CN113430233B - Nano-selenium synthetic active bacterial liquid of pseudomonas fluorescens, preparation method and application thereof - Google Patents
Nano-selenium synthetic active bacterial liquid of pseudomonas fluorescens, preparation method and application thereof Download PDFInfo
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- CN113430233B CN113430233B CN202011513436.2A CN202011513436A CN113430233B CN 113430233 B CN113430233 B CN 113430233B CN 202011513436 A CN202011513436 A CN 202011513436A CN 113430233 B CN113430233 B CN 113430233B
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Abstract
The invention discloses a nano-selenium synthetic active bacterial liquid of pseudomonas fluorescens, a preparation method and application thereof, belonging to the technical field of microorganisms and microbial preparations. The invention relates to a nano-selenium synthetic active bacterial liquid of fluorescent pseudomonas; after pseudomonas fluorescens is biologically fermented to synthesize nano selenium, mixed solution consisting of pseudomonas fluorescens and the synthesized nano selenium; 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 nano-selenium synthetic active bacterial liquid of the pseudomonas fluorescens has better control effect on crop virus diseases, and provides new microbial resources for control of the crop virus diseases; in addition, the fertilizer also has growth promoting effect on crops, and the effect is obvious.
Description
Technical Field
The invention belongs to the technical field of microorganisms and microbial preparations, and particularly relates to a nano-selenium synthetic active bacterium solution of pseudomonas fluorescens, and a preparation method 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 the organisms, regulating the metabolism, resisting oxidation, detoxifying, promoting the reproduction and the like, and becomes the best selenium supplement 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 research on crop pest control. 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 nano-selenium synthetic active bacterial liquid for biosynthesis of sodium selenite-tolerant pseudomonas fluorescens; the nano-selenium synthetic active bacterial liquid has a good control effect on crop virus diseases, and provides a new microbial resource for control of the crop virus diseases. In addition, the nano-selenium synthesized active bacterial liquid of the fluorescent pseudomonas has a growth promoting effect on crops, and the effect is obvious.
In order to achieve the purpose, the invention adopts the following technical scheme:
the nano-selenium synthetic active bacterial liquid of the fluorescent pseudomonas is a mixed liquid which is formed by the fluorescent pseudomonas and the synthesized nano-selenium after the fluorescent pseudomonas is biologically fermented to synthesize the nano-selenium;
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 Xilu-Beijing, chaoyang, and the preservation number of CGMCC No.20567, and is classified and named as Pseudomonas fluorescens.
The preparation method of the nano-selenium synthetic active bacterial liquid of the fluorescent pseudomonas comprises the following steps:
adding the activated strain CGMCC No.20567 into LB liquid culture medium containing 1mM sodium selenite in an inoculation amount of 1%, carrying out shaking culture at 28 ℃ at 150r/min, and observing red color in the bacterial liquid after 2-5 days, wherein the fermentation mixed liquid is the nano-selenium synthetic active bacterial liquid of the pseudomonas fluorescens.
The nano-selenium synthetic active bacterial liquid of the pseudomonas fluorescens is applied to preventing and treating crop virus diseases or promoting crop growth.
The nano-selenium synthetic active bacterial liquid of the pseudomonas fluorescens is applied to preparing antiviral agents for preventing and treating crop virus diseases or crop growth promoters.
A method for preventing and treating crop virus diseases comprises uniformly spraying the nano-selenium synthetic active bacterial liquid of the fluorescent pseudomonas on the front and back surfaces of crop leaves to enable the crops and a substrate to be thoroughly wetted; the bacterium content in the nano-selenium synthetic active bacterium liquid of the fluorescent pseudomonas is more than or equal to 10 8 cfu/mL; the concentration of the nano-selenium is more than or equal to 50mg/L.
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).
Diluting the nano-selenium synthetic active bacterial liquid of the fluorescent pseudomonas and then irrigating roots of crops, wherein the bacterial content in the diluted nano-selenium synthetic active bacterial liquid of the fluorescent pseudomonas is 2 multiplied by 10 5 cfu/mL～2×10 6 cfu/mL, the concentration of nano-selenium is 0.1 mg/L-1 mg/L; preferably, the bacterium content in the diluted nano-selenium synthetic active bacterium liquid of the pseudomonas fluorescens is 5 multiplied by 10 5 cfu/mL, nano-selenium concentration is 0.25mg/L.
On the basis of the scheme, the root irrigation treatment comprises the following steps: irrigating roots once during transplanting; and (3) after seedling rejuvenation for 3d, continuously irrigating the roots for 2 times at an interval of 3d, and 10mL of each plant.
The active ingredient of the crop antiviral agent comprises nano-selenium synthetic active bacterial liquid of the pseudomonas fluorescens; the content of pseudomonas fluorescens in the crop antiviral agent is more than or equal to 10 8 cfu/mL, and the concentration of the nano-selenium is more than or equal to 50mg/L.
A crop growth promoter comprises the active ingredient of nano-selenium synthetic active bacterial liquid of the fluorescent pseudomonas as well as a growth promoter; the content of fluorescent pseudomonas in the crop growth promoter is 2 multiplied by 10 5 cfu/mL～2×10 6 cfu/mL, and the concentration of the nano-selenium is 0.1 mg/L-1 mg/L.
The technical scheme of the invention has the advantages that:
the nano-selenium synthetic active bacterial liquid of the fluorescent pseudomonas can be applied to preventing and treating crop virus diseases. Biological determination of inoculated blight hosts shows that the nano-selenium synthetic active bacterium solution of pseudomonas fluorescens has better inhibition effect on TMV, the inhibition rate is 95.5%, and the bacterium solution has better control effect compared with the single use of pseudomonas fluorescens. Meanwhile, the nano-selenium synthetic active bacterium liquid of the pseudomonas fluorescens has good prevention effect on TMV, CMV, PVY and TSWV, and the prevention effect is more than 70.0%. The result shows that the fluorescent pseudomonas enhances the antiviral ability after the fluorescent pseudomonas biologically ferments and synthesizes active nano-selenium, and the fluorescent pseudomonas can be used as an antiviral agent in field production to effectively prevent field virus diseases. In addition, the nano-selenium synthetic active bacterial liquid of the fluorescent pseudomonas 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 on inhibiting the scorching spots of TMV (right side leaf: bacterial liquid for nano-selenium synthesis; 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 ddH 2 Performing shaking culture at 28 deg.C and 180rpm for 16h, taking out, standing for 30min, taking supernatant, streaking on LB solid medium, and placing in 28 deg.C incubatorAnd (5) culturing 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 is KBD-1, and is preserved in China general microbiological culture Collection center in 31 days at 08 months in 2020, with the preservation address being No. 3 of the Ministry of Xilu No. 1 of Beijing, chaoyang, the strain preservation number being CGMCC No.20567, and the classification name being Pseudomonas fluorescens.
EXAMPLE 2 biosynthesis of Nano-selenium by Strain KBD-1
Inoculating active strain KBD-1 to LB solid culture medium for purification, selecting a single colony with good growth, inoculating the single colony in LB liquid culture medium, performing shake culture at 28 ℃ and 150rpm for 20h, adding the single colony into LB liquid culture medium containing 1mM sodium selenite at an inoculum size of 1%, performing shake culture at 28 ℃ and 150r/min for 2-5d, observing red color in the bacterial liquid, indicating that the active strain KBD-1 can reduce the sodium selenite to generate nano-selenium (figure 1), diluting with water to obtain nano-selenium synthetic active bacterial liquid (the selenium content is 50mg/L, and the bacterial concentration is 10 mg/L) 8 cfu/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 taken 8 cfu/mL) and nano-selenium synthetic active bacterial liquid (selenium content is 50mg/L, bacterial concentration is 10) 8 cfu/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
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) 8 cfu/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 the virus juice, mixing for 15min, and 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 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 viruses by mixed inoculation of the bacterial liquid and the viruses is optimal, the bacterial liquid is firstly sprayed and then inoculated, and the bacterial strain KBD-1 can be biosynthesized into nano selenium used as an antiviral agent in actual production, so that 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
Example 5 growth promotion of Nano-selenium biosynthesis active bacterial liquid on crops
The filter paper was trimmed to size and placed in a petri dish. Respectively diluting the active bacterial liquid (selenium content is 50 mg/L) of nano-selenium biosynthesis by 0 time (selenium content is 50mg/L, bacterial concentration is 10) 8 cfu/mL), 50-fold, 100-fold, 200-fold, and 500-fold, and was dropped onto a filter paper to wet it. Then, round and plump mung beans are selected, 10 granules are placed in each culture dish, 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 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 dilution, the germination rate is only 53.3%, and the bud length is only 0.3cm. After diluted by 50 times, the plant growth inhibitor has an inhibiting effect on the rooting of mung beans, but has a promoting effect on the increase of plant height. 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 50 mg/L) during transplanting, continuously irrigating roots for 2 times after 3 days of seedling rejuvenation, performing root irrigation for 3 days at an interval of 3 days, performing 10mL of each plant every time, performing root irrigation with LB culture medium and clear water as controls, and repeating 3 times after treating 6 plants each time. 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 nano-selenium biosynthesis active bacterial liquid on mung bean
TABLE 4 growth promoting effect of active bacteria liquid for biosynthesis of nano-selenium on K326
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. 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 nanometer selenium synthetic active bacterial liquid of fluorescent pseudomonas is characterized in that after the fluorescent pseudomonas is subjected to biological fermentation to synthesize nanometer selenium, the fluorescent pseudomonas and the synthesized nanometer selenium form mixed liquid;
the fluorescent pseudomonas is fluorescent pseudomonas KBD-1; the strain is preserved in China general microbiological culture Collection center in 31 days 08 and 31 months in 2020, the preservation address is No. 3 of No. 1 Xilu Beijing of Beijing's North Chen Yangyang region, the preservation number of the strain is CGMCC No.20567, and the strain is classified and named as Pseudomonas fluorescens.
2. The method for preparing the nano-selenium synthetic active bacterial liquid of the fluorescent pseudomonas as described in claim 1, which is characterized by comprising the following steps:
adding the activated strain CGMCC No.20567 into an LB liquid culture medium containing 1mM sodium selenite in an inoculation amount of 1%, carrying out shaking culture at 28 ℃ at 150r/min, and observing the appearance of red in the bacterial liquid after 2-5 days, wherein the fermentation mixed liquid is the nano-selenium synthetic active bacterial liquid of the pseudomonas fluorescens.
3. The application of the nano-selenium synthetic active bacterial liquid of the fluorescent pseudomonas as claimed in claim 2 in preventing and treating crop virus diseases or promoting crop growth, wherein the crop virus diseases are caused by any one virus of tobacco mosaic virus, cucumber mosaic virus, potato virus Y and tomato spotted wilt virus; the growth-promoting crops in the crop growth promotion are tobacco or mung bean.
4. The use of the nano-selenium-synthesized active bacterial liquid of pseudomonas fluorescens of claim 2 in the preparation of an antiviral agent for preventing and treating crop virus diseases or a crop growth promoter, wherein the crop virus diseases are crop diseases caused by any one of tobacco mosaic virus, cucumber mosaic virus, potato virus Y and tomato spotted wilt virus; the growth-promoting crops in the crop growth promotion are tobacco or mung bean.
5. A method for preventing and treating crop virus diseases is characterized in that the nano-selenium synthetic active bacterial liquid of fluorescent pseudomonas as claimed in claim 2 is uniformly sprayed on the front and back surfaces of crop leaves to enable the crops and a substrate to be thoroughly wetted; the bacterium content in the nano-selenium synthetic active bacterium liquid of the fluorescent pseudomonas is more than or equal to 10 8 cfu/mL; the concentration of the nano-selenium is more than or equal to 50mg/L; the crop virus disease is a crop disease caused by any one of tobacco mosaic virus, cucumber mosaic virus, potato virus Y and tomato spotted wilt virus.
6. A method for promoting the growth of crops is characterized in that the nano-selenium synthetic active bacterial liquid of the fluorescent pseudomonas as claimed in claim 2 is diluted and then is subjected to root irrigation treatment, and the bacterial content in the diluted nano-selenium synthetic active bacterial liquid of the fluorescent pseudomonas is 2 x 10 5 cfu/mL～2×10 6 cfu/mL, and the concentration of the nano-selenium is 0.1 mg/L-1 mg/L; the crop is tobacco or mung bean.
7. The method for promoting the growth of a crop as claimed in claim 6, wherein the root irrigation treatment is: irrigating roots once during transplanting; and (4) after 3 days of seedling rejuvenation, continuously irrigating roots for 2 times at an interval of 3 days, and 10mL of each plant.
8. An antiviral agent for crops, characterized in that the effective component comprises nano-selenium synthetic active bacterium liquid of the fluorescent pseudomonas as claimed in claim 2; the content of pseudomonas fluorescens in the crop antiviral agent is more than or equal to 10 8 cfu/mL; the concentration of the nano-selenium is more than or equal to 50mg/L; the virus is any one of tobacco mosaic virus, cucumber mosaic virus, potato virus Y and tomato spotted wilt virus.
9. A crop growth promoting agent, characterized in that the effective component comprises nano-selenium synthetic active bacterial liquid of fluorescent Pseudomonas as claimed in claim 2; the content of fluorescent pseudomonas in the crop growth promoter is 2 multiplied by 10 5 cfu/mL～2×10 6 cfu/mL, the concentration of nano-selenium is 0.1 mg/L-1 mg/L; the crop is tobacco or mung bean.
10. The method according to claim 6, wherein the diluted nano-selenium synthetic active bacterial liquid of Pseudomonas fluorescens has a bacterial content of 5 x 10 5 cfu/mL, nano-selenium concentration is 0.25mg/L.
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| JPH104954A (en) * | 1996-06-25 | 1998-01-13 | Japan Tobacco Inc | Microorganism of genus pseudomonas capable of colonizing in rhizosphere of tobacco and soil disease injury controlling agent and control using the same |
| CN109234197A (en) * | 2018-09-29 | 2019-01-18 | 河南省农业科学院植物保护研究所 | One plant can prevent and treat tobacco black shank and root black rot and pseudomonas aeruginosa and its application with growth-promoting functions simultaneously |
| CN110438049A (en) * | 2019-08-26 | 2019-11-12 | 中国农业科学院烟草研究所 | One plant of tobacco mosaic viruses biological and ecological methods to prevent plant disease, pests, and erosion Yi Shi pseudoalteromonas bacterial strain and its application |
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| JPH104954A (en) * | 1996-06-25 | 1998-01-13 | Japan Tobacco Inc | Microorganism of genus pseudomonas capable of colonizing in rhizosphere of tobacco and soil disease injury controlling agent and control using the same |
| CN109234197A (en) * | 2018-09-29 | 2019-01-18 | 河南省农业科学院植物保护研究所 | One plant can prevent and treat tobacco black shank and root black rot and pseudomonas aeruginosa and its application with growth-promoting functions simultaneously |
| CN110438049A (en) * | 2019-08-26 | 2019-11-12 | 中国农业科学院烟草研究所 | One plant of tobacco mosaic viruses biological and ecological methods to prevent plant disease, pests, and erosion Yi Shi pseudoalteromonas bacterial strain and its application |
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