CN112352640A - Method for verifying endophytic symbiotic bacteria - Google Patents
Method for verifying endophytic symbiotic bacteria Download PDFInfo
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- CN112352640A CN112352640A CN202011172513.2A CN202011172513A CN112352640A CN 112352640 A CN112352640 A CN 112352640A CN 202011172513 A CN202011172513 A CN 202011172513A CN 112352640 A CN112352640 A CN 112352640A
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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
- A01G22/20—Cereals
- A01G22/22—Rice
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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
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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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- Botany (AREA)
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- Forests & Forestry (AREA)
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- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Pretreatment Of Seeds And Plants (AREA)
Abstract
The invention discloses a method for verifying endosymbiont bacteria, which comprises the following steps: s1: a device for verifying endosymbionts; a base device: by the glass wide-necked bottle, go up to have glass bell jar, glass wide-necked bottle, plastics bottle plug, standby material are place to the bottleneck: substances such as vaseline and the like at the mouth of the wide-mouth bottle can be tightly sealed; a drier used for extracting air in the bell jar. The microorganism of the invention is from the natural ecosystem of the organism but not the category of the soil microorganism, the microorganism to be tested is marked by various biological, physical and chemical methods, the general radioactive interference, antibiotic mark and the possible interferences such as pollution, drift and the like generated by gene mark are completely eliminated, the microorganism to be tested is proved to be completely and naturally planted, propagated and transferred in the organism, the microorganism is marked by radioactive substances, the microorganism is simultaneously cultured to have antagonistic performance to more than two antibiotics, and the existence of endophytic symbiotic bacteria in the organism is verified.
Description
Technical Field
The invention relates to the technical field of plant pathology, in particular to a method for verifying endosymbiont bacteria.
Background
"endogenetic" (endobiotic) in phytopathology is a term referring to pathogenic bacteria which are endophytes in plants. Some pathogenic bacteria establish a 'symbiotic' relationship with plants, and the endophytic symbiosis is not directly proved by tests.
Biological individuals are composed of individual cells, tissues and microorganisms in vivo. These microorganisms can be classified as environmental microorganisms, endogenous microorganisms, and endogenous symbiotic microorganisms. At present, the traditional mode cannot well prove the endogenous symbiotic technology that certain microorganisms can be planted, propagated and transferred in plants, so that the verification of the existence of endogenous symbiotic bacteria in organisms is very important.
To this end, a method of verifying endosymbionts is proposed.
Disclosure of Invention
The present invention aims to provide a method for verifying endosymbiont bacteria, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a method of validating endosymbionts, comprising the steps of:
s1: a device for verifying endosymbionts;
a base device: the glass jar is provided with a glass bell jar and a plastic bottle stopper;
preparing a standby material: substances such as vaseline and the like at the mouth of the wide-mouth bottle can be tightly sealed; a drier used for extracting air in the bell jar.
S2: cultivating sterile rice fungi;
washing the rice seeds, and then washing the rice seeds with sterile water for three times;
s3: carrying out radioactive double-antibody treatment on the test strain: the test strain is radiated by cobalt 60 and cultured in the antibiotic streptomycin rifampicin from low concentration to the concentration of two antibiotics up to 200 ppm;
s4: placing the sterile rice seedlings in a sterilized wide-mouth bottle, adding bacillus containing nutrient solution for testing the radiogram anti-two antibiotics into the wide-mouth bottle, and enabling the nutrient solution to overflow the roots of the rice;
s5: tightly sealing the mouth of the jar to be tested to avoid the existence of a water film and prevent bacillus in the jar from transferring to the upper part of the bottle stopper from the lower part through the surface of the stem and the bottle stopper;
s6: in a bell jar at the upper part of the bottle stopper, air is pumped and dried, and the stem leaves at the upper part are not allowed to have any water film, and the test bacteria are not allowed to have water films on the surfaces of the stem leaves;
s7: after five days, the test bacillus could be examined on the tip of the test rice seedling.
(1) Radiodual resistant bacillus shares a cobalt 60 radiolabel and grows on 200ppm streptomycin rifampicin two antibiotic cultured reagents;
(2) the test was conducted after the supply was killed and the control was made. The control had no radiolabel at the upper leaf tip and was resistant to bacillus for both antibiotics.
Preferably, in the step S2, the washed rice seeds are placed in a sterile nutrient solution to be cultured into seedlings at room temperature for later use.
Preferably, the test strain in said step S3 is bacillus.
Compared with the prior art, the invention has the beneficial effects that:
the microorganism of the invention is from the natural ecosystem of the organism but not the category of the soil microorganism, the microorganism to be tested is marked by various biological, physical and chemical methods, the general radioactive interference, antibiotic mark and the possible interferences such as pollution, drift and the like generated by gene mark are completely eliminated, the microorganism to be tested is proved to be completely and naturally planted, propagated and transferred in the organism, the microorganism is marked by radioactive substances, the microorganism is simultaneously cultured to have antagonistic performance to more than two antibiotics, and the existence of endophytic symbiotic bacteria in the organism is verified.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic view of a control sample of the present invention.
Detailed Description
In the following, the technical solutions in the embodiments of the present invention will be clearly and completely described with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a technical scheme that:
a method of validating endosymbionts, comprising the steps of:
s1: a device for verifying endosymbionts;
a base device: the glass jar is provided with a glass bell jar and a plastic bottle stopper;
preparing a standby material: substances such as vaseline and the like at the mouth of the wide-mouth bottle can be tightly sealed; a dryer for exhausting air inside the bell jar;
s2: cultivating sterile rice fungi;
washing rice seeds, then washing the rice seeds with sterile water for three times, and placing the rice seeds in sterile nutrient solution to culture the rice seeds into seedlings for later use;
s3: carrying out radioactive double-antibody treatment on the test strain: the test strain is bacillus, the test strain is radiated by cobalt 60, and the test strain is gradually cultured in the antibiotic streptomycin rifampicin from low concentration to the concentration of two antibiotics reaching 200 ppm;
s4: placing the sterile rice seedlings in a sterilized wide-mouth bottle, adding bacillus containing nutrient solution for testing the radiogram anti-two antibiotics into the wide-mouth bottle, and enabling the nutrient solution to overflow the roots of the rice;
s5: tightly sealing the mouth of the jar to be tested to avoid the existence of a water film and prevent bacillus in the jar from transferring to the upper part of the bottle stopper from the lower part through the surface of the stem and the bottle stopper;
s6: in a bell jar at the upper part of the bottle stopper, air is pumped and dried, and the stem leaves at the upper part are not allowed to have any water film, and the test bacteria are not allowed to have water films on the surfaces of the stem leaves;
s7: after five days, the test bacillus can be detected on the tip of the top end of the test rice seedling;
(1) radiodual resistant bacillus shares a cobalt 60 radiolabel and grows on 200ppm streptomycin rifampicin two antibiotic cultured reagents;
(2) the test was conducted after the supply was killed and the control was made. The control had no radiolabel at the upper leaf tip and was resistant to bacillus for both antibiotics.
The microorganism of the invention is from the natural ecosystem of the organism but not the category of the soil microorganism, the microorganism to be tested is marked by various biological, physical and chemical methods, the general radioactive interference, antibiotic mark and the possible interferences such as pollution, drift and the like generated by gene mark are completely eliminated, the microorganism to be tested is proved to be completely and naturally planted, propagated and transferred in the organism, the microorganism is marked by radioactive substances, the microorganism is simultaneously cultured to have antagonistic performance to more than two antibiotics, and the existence of endophytic symbiotic bacteria in the organism is verified.
The invention follows the following principles: the microorganism with radioactivity and double resistance is placed in a nutrient solution to inoculate roots, the rhizome part is strictly isolated from the stem leaf on the upper part, the stem leaf on the upper part is placed in a bell jar, the bell jar is strictly pumped and dried, the surface of the stem leaf part is controlled to be free from any water film, the microorganism to be tested cannot exist on the surface of the organism, finally, the radioactive antimicrobial is fixedly planted, propagated and transferred in the organism, the radioactive and double antimicrobial exists on the top of the organism to be tested, a dead bacteria control (shown in figure 1) is additionally arranged, and dead bacteria only exist in the root part and can never exist on the upper part of the stem leaf.
The invention must meet the following requirements:
1. the test microorganism is inoculated to the root and stem part at the lower part of the test material plant, and is strictly isolated and sealed with the stem and stem part at the upper part, so that the test microorganism can only be planted, propagated and transferred in the plant body.
2. The method is characterized in that the lower rhizome of the plant is inoculated with the test microorganism, the test microorganism is completely isolated and blocked from the upper stem and leaf, the upper stem and leaf is placed in a bell jar, and the upper stem and leaf cannot have any water film through measures such as strict air exhaust and drying, so that the test microorganism cannot be planted, propagated and transferred on the surfaces of the stem and leaf, and the test microorganism can only be planted, propagated and transferred in the upper stem and leaf.
3. The complete marker of the lower rhizome marker of the plant, namely the marker with radioactivity and double resistance can be detected on the uppermost stem leaf part of the upper stem leaf of the plant.
The first embodiment is as follows:
examples of the verification of the M-33 strain of endosymbiont Bacillus:
radioactive cobalt 60 calibration is carried out on M-33 strain in bacillus, and simultaneously strains of streptomycin and rifampicin resistant antibiotics are cultivated;
and inoculating the marked M-33 strain to the root of the cultured rice seedling, placing the inoculated M-33 strain in a verification endosymbiotic device, and strictly sealing the device, wherein the M-33 strain cannot be fixedly planted, propagated and transferred on the surface of the rice seedling. The M-33 strain can only be planted, propagated and transferred in the rice body. Within 10 days, in the rice seedling cell tissue, the tested M-33 strain can exist in the top leaf of the rice seedling, which proves that the endosymbiotic strain M-33 really exists in the rice seedling cell tissue, and proves that the M-33 strain is really planted, propagated and transferred in the rice seedling. Completely and autonomously transferring the rootstock part to the top stem leaf;
the M-33 strain is indeed an endosymbiont.
Example two:
verifying that the endosymbiont bacillus M-33 can prevent cotton wilt:
the endophytic symbiotic bacillus has a radioactive double-resistant M-33 strain and is used for preventing and treating cotton wilt.
When the radioactive double-resistant strain M-33 is used for dressing seeds of cotton, the cotton is inoculated by dressing the seeds, and the control effect on cotton wilt reaches 55% -65%.
1. Inoculation:
during cotton inoculation, the radioactive double-resistant strain M-33 is used for dressing cotton seeds.
The M-33 strain to be tested is planted, propagated and transferred on the rhizome part.
The wilt is delayed for 10-15 days at the initial stage of rhizome dip-dyeing; the nutritional and hair-bundle disease index of the rhizome part is reduced by 50 to 55 percent.
2. And (3) a ring-up period:
the wilt disease is caused by the fact that when cotton buds bloom, blight bacteria have spread to petioles of bolls from roots.
The petioles were inoculated (sprayed, dipped) with the radiodouble resistant strain every 10 days at this time. The petiole part is used for emitting double-antibody M-33 to prevent the wilt from expanding from a rhizome part to the petiole part, and the onset time is delayed for 15-20 days. The disease index is reduced by about 65 percent. Basically ensures that cotton bolls are formed and bolls are opened, and the yield is kept about 85 percent.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (3)
1. A method for verifying endosymbionts, comprising the steps of:
s1: a device for verifying endosymbionts;
a base device: the glass jar is provided with a glass bell jar and a plastic bottle stopper;
preparing a standby material: substances such as vaseline and the like at the mouth of the wide-mouth bottle can be tightly sealed; a dryer for exhausting air inside the bell jar;
s2: cultivating sterile rice fungi;
washing the rice seeds, and then washing the rice seeds with sterile water for three times;
s3: carrying out radioactive double-antibody treatment on the test strain: the test strain is radiated by cobalt 60 and cultured in the antibiotic streptomycin rifampicin from low concentration to the concentration of two antibiotics up to 200 ppm;
s4: placing the sterile rice seedlings in a sterilized wide-mouth bottle, adding bacillus containing nutrient solution for testing the radiogram anti-two antibiotics into the wide-mouth bottle, and enabling the nutrient solution to overflow the roots of the rice;
s5: tightly sealing the mouth of the jar to be tested to avoid the existence of a water film and prevent bacillus in the jar from transferring to the upper part of the bottle stopper from the lower part through the surface of the stem and the bottle stopper;
s6: in a bell jar at the upper part of the bottle stopper, air is pumped and dried, and the stem leaves at the upper part are not allowed to have any water film, and the test bacteria are not allowed to have water films on the surfaces of the stem leaves;
s7: after five days, the test bacillus could be examined on the tip of the test rice seedling.
(1) Radiodual resistant bacillus shares a cobalt 60 radiolabel and grows on 200ppm streptomycin rifampicin two antibiotic cultured reagents;
(2) the test was conducted after the supply was killed and the control was made. The control had no radiolabel at the upper leaf tip and was resistant to bacillus for both antibiotics.
2. The method for verifying endosymbiont bacteria according to claim 1, wherein: and in the step S2, putting the cleaned rice seeds into a sterile nutrient solution, and culturing the rice seeds into seedlings at room temperature for later use.
3. The method for verifying endosymbiont bacteria according to claim 1, wherein: the test strain in the step S3 is bacillus.
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| CN202011172513.2A CN112352640A (en) | 2020-10-28 | 2020-10-28 | Method for verifying endophytic symbiotic bacteria |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997024433A1 (en) * | 1995-12-29 | 1997-07-10 | The United States Of America, Represented By The Secretary Of Agriculture | The use of enterobacter cloacae as an endophyte for the control of diseases caused by fungi |
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2020
- 2020-10-28 CN CN202011172513.2A patent/CN112352640A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997024433A1 (en) * | 1995-12-29 | 1997-07-10 | The United States Of America, Represented By The Secretary Of Agriculture | The use of enterobacter cloacae as an endophyte for the control of diseases caused by fungi |
Non-Patent Citations (7)
| Title |
|---|
| 刘佳等: "内生成团泛菌HAUM1对宿主水稻的定殖及促生作用", 《湖北农业科学》 * |
| 刘忠梅等: "有益内生细菌B946在小麦体内的定殖规律", 《中国生物防治》 * |
| 张则君等: "植物内生细菌分离鉴定方法概述", 《农业技术与装备》 * |
| 文才艺等: "内生细菌EBS05在小麦体内的定殖动态及其对小麦纹枯病的防治作用", 《植物保护学报》 * |
| 梅汝鸿等: "《植物微生态学 第一版》", 30 September 1998 * |
| 罗明等: "内生拮抗细菌在哈密瓜植株体内的传导定殖和促生作用研究", 《西北植物学报》 * |
| 肖相政等: "短小芽孢杆菌BX-4抗生素标记及定殖效果研究", 《农业环境科学学报》 * |
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Application publication date: 20210212 |