | 2020-07-04 | 2020-11-13 | 菲吉乐科(南京)生物科技有限公司 | Microecological preparation prepared by coupling fermentation of bacteriophage and probiotics and preparation method |
| CN113444693A (en) * | 2021-06-04 | 2021-09-28 | 山东宝来利来生物工程股份有限公司 | Combined fermentation process of bacteriophage and probiotics |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110592029A (en) * | 2019-10-18 | 2019-12-20 | 贵州省烟草科学研究院 | Method for expanding propagation of ralstonia solanacearum bacteriophage by utilizing ralstonia solanacearum complex properties |
| CN111789132A (en) * | 2020-07-04 | 2020-10-20 | 菲吉乐科(南京)生物科技有限公司 | Novel composite preparation and application thereof in bacterial diseases |
| CN111925995A (en) * | 2020-07-04 | 2020-11-13 | 菲吉乐科(南京)生物科技有限公司 | Microecological preparation prepared by coupling fermentation of bacteriophage and probiotics and preparation method |
| CN111925995B (en) * | 2020-07-04 | 2022-05-17 | 菲吉乐科(南京)生物科技有限公司 | Microecological preparation prepared by coupling fermentation of bacteriophage and probiotics and preparation method |
| CN113444693A (en) * | 2021-06-04 | 2021-09-28 | 山东宝来利来生物工程股份有限公司 | Combined fermentation process of bacteriophage and probiotics |
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