CN113337415A - Sodium selenite-tolerant pseudomonas fluorescens and application thereof - Google Patents

Sodium selenite-tolerant pseudomonas fluorescens and application thereof Download PDF

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CN113337415A
CN113337415A CN202011509076.9A CN202011509076A CN113337415A CN 113337415 A CN113337415 A CN 113337415A CN 202011509076 A CN202011509076 A CN 202011509076A CN 113337415 A CN113337415 A CN 113337415A
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pseudomonas
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pseudomonas fluorescens
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CN113337415B (en
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杨金广
吴福如
袁莲莲
李莹
宋丽云
申莉莉
焦玉冰
王凤龙
黄坤
王勇
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SICHUAN TOBACCO Corp LIANGSHANZHOU BRANCH
Yunnan Tobacco Co Honghe Prefecture Co
Tobacco Research Institute of CAAS
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SICHUAN TOBACCO Corp LIANGSHANZHOU BRANCH
Yunnan Tobacco Co Honghe Prefecture Co
Tobacco Research Institute of CAAS
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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
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/27—Pseudomonas
    • C—CHEMISTRY; METALLURGY
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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Abstract

The invention discloses a sodium selenite-tolerant pseudomonas fluorescens and application thereof, belonging to the technical field of microorganisms and microbial preparations. The fluorescent pseudomonas is fluorescent pseudomonas KBD-1; the strain is preserved in China general microbiological culture Collection center (CGMCC) at 31.08.2020, and the preservation number of the strain is CGMCC No. 20567. The strain KBD-1 can inhibit infection of crop viruses, and the biocontrol experiment result shows that the biocontrol strain has good control effect on the crop viruses. Meanwhile, the strain KBD-1 can also synthesize nano selenium, and the nano selenium synthetic active bacterial liquid prepared from the strain has high application value in the aspects of growth promotion and crop virus disease prevention and treatment.

Description

Sodium selenite-tolerant pseudomonas fluorescens and application thereof
Technical Field
The invention belongs to the technical field of microorganisms and microbial preparations, and particularly relates to sodium selenite-tolerant pseudomonas fluorescens and application thereof.
Background
Plant viruses are in a large variety, and the number of the virus diseases discovered at present is hundreds, and is only lower than that of fungal diseases, so that the plant viruses cause great loss every year. The prevention and the treatment of virus diseases mainly aim at early prevention, and virus diseases are usually prevented and treated by screening disease-resistant varieties, preventing insects and detoxifying, cultivating management and the like in field production, but the effect is very little. Selenium is used as a 'vital element', is necessary for the growth of organisms, has important physiological functions in the aspects of improving the immunity of organisms, regulating metabolism, resisting oxidation, detoxifying, promoting reproduction and the like, and becomes the best selenium supplementing form due to low toxicity and high activity of nano selenium. The nano-selenium synthesized by microbial transformation has the advantages of mild condition, stable structure, good dispersibility and good application prospect.
In recent years, efficient and safe microbial preparations have been the focus of crop pest control research. However, at present, few research achievements with good disease resistance and growth promotion effects exist, and the popularization and application are lacked. The research on biological control is important in the current research, and the screening of microbial agents with growth promotion and excellent control effect for biological control of crop virus diseases is important.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a sodium selenite-tolerant pseudomonas fluorescens strain, the sodium selenite-tolerant pseudomonas fluorescens strain has a good control effect on crop virus diseases, and a new microbial resource is provided for control of the crop virus diseases. In addition, the fluorescent pseudomonas tolerant to sodium selenite can also biologically synthesize nano-selenium; has growth promoting effect on crops and has obvious effect.
In order to achieve the purpose, the invention adopts the following technical scheme:
the fluorescent pseudomonas tolerant to sodium selenite is KBD-1; the strain is preserved in China general microbiological culture Collection center at 31.08.2020, with the preservation address of No. 3 of West Lu No. 1 of Beijing university of Chaoyang, the preservation number of the strain is CGMCC No.20567, and the classified name of the strain is Pseudomonas fluorescens.
The application of the pseudomonas fluorescens in preventing and treating crop virus diseases.
The method for preventing and treating crop virus diseases is characterized by uniformly spraying the bacterial liquid of the pseudomonas fluorescens on the front and back surfaces of crop leaves to ensure that crops and matrixes are thoroughly wetted.
At the upper partOn the basis of the scheme, the bacterium content in the bacterium liquid of the fluorescent pseudomonas is not less than 108cfu/mL。
On the basis of the above scheme, the virus disease includes a crop disease caused by any one of Tobacco Mosaic Virus (TMV), Cucumber Mosaic Virus (CMV), Potato Virus Y (PVY) and Tomato Spotted Wilt Virus (TSWV).
The application of the pseudomonas fluorescens in preparing antiviral agents for preventing and treating crop virus diseases.
An antiviral agent for crops, which comprises the above Pseudomonas fluorescens as an active ingredient; the content of the pseudomonas fluorescens in the crop antiviral agent is more than or equal to 108cfu/mL。
The application of the fluorescent pseudomonas in biosynthesis of nano-selenium.
The method for biologically synthesizing the nano-selenium by adopting the pseudomonas fluorescens comprises the following steps:
adding the activated strain CGMCC No.20567 into LB liquid culture medium containing 1mM sodium selenite at an inoculation amount of 1%, performing shaking culture at 28 deg.C at 150r/min, and observing red color in bacterial liquid after 2-5 days to obtain the nano selenium generated by the strain reducing sodium selenite.
The application of the fluorescent pseudomonas in promoting the growth of crops.
The technical scheme of the invention has the advantages that:
the fluorescent pseudomonas strain can be applied to preventing and treating crop virus diseases. Biological determination of inoculated blight host shows that the KBD-1 bacterial liquid has good inhibition effect on TMV, and the inhibition rate is 89.2%. The antiviral agent can be used for field production to effectively prevent field viral diseases. In addition, the KBD-1 can biologically synthesize the nano selenium, has good growth promoting effect and has certain practical application value in production.
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FIG. 1 shows the effect of the bacterial strain KBD-1 in the biosynthesis of nano-selenium (left: active bacterial liquid for nano-selenium biosynthesis, the bacterial liquid is red; right: KBD-2 bacterial liquid, the bacterial liquid is light yellow);
FIG. 2 is a graph showing the effect of the bacterial liquid of nano-selenium biosynthesis on the inhibition of the scorching of TMV (right side leaf: nano-selenium synthetic active bacterial liquid; left side leaf: blank control).
Detailed Description
Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.
The present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
Example 1 isolation, purification and characterization of the strains
Collecting rhizosphere soil samples of non-diseased tobacco plants from high-diseased tobacco planting areas with the disease of the Baoshan virus, weighing 2g of collected soil samples, adding 20mL of ddH2Performing shaking culture at 28 ℃ and 180rpm for 16h, taking out and standing for 30min, taking the supernatant out and streaking on an LB solid culture medium, and putting the supernatant into an incubator at 28 ℃ for culture to obtain the strain. DNA extraction was performed according to the TIANGEN TIANAmp BACTERIA DNA Kit, and then PCR amplification was performed according to the 16S rDNA Bacterial Identification PCR Kit of TaKaRa, and 16S rDNA sequencing was performed by Takara Bio Inc. (Dagan). The determined sequence was aligned with the sequence in GenBank by BLAST program on NCBI website, and the strain was determined to be pseudomonas fluorescens. The strain has a code number of KBD-1, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms at 31.08.2020, the preservation address is No. 3 of West Lu No. 1 of Beijing, Chaoyang, and the strain preservation number is CGMCC No.20567, and is classified and named as Pseudomonas fluorescens.
EXAMPLE 2 biosynthesis of Nano-selenium by Strain KBD-1
Inoculating active strain KBD-1 to LB solid medium for purification, selecting a single colony with good growth, inoculating the single colony in LB liquid medium, performing shake culture at 28 ℃ and 150rpm for 20h, adding the single colony into LB liquid medium containing 1mM sodium selenite at an inoculum size of 1%, performing shake culture at 28 ℃ and 150r/min for 2-5d, and observing red color in bacterial liquid, which shows that the active strain KBD-1 can reduce sodium selenite to generate nano selenium (figure 1), diluting with water to obtain nano seleniumRice selenium synthetic active bacterial liquid (selenium content 50mg/L, bacterial concentration 10)8cfu/mL)。
Example 3 inhibition of TMV by Strain KBD-1 and Nano-selenium synthetic active bacterial solutions
The activity was measured by the hemiphyllic method, 10mL of KBD-1 bacterial solution (10% concentration) was taken8cfu/mL) and nano-selenium synthetic active bacterial liquid (selenium content is 50mg/L, bacterial concentration is 10)8cfu/mL) and an equal volume of 40-fold TMV juice (tobacco lamina with TMV was freeze-ground to a powder, as 1: 40 deionized water and gauze filtration) for 15min, respectively rubbing to inoculate 3 three raw tobaccos, inoculating 2 leaves on the upper part of each raw tobacco, simultaneously setting a blank control (LB liquid culture medium is mixed with TMV juice with the same volume of 40 times), and investigating the number of dry spots in 3-5 d. Experimental results show that the KBD-1 bacterial liquid and the nano-selenium synthetic active bacterial liquid have good passivation effects on TMV, the inhibition rates are 89.2% and 95.5% respectively (Table 1), and the inhibition effect on TMV is enhanced after nano-selenium is biosynthesized.
TABLE 1 inhibition of TMV by KBD-1 strain and nano-selenium-synthesized active bacterial liquid
Figure BDA0002845832110000031
Example 4 prevention of viral diseases in crops in the field by active bacterial liquid of Nano selenium biosynthesis
(1) And (6) treating bacterial liquid. 50L of nano-selenium biosynthetic active bacterial liquid fermented in laboratory (selenium content is 50mg/L, bacterial concentration is 10)8cfu/mL) and 40-fold virus (TMV, CMV, PVY, TSWV) juice for later use. After the non-toxic K326 tobacco seedlings are heeled in, inoculation is carried out in the 5-6 leaf stage. 3, treatment: treating the bacterial liquid I and mixing the viral juice for 15min and then inoculating; spraying a bacterium solution for 2 hours and then inoculating virus juice; and (3) inoculating virus juice, and spraying the bacterial liquid after 2 hours. When spraying the pesticide, the front and back surfaces of the leaf blade are uniformly sprayed, so that the spraying amount of each treatment is basically consistent, and the spraying amount is suitable for the tobacco seedlings and the matrix to be thoroughly wetted.
(2) And setting a cell. The anti-TMV, CMV, PVY and TSWV virus tests are respectively carried out on the ink test base of the tobacco institute of Chinese academy of agricultural sciences in 2020, 4 and 28 days. 3 treatments are set for each antiviral experiment, an LB culture medium and an equal volume of virus juice are mixed to serve as a positive control, each treatment is repeated for 3 times, 50 strains are obtained in each cell, and a protection row is set between the cells.
The disease condition is investigated after the tobacco plant is transplanted for 30 days, and the results show that the nano-selenium biosynthesis active bacterial liquid has better control effect on 4 viruses (Table 2). The mixed inhibition effect with the virus juice is the best, the bacterial liquid can passivate the activity of most viruses, the inhibition effect on 4 viruses is over 85.0 percent, and the highest TMV inhibition rate is 92.7 percent; secondly, spraying a bacterial liquid, inoculating viruses after 2 hours, wherein the bacterial liquid can colonize the surface of the tobacco leaf to prevent the infection of the viruses, and the control effect on 4 viruses is 71.8-73.3%; the prevention and treatment effect of the inoculated sprayed bacterium liquid on 4 viruses is not enough 15.0 percent, and the morbidity of a control group which is not treated by the bacterium liquid reaches more than 97.7 percent. Therefore, the effect of inhibiting the virus by the mixed inoculation of the bacterial liquid and the virus is optimal, and then the bacterial liquid is sprayed and inoculated, so that the bacterial strain KBD-1 can be biosynthesized into the nano selenium used as an antiviral agent in actual production, and the field virus disease can be effectively prevented.
TABLE 2 inhibitory Activity of the active bacterial liquid for biosynthesis of nano-selenium on 4 common viruses in the field
Figure BDA0002845832110000041
Example 5 growth promoting action of active bacterial liquid for biosynthesis of Nano selenium on crops
The filter paper was trimmed to size and placed in a petri dish. Biologically synthesizing active bacterial liquid containing nano selenium (selenium content 50mg/L, bacterial concentration 10)8cfu/mL) was diluted 0-fold, 50-fold, 100-fold, 200-fold, and 500-fold, respectively, and dropped onto a filter paper to wet the filter paper. Then, round and plump mung beans are selected, 10 granules are placed in each culture dish, a sterilized LB liquid culture medium is used as a control, and the filter paper is observed every day and is guaranteed to be wet. The result shows that the fresh weight and the plant height of the mung beans after being treated by the diluted nano-selenium biosynthesis active bacterial liquid are obviously higher than those of the mung beans treated by the LB culture medium, the mung beans are inhibited from growing without being diluted, the germination rate is only 53.3%, and the bud length is only 0.3 cm. Diluting semen Phaseoli Radiati 50 timesThe rooting has the inhibiting effect, but the plant height increasing has the promoting effect. In general, the growth promoting effect on the mung beans is the best after 200 times of dilution.
Culturing K326 to 5-6 true leaves in a seedling culture medium, irrigating roots with 200 times of nano-selenium biosynthesis active bacterial liquid (selenium content is 50mg/L) during transplanting, continuously irrigating the roots for 2 times after 3 days of seedling revival, performing root irrigation for 3 days at intervals, performing 10mL each plant, using LB culture medium and clean water to irrigate the roots as controls, and repeating 3 times after treating 6 plants. The fresh weight and the maximum leaf length and width were investigated after 3 days of root irrigation at the last time. As can be seen from Table 4, after the nano-selenium biosynthesis active bacterial liquid is continuously applied for three times, the fresh weight and the maximum leaf length and width of the tobacco plant are both higher than those of the tobacco plant treated by LB culture medium and clear water, and the growth promotion effect is obvious.
TABLE 3 growth promoting effect of active bacterial liquid for biosynthesis of nano-selenium on mung bean
Figure BDA0002845832110000051
TABLE 4 growth promoting effect of active bacteria liquid for biosynthesis of nano-selenium on K326
Figure BDA0002845832110000052
The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

Claims (10)

1. The fluorescent pseudomonas tolerant to sodium selenite is characterized in that the fluorescent pseudomonas is fluorescent pseudomonas KBD-1; the strain is preserved in China general microbiological culture Collection center at 31.08.2020, with the preservation address of No. 3 of West Lu No. 1 of Beijing university of Chaoyang, the preservation number of the strain is CGMCC No.20567, and the classified name of the strain is Pseudomonas fluorescens.
2. Use of the pseudomonas fluorescens of claim 1 for controlling crop virus diseases.
3. A method for preventing and treating crop virus diseases, which is characterized in that the pseudomonas fluorescens bacterial liquid of claim 1 is uniformly sprayed on the front and back surfaces of crop leaves to enable the crops and a substrate to be thoroughly wetted.
4. The method for preventing and treating crop virus diseases according to claim 3, wherein the bacterium content in the bacterium liquid of Pseudomonas fluorescens is not less than 108cfu/mL。
5. The method for controlling crop virus diseases according to claim 3 or 4, wherein the virus diseases comprise crop diseases caused by any one of tobacco mosaic virus, cucumber mosaic virus, potato virus Y and tomato spotted wilt virus.
6. Use of the pseudomonas fluorescens of claim 1 in the preparation of an antiviral agent for the control of a viral disease in a crop.
7. An antiviral agent for crops, characterized in that the effective ingredient comprises the fluorescent pseudomonas bacterium of claim 1; the content of the pseudomonas fluorescens in the crop antiviral agent is more than or equal to 108cfu/mL。
8. The use of the fluorescent pseudomonas of claim 1 in the biosynthesis of nanoselenium.
9. The method for biosynthesizing nano-selenium by adopting the pseudomonas fluorescens as claimed in claim 1, which is characterized by comprising the following steps of:
adding the activated strain CGMCC No.20567 in the claim 1 with the inoculation amount of 1% into LB liquid medium containing 1mM sodium selenite, carrying out shaking culture at 28 ℃ at 150r/min, and observing red color in bacterial liquid after 2-5 days, wherein the bacterial liquid is the nano selenium generated by reducing the sodium selenite by the strain in the claim 1.
10. Use of the fluorescent pseudomonas strain of claim 1 for crop growth promotion.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0325400A1 (en) * 1988-01-22 1989-07-26 Mycogen Corporation Novel hybrid bacillus thuringiensis gene, plasmid, and transformed pseudomonas fluorescens
CN109574745A (en) * 2018-12-10 2019-04-05 深圳市芭田生态工程股份有限公司 A kind of selenium Liquid Fertilizer and preparation method thereof
CN110923144A (en) * 2019-10-21 2020-03-27 湖北大学 High selenate tolerant bacteria and screening method and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0325400A1 (en) * 1988-01-22 1989-07-26 Mycogen Corporation Novel hybrid bacillus thuringiensis gene, plasmid, and transformed pseudomonas fluorescens
CN109574745A (en) * 2018-12-10 2019-04-05 深圳市芭田生态工程股份有限公司 A kind of selenium Liquid Fertilizer and preparation method thereof
CN110923144A (en) * 2019-10-21 2020-03-27 湖北大学 High selenate tolerant bacteria and screening method and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MICHIHIKO IKE: "Selenate reduction by bacteria isolated from aquatic environment free from selenium contamination", 《WATER RESEARCH》 *
NIDHI SINGH: "Biogenic Strain of Silver and Selenium Nanoparticles by Pseudomonas fluorescens and Cladosporium sp. JAPSK3 Isolated from Coal Mine Samples and Their Antimicrobial Activity", 《INTERNATIONAL JOURNAL OF NANOSCIENCE》 *
李吉祥: "细菌生物合成纳米硒的研究进展", 《农业资源与环境学报》 *

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