CN110699305A - Burkholderia and application thereof - Google Patents
Burkholderia and application thereof Download PDFInfo
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- CN110699305A CN110699305A CN201911185814.6A CN201911185814A CN110699305A CN 110699305 A CN110699305 A CN 110699305A CN 201911185814 A CN201911185814 A CN 201911185814A CN 110699305 A CN110699305 A CN 110699305A
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Abstract
The invention relates to a Burkholderia and application thereof, the bacteria is Burkholderia, Latin is Burkholderia arboris J211; the preservation number is CCTCC NO: m2019747; separating from Yunnan Kunming tobacco field soil; the culture medium is beef extract peptone culture medium; the application is for preventing and treating plant nematode diseases. The invention has the beneficial effects that 1, the ability of field crop against nematodiasis is obviously improved; 2. improving the commodity quality of crops; 3. the yield of the crops is improved. The product can also be used in combination with other nematode biocontrol microbial inoculum to further enhance the control effect of the microbial inoculum.
Description
Technical Field
The invention belongs to the technical field of methods for bacterial culture and fermentation application.
Background
Nematodes are a very common plant pest in the growth process of crops. Some plant parasitic nematodes destroy the tissue of plant roots, such as root-knot nematodes, which can cause stunted root development, impaired nutrient uptake, and some nematodes can transmit other diseases. Nematodes are estimated to cause losses equivalent to $ 1000 billion per year to crops. Root-knot nematodes (Meloidogyne spp.) and cyst nematodes (Heterodera spp.) are two main types of nematodes which can seriously harm various important crops, and are widely distributed and seriously harmful all over the world.
The common nematode preventing and controlling method mainly comprises fumigating, using chemical pesticide and also using microbial inoculum with nematicidal function. The fumigation has a great destructive effect on the environment, and the use of the fumigation is forbidden by the state. The chemically synthesized pesticide has various hazards to human body, environment and ecological balance due to residues. Although nematode diseases of some plants can be controlled by traditional rotation, soil improvement and other ways, rotation disease prevention is difficult to implement or even impossible to implement for some economic crops and forest trees. Biological control is more and more concerned by people because of the advantages of removing harm and increasing yield, protecting ecological balance, reducing environmental pollution and the like. The microorganism has the advantages of nature, no residue and the like, and becomes the method with the greatest development prospect. The most common nematicidal microorganisms are paecilomyces lilacinus, verticillium and other fungi, but the field control effect is not ideal.
Bacteria are also important biological types for controlling nematodes, but the types and reports of application in industry are few. Wangnan et al reported that a strain of Burkholderia vietnamiensis B418 reported efficacy in controlling nematodes. Other Burkholderia (Burkholderia) nematicidal activities have not been reported.
Disclosure of Invention
The invention provides a Burkholderia and application thereof in order to solve the defects of the problems. The Burkholderia arborvitae J211 is unreported Burkholderia arborvitae and has high-efficiency nematicidal activity, and the Burkholderia arborvitae J211 can achieve the effects of efficiently preventing and controlling plant nematode diseases in fields and improving plant quality.
The invention is realized by adopting the following technical scheme.
A bacterium which is Burkholderia, Burkholderia arboris J211; the preservation number is CCTCC NO: m2019747. Now, the strain is preserved in a preservation unit (the name of the preservation unit is China center for type culture Collection, the preservation date is 10 months and 10 days in 2019, the preservation number is CCTCC NO: M2019747, the address of the preservation unit is Wuhan, Lojia mountain, Wuhan university and postal code 430072) appointed by the State intellectual property office.
Furthermore, the bacteria are separated from Yunnan Kunming tobacco field soil.
Furthermore, the culture medium of the bacterium is a beef extract peptone culture medium.
Further, the bacterium of the present invention is useful for controlling plant nematode disease.
Further, the fermentation broth of the bacterium of the present invention is used for controlling plant nematode disease. Has obvious effect of preventing and controlling plant nematode disease in field.
The application of the bacteria of the invention, the preparation method of the fermentation liquor of the bacteria comprises the following steps: preparing beef extract peptone liquid culture medium → sterilizing → cooling → inoculating → fermenting for 12-72 hours by using a shaking table at 200 and 300rpm → collecting the bacterial liquid.
Further, the method for applying the fermentation broth of the bacterium of the present invention comprises:
A. when crops are cultivated, digging cultivation holes corresponding to the sizes of seedling roots in soil, placing seedlings, pouring fermented bacterial liquid on the roots of the plants, wherein the usage amount of the bacterial liquid is 10ml-100ml per plant according to the sizes of the roots; or the like, or, alternatively,
B. mixing the fermented bacteria liquid with watering water in any proportion, and watering the mixture into a cultivation hole when crops are cultivated, or watering the mixture after soil covering; or the like, or, alternatively,
C. adding the fermented bacteria liquid into sterilized wheat bran, drying at low temperature to obtain bacteria powder, and applying the dried bacteria powder to field during plant cultivation; or the like, or, alternatively,
D. adding the fermented bacteria liquid into sterilized wheat bran, drying at low temperature to obtain bacteria powder, activating the bacteria powder in nutrient solution, and pouring into plant root during cultivation; or the like, or, alternatively,
E. freeze-drying the fermented bacteria liquid to prepare pure bacteria powder, and directly applying the pure bacteria powder when the plants are cultivated; or the like, or, alternatively,
F. freeze-drying the fermented bacteria liquid to prepare pure bacteria powder, and mixing the pure bacteria powder with other substances for application; or the like, or, alternatively,
G. freeze drying the fermented bacteria liquid to obtain pure bacteria powder, activating the bacteria powder and pouring the bacteria liquid containing activated bacteria onto the root of plant.
Further, the method of applying the bacterial fermentation broth of the present invention does not include applying a bactericide drug to the roots.
Furthermore, the application method of the fermentation liquor of the bacteria comprises the compound use with other nematode biological control agents.
The invention has the beneficial effects that 1, the ability of resisting the nematode disease of the field crops is obviously improved; 2. improving the commodity quality of crops; 3. the yield of the crops is improved. The product can also be used in combination with other nematode biocontrol microbial inoculum to further enhance the control effect of the microbial inoculum.
The invention is further explained below with reference to the drawings and the detailed description.
Drawings
FIG. 1 is a photograph of a colony of the present invention on a beef extract peptone medium plate in front;
FIG. 2 is a photograph of a colony of the present invention on the reverse side of a beef extract peptone medium plate;
FIG. 3 is a dendrogram of the J211 strain of the invention;
FIG. 4 is a diagram of genetically linked markers of BCESM genes that may be present in some species of Burkholderia cepacia;
FIG. 5 is a graph showing the effect of the J211 bacterial liquid on the root of tobacco according to the present invention;
FIG. 6 is a graph showing the effect of the bacterial suspension J211 of the present invention on the root of tobacco without being applied thereto.
Detailed Description
As shown in fig. 3. A bacterium which is Burkholderia, latin being Burkholderia arborisJ 211; the preservation number is CCTCC NO: m2019747. Now, the cells are preserved in a preservation unit (the name of the preservation unit is China center for type culture Collection, the preservation date is 10 months and 10 days in 2019, the preservation number is CCTCC NO: M2019747, the address of the preservation unit is Wuhan, Lodok mountain, Wuhan university, and the postal code is 430072) appointed by the State intellectual Property office.
Furthermore, the bacteria are separated from Yunnan Kunming tobacco field soil.
See fig. 1 and fig. 2. Furthermore, the culture medium of the bacterium is a beef extract peptone culture medium.
Further, the bacterium of the present invention is useful for controlling plant nematode disease.
Further, the fermentation broth of the bacterium of the present invention is used for controlling plant nematode disease. Has obvious effect of preventing and controlling plant nematode disease in field.
The application of the bacteria of the invention, the preparation method of the fermentation liquor of the bacteria comprises the following steps: preparing beef extract peptone liquid culture medium → sterilizing → cooling → inoculating → fermenting for 12-72 hours by using a shaking table at 200 and 300rpm → collecting the bacterial liquid.
As shown in fig. 5 and 6; further, the method for applying the fermentation broth of the bacterium of the present invention comprises:
A. when crops are cultivated, digging cultivation holes corresponding to the sizes of seedling roots in soil, placing seedlings, pouring fermented bacterial liquid on the roots of the plants, wherein the usage amount of the bacterial liquid is 10ml-100ml per plant according to the sizes of the roots; or the like, or, alternatively,
B. mixing the fermented bacteria liquid with watering water in any proportion, and watering the mixture into a cultivation hole when crops are cultivated, or watering the mixture after soil covering; or the like, or, alternatively,
C. adding the fermented bacteria liquid into sterilized wheat bran, drying at low temperature to obtain bacteria powder, and applying the dried bacteria powder to field during plant cultivation; or the like, or, alternatively,
D. adding the fermented bacteria liquid into sterilized wheat bran, drying at low temperature to obtain bacteria powder, activating the bacteria powder in nutrient solution, and pouring into plant root during cultivation; or the like, or, alternatively,
E. freeze-drying the fermented bacteria liquid to prepare pure bacteria powder, and directly applying the pure bacteria powder when the plants are cultivated; or the like, or, alternatively,
F. freeze-drying the fermented bacteria liquid to prepare pure bacteria powder, and mixing the pure bacteria powder with other substances for application; or the like, or, alternatively,
G. freeze drying the fermented bacteria liquid to obtain pure bacteria powder, activating the bacteria powder and pouring the bacteria liquid containing activated bacteria onto the root of plant.
Further, the method of applying the bacterial fermentation broth of the present invention does not include applying a bactericide drug to the roots.
Furthermore, the application method of the fermentation liquor of the bacteria comprises the compound use with other nematode biological control agents.
In FIG. 5, after the J211 bacterial liquid is applied, although the tobacco is infected by mild root-knot nematode, fibrous root systems are still obvious.
FIG. 6 shows that the tobacco root system is seriously infected by the root-knot nematode and the root system is obviously expanded in the blank control without applying the bacterial liquid.
As shown in fig. 4. Some species in burkholderia cepacia may have genetically linked markers of the BCESM gene, which virulence genes are present only in pathogenic strains. Designing a primer BCESM1 aiming at the BCESM gene: 5'-CCACGGACGTGACTAACA-3', respectively; BCESM 2: 5'-CGTCCATCCGAACACGAT-3', the length of the amplified fragment is 1418 bp. The electrophoresis result shows that no target band appears at the position of about 1400bp, which proves that J211 does not contain virulence gene BCESM. M: 2000bp marker, 1-4: j211
It is known that the strain of the present invention is very safe and does not contain a gene causing diseases to humans.
The above experimental conclusion is obtained by the following experimental procedure, but the following examples should not be construed as limiting the scope of the present invention. The main key technical points of the invention are as follows: 1. burkholderia arabidopsis strain J211; 2. the strain is safe and does not contain genes causing diseases to human; 3. the nematode preventing and controlling field has good use effect.
Example one
And (5) finishing the tobacco field soil according to the conventional requirement. Selecting strong and tidy tobacco seedlings with the height of about 10cm, the plant distance of 50cm and the pit depth of 15 cm. In each hole for tobacco seedling cultivation, 10ml of Burkholderia arboris J211 strain liquid is applied, then tobacco is managed according to tobacco seedling cultivation specifications through measures of soil covering, watering and the like, and the tobacco seedlings can grow normally. The effect of nematode control can be seen after the tobacco plants grow.
Example two
Selecting tomato seedlings growing normally, digging small holes beside roots and making the holes deep to lateral roots. 50ml of Burkholderia arboris J211 strain liquid is applied to each hole, then soil covering is carried out, and management is carried out according to a normal vegetable seedling management method, so that the normal growth of the vegetable seedlings can be seen.
EXAMPLE III
And (5) finishing the tobacco field soil according to the conventional requirement. Selecting strong and tidy tobacco seedlings with the height of about 10cm, the plant distance of 50cm and the pit depth of 15 cm. 100ml of Burkholderia arboris J211 strain liquid is applied to each tobacco seedling cultivation hole, then the tobacco is managed by measures such as soil covering, watering and the like according to the tobacco seedling cultivation standard, and the tobacco seedlings can grow normally. The effect of nematode control can be seen after the tobacco plants grow.
Example four
And (5) finishing the tobacco field soil according to the conventional requirement. Selecting strong and tidy tobacco seedlings with the height of about 10cm, the plant distance of 50cm and the pit depth of 15 cm. In each hole for cultivating tobacco seedlings, 1 g of Burkholderia arboris J211 strain liquid freeze-dried bacterial powder is dissolved in 1L of root watering water, and then the tobacco is managed by measures such as soil covering, watering and the like according to the tobacco seedling cultivation specification, so that the tobacco seedlings can grow normally. The nematode control effect can be seen after the tobacco plant grows.
EXAMPLE five
And (5) finishing the tobacco field soil according to the conventional requirement. Selecting strong and tidy tobacco seedlings with the height of about 10cm, the plant distance of 50cm and the pit depth of 15 cm. Then, the tobacco is managed by measures such as soil covering, watering and the like according to the tobacco seedling cultivation standard, and the tobacco seedlings can grow normally. In each hole for cultivating tobacco seedlings, 20ml of Burkholderia arboris J211 bacterial liquid is applied firstly, after the tobacco plants are fixedly planted, 10ml of bacterial liquid is diluted by 90ml of clear water each time and then is irrigated to roots, and the rest are managed according to the normal management method of the tobacco plants.
The foregoing is merely a specific embodiment of the present invention and common general knowledge of known specific features of the scheme is not described herein in any greater detail. It should be noted that the above-mentioned embodiments do not limit the present invention in any way, and all technical solutions obtained by means of equivalent substitution or equivalent transformation for those skilled in the art are within the protection scope of the present invention. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
<110> university of Yunnan
<120> a burkholderia and application thereof
<160>2
<210>1
<211>18
<212>DNA
<213> Artificial sequence
<400>1
CCACGGACGTGACTAACA
<210>2
<211>18
<212>DNA
<213> Artificial sequence
<400>2
CGTCCATCCGAACACGAT
Claims (10)
1. A bacterium which is Burkholderia arboris J211; the preservation number is CCTCC NO: m2019747.
2. The bacterium of claim 1, wherein the bacterium is isolated from Yunnan Kunming tobacco field soil.
3. The bacterium according to claim 1, wherein the culture medium of the bacterium is a beef extract peptone medium.
4. Use of a bacterium according to any one of claims 1 to 3 for the control of plant nematode disease.
5. Use according to claim 4, characterized in that the fermentation broth of the bacterium is used for the control of plant nematode disease.
6. The use according to claim 5, wherein the bacterial fermentation broth is prepared by a process comprising: preparing beef extract peptone liquid culture medium → sterilizing → cooling → inoculating → fermenting for 12-72 hours by using a shaking table at 200 and 300rpm → collecting the bacterial liquid.
7. The use according to claim 5, wherein the method of administering the bacterial fermentation broth comprises:
A. when crops are cultivated, digging cultivation holes corresponding to the sizes of seedling roots in soil, placing seedlings, pouring fermented bacterial liquid on the roots of the plants, wherein the usage amount of the bacterial liquid is 10ml-100ml per plant according to the sizes of the roots; or the like, or, alternatively,
B. mixing the fermented bacteria liquid with water for root watering in any proportion, and watering the mixture into a cultivation hole when crops are cultivated, or watering the mixture after soil covering.
8. The use according to claim 5, wherein the method of administering the bacterial fermentation broth comprises:
C. adding the fermented bacteria liquid into sterilized wheat bran, drying at low temperature to obtain bacteria powder, and applying the dried bacteria powder to field during plant cultivation; or the like, or, alternatively,
D. adding the fermented bacteria liquid into sterilized wheat bran, drying at low temperature to obtain bacteria powder, activating the bacteria powder in nutrient solution, and pouring into plant root during cultivation; or the like, or, alternatively,
E. freeze-drying the fermented bacteria liquid to prepare pure bacteria powder, and directly applying the pure bacteria powder when the plants are cultivated; or the like, or, alternatively,
F. freeze-drying the fermented bacteria liquid to prepare pure bacteria powder, and mixing the pure bacteria powder with other substances for application; or the like, or, alternatively,
G. freeze drying the fermented bacteria liquid to obtain pure bacteria powder, activating the bacteria powder and pouring the bacteria liquid containing activated bacteria onto the root of plant.
9. Use according to claim 7 or 8, characterized in that the method of application of the bacterial fermentation broth does not comprise the application of a bactericide drug to the roots.
10. The use according to claim 7 or 8, wherein the method of applying the bacterial broth comprises the use of a complex with other nematode biocontrol agents.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115960765A (en) * | 2022-11-02 | 2023-04-14 | 云南大学 | Flavobacterium bacteria and application thereof in low-temperature composting |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115960765A (en) * | 2022-11-02 | 2023-04-14 | 云南大学 | Flavobacterium bacteria and application thereof in low-temperature composting |
| CN115960765B (en) * | 2022-11-02 | 2024-04-26 | 云南大学 | Flavobacterium strain and application thereof in low-temperature composting |
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