Pseudomonas defense and uses thereof

Abstract

The application discloses a kind of defense pseudomonas and its application, belong to the field of biotechnology.The defense pseudomonas KBD-3 has been preserved in China General Microbiological Culture Collection Center on November 14, 2022, and its preservation number is CGMCC No.26145.The defense pseudomonas KBD-3 of the application has tobacco mosaic virus and tobacco bacterial wilt pathogen antagonistic activity, and also has selenium reduction capacity, so it can be better used for crop selenium enrichment planting, and also provides new microbial resources for crop disease control.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a Pseudomonas protegens and application thereof. BACKGROUND

[0002] In nature, plants are closely related to microorganisms in their growing environment, and the use of biocontrol agents to prevent and control plant diseases takes advantage of the relationship between plants and microorganisms and between microorganisms, and uses one or more beneficial microorganisms, i.e., biocontrol agents, to reduce the number of pathogenic microorganisms or reduce the pathogenic activity of pathogenic microorganisms, thereby achieving the purpose of reducing the occurrence of plant diseases. Biocontrol agents for preventing and controlling plant diseases have become the focus of research due to their green safety, difficulty in developing resistance, and strong selectivity.

[0003] As a "life element", selenium is essential for the growth of organisms, and has important physiological functions in improving immunity, regulating metabolism, antioxidant, detoxification, and promoting reproduction. In recent years, with people paying more and more attention to health and health preservation, various selenium-rich products have emerged, and farmers spray inorganic selenium (sodium selenate, sodium selenite, etc.) to increase the selenium content in the product during the planting process to improve the selling point. However, inorganic selenium is highly toxic and is classified as a sixth-class toxic substance in the Classification and Marking of Hazardous Chemicals (GB13690-2009). The use of inorganic selenium has seriously harmed the environment and human health. Nano-selenium has become the best form of selenium supplementation due to its low toxicity and high activity. Microbial transformation of nano-selenium has the advantages of mild conditions, stable structure, and good dispersibility, and has good application prospects.

[0004] Highly efficient and safe microbial agents have always been the focus of crop disease control research, but there are few research results on multiple prevention effects and selenium-enriched crops, and there is a lack of popularization and application. The present application will explore biological technology to screen for microbial agents that can enrich crops with selenium and have excellent prevention effects for application in the growth process of crops. SUMMARY

[0005] The present application provides a Pseudomonas protegens, named KBD-3, which is preserved in the China General Microbiological Culture Collection Center, with a preservation number of CGMCC No.26145 and a preservation date of November 14, 2022.

[0006] The above-mentioned Pseudomonas protegens KBD-3 has inhibitory activity against tobacco mosaic virus and Ralstonia solanacearum. Therefore, it can be applied to the prevention and / or treatment of plant diseases caused by tobacco mosaic virus or Ralstonia solanacearum; specifically, the above-mentioned Pseudomonas protegens KBD-3 can be prepared into a microbial agent to prevent and / or treat plant diseases caused by tobacco mosaic virus or Ralstonia solanacearum, such as tobacco mosaic disease and tobacco bacterial wilt.

[0007] The present application provides a kind of microbial preparation, containing above-mentioned defense pseudomonas KBD-3.

[0008] In the research of defense pseudomonas KBD-3, we found that KBD-3 has strong reduction and conversion ability to selenium element, which can convert inorganic selenium into selenium single element. For this reason, the present application also provides the application of above-mentioned defense pseudomonas KBD-3 in inorganic selenium reduction or selenium single element preparation.

[0009] The present application provides a kind of preparation method of nano selenium active bacteria liquid, steps as follows:

[0010] The above-mentioned defense pseudomonas KBD-3 bacteria liquid is inoculated in the culture medium containing inorganic selenium, and cultured under suitable conditions until the red material is enriched in the bacteria liquid, and the inorganic selenium is reduced to red nano selenium single element. The bacteria liquid is diluted to obtain the nano selenium active bacteria liquid.

[0011] In the above-mentioned preparation method of nano selenium active bacteria liquid, the inoculation amount of defense pseudomonas KBD-3 bacteria liquid is 1%, wherein the content of defense pseudomonas KBD-3 in the bacteria liquid is ≥10 8 cfu / mL. The defense pseudomonas KBD-3 bacteria liquid can be prepared by the following method:

[0012] The defense pseudomonas KBD-3 strain is inoculated on LB solid culture medium for purification, and the single colony with good growth is inoculated in LB liquid culture medium and cultured at 28℃, 150rpm for 20h to obtain the defense pseudomonas KBD-3 fermentation bacteria liquid.

[0013] In the above-mentioned preparation method of nano selenium active bacteria liquid, the culture medium can be selected from liquid culture medium, such as LB liquid culture medium, etc.

[0014] In the present application, the suitable temperature condition for the growth and reproduction of defense pseudomonas KBD-3 is 28-37℃, preferably 28℃. In the culture process, the shaking culture can also be selected, and the shaking rate should be suitable for defense pseudomonas KBD-3, for example, it can be 150r / min.

[0015] In the above-mentioned preparation method of nano selenium active bacteria liquid, the dilution of bacteria liquid can be diluted according to the requirement.

[0016] The present application provides a kind of preparation method of nano selenium, steps as follows:

[0017] The above-mentioned nano selenium active bacteria liquid is centrifuged, and the precipitate is the mixture of bacteria and nano selenium. Lysozyme is added to the precipitate and incubated fully. Then it is placed on ice for ultrasonic treatment. The solution after ultrasonic treatment is centrifuged, and the bacteria precipitate is discarded, and the supernatant is recovered. The supernatant is centrifuged, and the supernatant is discarded. The precipitate is washed to obtain nano selenium.

[0018] The nano selenium or the nano selenium active bacteria liquid prepared by the method can be used for selenium-enriched planting of crops.

[0019] In the present application, the inorganic selenium includes, but is not limited to, one or more of selenate, selenite and selenium oxide, and specifically can be sodium selenate, sodium selenite and the like.

[0020] The present application has the following beneficial effects:

[0021] The present application provides a defense pseudomonas with tobacco mosaic virus and ralstonia solanacearum antagonistic activity, which has selenium element reduction capacity, can be better used for selenium-enriched planting of crops, and provides a new microbial resource for crop disease control. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is a selenium element reduction test diagram, wherein the left diagram is KBD-3 fermentation bacteria liquid, and the right diagram is nano selenium active bacteria liquid;

[0023] Figure 2 It is a tobacco bacterial wilt pathogen inhibition zone test diagram, wherein the left diagram is KBD-3 bacteria liquid, and the right diagram is a control;

[0024] Figure 3 It is a KBD-3 fermentation bacteria liquid on TMV dry spot inhibition effect diagram. DETAILED DESCRIPTION

[0025] The KBD-3 strain is separated from a tobacco area soil sample in Baoshan, and the culture conditions of the strain are 28 DEG C, LB culture medium and pH 7.0, which is identified as Pseudomonas protegens and preserved in the China General Microbiological Culture Collection Center, with a preservation number of CGMCC No. 26145, a preservation date of November 14, 2022, and a preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing.

[0026] In the present application, the terms used herein have meanings generally understood by those of ordinary skill in the art, unless otherwise specified. The present application is further described in detail below in conjunction with specific examples and by referring to the data. The following examples are only for illustrating the present application, and do not limit the scope of the present application in any way.

[0027] Example 1

[0028] The separation, purification and identification test of the strain are as follows:

[0029] The rhizosphere soil sample of a non-diseased tobacco plant was collected from a serious tobacco planting area in Baoshan. 2 g of the collected soil sample was added to 20 mL of ddH2O, and the mixture was incubated at 28°C with 180 rpm shaking for 16 h. After standing for 30 min, the supernatant was streaked on LB solid medium and incubated in a 28°C incubator to obtain the strain. A single colony was picked and streaked on LB solid medium and incubated at 28°C to obtain a purified strain. The morphological characteristics of the strain are as follows: round colony, smooth surface, moist, shiny, creamy or translucent. It is preliminarily identified as Pseudomonas protegens.

[0030] DNA extraction and PCR amplification were performed according to the kit instructions, and 16S rDNA sequencing was performed by Qingdao Pisengnolgen Biotechnology Co., Ltd.

[0031] The sequencing results are as follows:

[0032]

[0033]

[0034] The determined sequence was compared with the sequences in GenBank through the BLAST program on the NCBI website, and the similarity was 99.93%. It was finally identified as Pseudomonas protegens, named KBD-3. The strain was preserved in the China General Microbiological Culture Collection Center on November 14, 2022, and the strain preservation number was CGMCC No. 26145.

[0035] Example 2

[0036] Selenium element reduction test:

[0037] Since the reduction of selenite is accompanied by the generation of red elemental selenium, a visual observation method was used for preliminary qualitative determination of the selenium-producing strain. Specifically as follows:

[0038] Pseudomonas protegens KBD-3 was inoculated on LB solid medium for purification, and a single colony with good growth was inoculated in LB liquid medium and incubated at 28°C with 150 rpm shaking for 20 h to obtain a fermentation broth ( Figure 1 , left panel). Then, the fermentation broth was inoculated into LB liquid medium containing 1 mM sodium selenite at an inoculation amount of 1%, and incubated at 28°C with 150 r / min shaking. After 2-5 days, red color was observed in the broth ( Figure 1 , right panel), indicating that Pseudomonas protegens KBD-3 reduced sodium selenite to nano-selenium.

[0039] Example 3

[0040] The tobacco bacterial wilt pathogen inhibition zone test is as follows:

[0041] In the center of the NA medium, 20 μL of P. protegens KBD-3 fermentation broth (concentration of 10 8 cfu / mL) was added, and after the fermentation broth was fully absorbed by the medium, it was cultured in an inverted position in a 28°C incubator for 48 h, and then a bacterial suspension of R. solanacearum (OD 600 = 0.3) was sprayed using a watering can, and it was cultured in an inverted position in a 28°C incubator. A control was set up, and the results were observed after 48 h. The test results are shown in Figure 2 Fig. 1. The KBD-3 bacterial liquid had a good inhibitory effect on R. solanacearum, with a diameter of the inhibition zone of 6.2 cm (Fig. 1, left), while the medium of the control group was full of R. solanacearum (Fig. 1, right). Figure 2 Figure 2 Example 4

[0042] Prevention test of P. protegens KBD-3 on tobacco bacterial wilt, as follows:

[0043] Test group: K326 seeds were sown in a seedling tray, and when the tobacco seedlings stretched 4-5 leaves, they were transplanted into flowerpots containing sterile soil. After the seedlings were acclimated, the KBD-3 fermentation broth (concentration of 10 8 cfu / mL) was used for the first time to irrigate the roots of the tobacco seedlings, and 5 days later, it was used again. Two days after the second irrigation, a bacterial suspension of R. solanacearum was inoculated into the rhizosphere soil of the tobacco seedlings, 10 mL per plant.

[0044] Control group: CK1 was a positive control, and 72% agricultural streptomycin was applied. CK2 was a disease control, and only R. solanacearum was inoculated. CK3 was a blank control, and no bacteria were inoculated. The other treatments were the same as the test group. Treatment Morbidity (%) Each group had 5 plants, and 3 repetitions were made. After 7 days of inoculation of R. solanacearum, the incidence rate (%), disease index, and relative control effect (%) of each tobacco seedling were investigated. Disease index Relative control efficiency (%) The test results are shown in Table 1. The incidence rate of tobacco bacterial wilt in the test group was 40.24%, the disease index was relatively low, the control effect reached 61.46%, which was higher than that of the positive control group, and the incidence rate of tobacco bacterial wilt in the disease control group was as high as 89.31%. Test group 40.24 Table 1 22.53 61.46 Treatment Morbidity (%) Disease index Relative control efficiency (%) Test group 40.24 22.53 61.46 CK1 53.86 30.21 48.32 CK2 89.31 58.46 - CK3 0 0 - 0 - Example 5

[0050] Tobacco mosaic virus inhibition test, as follows:

[0051]

[0052] ​​The semi-leaf method was used for the test. 10 mL of KBD-3 fermentation bacterial liquid (concentration of 10 8 cfu / mL) was mixed with the same volume of 40 times TMV juice (TMV-containing tobacco leaves were frozen and ground into powder, 1:40 was added to deionized water, and gauze was filtered) for 15 min, and 3 strains of Nicotiana tabacum were rubbed and inoculated with 2 leaves on the upper part, and a blank control (10 mL of LB liquid medium mixed with the same volume of 40 times TMV juice) was set up, and the number of dry spots was investigated 3-5 days later. The test results are shown in Table 2 and Figure 3 Figure 1, KBD-3 fermentation bacterial liquid has a good passivation effect on TMV Figure 3 , and the inhibition rate is as high as 95.8% (Table 2).

[0053] Table 2

[0054] Treatment Number of dry spots Dry spot inhibition rate (%) KBD-3 fermented bacterial solution 3.3 95.8 Blank control (CK) 78.6 -

[0055] Example 6

[0056] Preparation of nano-selenium:

[0057] The defense Pseudomonas KBD-3 bacterial liquid was inoculated into the LB liquid medium containing 1 mM sodium selenite at an inoculation amount of 1%, and was cultured at 28°C and 150 r / min for 2-5 days to obtain the nano-selenium active bacterial liquid. The nano-selenium active bacterial liquid was centrifuged at 13,000 x g for 10 min, and the precipitate was a mixture of bacteria and nano-selenium. Lysozyme was added to the precipitate to a final concentration of 20 mg / mL, and was incubated at 37°C for 20 min. During the incubation, the solution was inverted every 5 min to mix well. Then it was ultrasonicated on ice for 30 min (ultrasonic for 30 s, stop for 40 s). The ultrasonicated solution was centrifuged at 6,000 x g for 10 min, and the bacterial precipitate was discarded, and the supernatant was recovered. The supernatant was centrifuged at 13,000 x g for 10 min, and the supernatant was discarded, and the precipitate was dissolved and washed twice with ultrapure water to obtain nano-selenium.

[0058] Example 7

[0059] The selenium-rich treatment test is as follows:

[0060] The green beans with bright color, full grains, no worm and no mildew are selected as seed beans, soaked in 50℃ hot water for 30min, then transferred to a clean beaker after draining water. Under room temperature, 30mL 15μg / mL nano-selenium solution is added to the beaker, soaked overnight, then taken out and drained, and placed in a seedling tray covered with 4 layers of gauze, the upper part is covered with 4 layers of wet gauze, 10mL 15μg / mL nano-selenium solution is sprayed every 12h, at the same time, the green bean sprouts without selenium enrichment are used as blank control test (CK). After 4d, the green bean sprouts cultured under different conditions are taken, the seed coat is removed, and the surface attached selenium is removed by repeatedly rinsing with deionized water, then placed in a 60℃ oven for drying (about 6h), crushed through a 60 mesh sieve, then the selenium content in the sample is determined by using a hydride atomic fluorescence spectrophotometer. The test results are shown in Table 3, the selenium content in the green bean sprouts treated by nano-selenium solution is 45.35μg / g, compared with the control, the selenium content is greatly improved, which can meet the safe demand of human body for selenium.

[0061] Table 3

[0062] Treatment Sample mass (g) Selenium content (μg / g) Nano-selenium solution 0.15 45.35 CK 0.15 0.00

[0063] The above only describes preferred embodiments of the present application and is not intended to limit the present application in other forms. Any skilled person in the art can modify or change the above disclosed technical content to equivalent embodiments. However, any simple modification, equivalent change and modification made to the above embodiments without departing from the technical solution of the present application, and according to the technical essence of the present application, still belongs to the protection scope of the technical solution of the present application.

Claims

1. A defense against Pseudomonas (Pseudomonas spp.) characterized by, Pseudomonas protegens The bacterial strain is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 26145, and the preservation date is November 14, 2022. ​ 2. Use of the Pseudomonas protegens in claim 1 in the preparation of a microbial preparation for preventing and treating plant diseases caused by tobacco mosaic virus and / or Ralstonia solanacearum.

3. A microbial preparation, characterized in that, The microbial preparation contains the Pseudomonas protegens in claim 1.

4. Use of the Pseudomonas protegens in claim 1 in the reduction of inorganic selenium or the preparation of elemental selenium.

5. A method for preparing a nano-selenium active bacteria solution, characterized in that, The steps are as follows: The bacterial liquid of the Pseudomonas protegens in claim 1 is inoculated into a culture medium containing inorganic selenium, and cultured under suitable conditions until the bacterial liquid is enriched with red substances, and the inorganic selenium is reduced to red elemental nanose.

6. The production method according to claim 5, characterized by, The inoculation amount of the Pseudomonas protegens bacterial liquid is 1%, and the suitable conditions are 28-37℃.

7. A method for preparing nano-selenium, characterized in that, The steps are as follows: The nanose active bacterial liquid prepared in claim 5 is centrifuged, and the precipitate is a mixture of bacteria and nanose; lysozyme is added to the precipitate and incubated thoroughly; and then ultrasonic is performed on ice; The solution after ultrasonic is centrifuged, and the bacterial precipitate is discarded, and the supernatant is recovered; the supernatant is centrifuged, and the supernatant is discarded, and the precipitate is washed to obtain nanose.

8. The use according to claim 4, or the preparation method of nano-selenium active bacteria solution according to any one of claims 5-6, or the preparation method of nano-selenium according to claim 7, characterized in that, The inorganic selenium is selected from one or more of selenate, selenite and selenium oxide.

9. The nanose active bacterial liquid prepared by the method in any one of claims 5-6, or the nanose prepared by the method in claim 7.

10. Use of the nanose active bacterial liquid or nanose in claim 9 in selenium-enriched planting of crops.

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