阅读说明：本技术 荧光假单胞杆菌的纳米硒合成活性菌液、其制备方法及其应用 (Nano-selenium synthetic active bacterial liquid of pseudomonas fluorescens, preparation method and application thereof ) 是由 杨金广 袁莲莲 宋丽云 焦玉冰 李莹 申莉莉 王凤龙 黄坤 夏振远 于 2020-12-18 设计创作，主要内容包括：本发明公开了一种荧光假单胞杆菌的纳米硒合成活性菌液、其制备方法及其应用,属于微生物及微生物制剂技术领域。本发明的荧光假单胞杆菌的纳米硒合成活性菌液；是由荧光假单胞杆菌进行生物发酵合成纳米硒后,由荧光假单胞杆菌及其合成的纳米硒组成的混合液；所述荧光假单胞杆菌为荧光假单胞杆菌KBD-1；该菌株于2020年08月31日保藏于中国微生物菌种保藏管理委员会普通微生物中心,菌种保藏号为CGMCC No.20567。本发明荧光假单胞杆菌的纳米硒合成活性菌液对作物病毒病具有较好的防治效果,为作物病毒病的防治提供新的微生物资源；此外,还对作物具有促生效果,且效果显著。(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; is a mixed solution which is formed by fluorescent pseudomonas and 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 (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.)

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 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. 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 LB liquid culture medium containing 1mM sodium selenite with the inoculation amount of 1%, carrying out shaking culture at 28 ℃ at 150r/min, and observing the bacterial liquid to be red 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 pseudomonas fluorescens in claim 2 in preventing and treating crop virus diseases or promoting crop growth.

4. The use of the nano-selenium-synthesized active bacterial liquid of pseudomonas fluorescens as claimed in claim 2 in the preparation of antiviral agents for preventing and treating crop virus diseases or crop growth promoters.

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 10⁸cfu/mL; the concentration of the nano-selenium is more than or equal to 50 mg/L.

6. The method for controlling crop virus diseases according to claim 5, 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.

7. A method for promoting the growth of crops, which is characterized in that the nano-selenium synthetic active bacterial liquid of the fluorescent pseudomonas as claimed in claim 2 is diluted and then the root of the crops is irrigatedThe bacterium content in the diluted nano-selenium synthetic active bacterium liquid of the pseudomonas fluorescens is 2 multiplied by 10⁵cfu/mL～2×10⁶cfu/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 10⁵cfu/mL, nano-selenium concentration is 0.25 mg/L.

8. The method for promoting the growth of a crop according to claim 7, wherein the root irrigation treatment is: 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.

9. An antiviral agent for crops, characterized in that the effective component comprises nano-selenium synthetic active bacterium liquid of pseudomonas fluorescens of claim 2; the content of pseudomonas fluorescens in the crop antiviral agent is more than or equal to 10⁸cfu/mL, and the concentration of the nano-selenium is more than or equal to 50 mg/L.

10. A crop growth promoter, 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 10⁵cfu/mL～2×10⁶cfu/mL, and the concentration of the nano-selenium is 0.1 mg/L-1 mg/L.

Technical Field

The invention belongs to the technical field of microorganisms and microbial preparations, and particularly relates to a nano-selenium synthetic active bacterial liquid of pseudomonas fluorescens, 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 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 nano-selenium synthetic active bacterial liquid for resisting the 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 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 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 with the inoculation amount of 1%, carrying out shaking culture at 28 ℃ at 150r/min, and observing the bacterial liquid to be red 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 10⁸cfu/mL; the concentration of the nano-selenium is more than or equal to 50 mg/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 10⁵cfu/mL～2×10⁶cfu/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 10⁵cfu/mL, nano-selenium concentration is 0.25 mg/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.

An antiviral agent for crops, whose effective component contains the above-mentioned fluorescentNano-selenium synthesized active bacterial liquid of pseudomonas photaenis; the content of pseudomonas fluorescens in the crop antiviral agent is more than or equal to 10⁸cfu/mL, and the concentration of the nano-selenium is more than or equal to 50 mg/L.

A crop growth promoter comprises the effective component of the nano-selenium synthetic active bacterial liquid of the fluorescent pseudomonas; the content of fluorescent pseudomonas in the crop growth promoter is 2 multiplied by 10⁵cfu/mL～2×10⁶cfu/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.

Drawings

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 China general microbiological culture Collection center at 31.08.2020 for 31.7.4.1. the preservation address is No. 3 of West Lu No. 1 of Beijing city Kogyo area, the preservation number of the strain is CGMCC No.20567, and the strain 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 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)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 times the TMV sap (tobacco leaves with TMV were 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)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

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. Respectively diluting the active bacterial liquid (selenium content is 50mg/L) of nano-selenium biosynthesis by 0 time (selenium content is 50mg/L, bacterial concentration is 10)⁸cfu/mL), 50 times, 100 times, 200 times, and 500 times, 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, 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. After being diluted by 50 times, the green bean rooting promoter has an inhibiting effect on the rooting of the green 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 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

TABLE 4 growth promoting effect of active bacteria liquid for biosynthesis of nano-selenium on K326

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.