CN114717160A

CN114717160A - Enterobacter and microbial inoculum and application thereof

Info

Publication number: CN114717160A
Application number: CN202210516679.4A
Authority: CN
Inventors: 孙艳梅; 王利平; 魏福; 夏思哲; 郭子晟; 王世伟
Original assignee: Northwest University
Current assignee: Enshi Benxin Agricultural Technology Co ltd
Priority date: 2022-05-12
Filing date: 2022-05-12
Publication date: 2022-07-08
Anticipated expiration: 2042-05-12
Also published as: CN114717160B

Abstract

The invention relates to the technical field of microbiology and plant selenium enrichment, in particular to a selenium-enriched strain, a microbial inoculum containing the selenium-enriched strain and application of the selenium-enriched strain and the microbial inoculum. The enterobacter has a preservation number of CGMCCNo.24655. Compared with the existing selenium-rich mode, the selenium-rich microbial inoculum provided by the invention has the advantages that the selenium content in plants can be greatly increased and the environmental pollution risk of selenium salt is eliminated through the absorption promotion effect of high-selenium-rich microorganisms on inorganic selenium salt. The selenium-enriched strain provided by the invention can tolerate selenite with high concentration, so that the applicable window of the strain to the selenium salt environment is increased. The selenium-rich strain provided by the invention can be colonized in plants, particularly in the rhizosphere and leaves of the plants, thereby providing a basis for cultivating the plants rich in selenium and selenium-rich plants. The selenium-enriched strain provided by the invention can also influence the diversity of endophytic microorganisms of plants, for example, the ratio of Alcaligenes in plant leaves can be increased, so that the selenium-enriched level is further increased.

Description

Enterobacter and microbial inoculum and application thereof

Technical Field

The invention relates to the technical field of microbiology and plant selenium enrichment, in particular to a selenium-enriched strain, a microbial inoculum containing the selenium-enriched strain and application of the selenium-enriched strain and the microbial inoculum.

Background

Selenium is a trace element necessary for human bodies, the Chinese society of nutrition has listed selenium as one of 15 nutrients necessary for human bodies, but the ingestion of high-dose selenium has a toxic effect, and the selenium supplement mode is considered to be the safest selenium supplement mode when people eat natural selenium-rich plants. Therefore, the market demand of selenium fertilizers for enriching and transforming selenium for crops, fruits and vegetables is increasing. The traditional selenium-rich fertilizer mainly has two application modes, namely, inorganic selenite or selenate is used, so that the absorption of plants is less, and soil and water pollution is easily caused; secondly, organic selenium fertilizer, such as selenium-enriched yeast or some selenium-enriched plants, is used, needs biotransformation, and has long time, high cost and incomplete plant absorption.

Therefore, a safe, reliable and high-bioavailability selenium enrichment mode is urgently needed to be developed, so that the selenium content in plants is greatly increased, the environmental pollution risk of selenium salt is eliminated, and the defects that the traditional fertilizer is reduced in benefit and serious in non-point source pollution are overcome.

Disclosure of Invention

In order to realize the aim, the invention provides a selenium-enriched strain, a microbial inoculum containing the selenium-enriched strain and application of the selenium-enriched strain in selenium metabolism and selenium enrichment. The enterobacter provided by the invention can promote the absorption and transformation of inorganic selenium salt, increase the selenium content in plants, has higher selenium salt tolerance, can be colonized on the roots and leaves of the plants, and influences the diversity of microorganisms in the plants.

Therefore, in the first aspect of the present invention, an Enterobacter (Enterobacter hormecei) strain with a preservation number of CGMCC No.24655 is provided.

In a second aspect, there is provided a microbial preparation comprising the Enterobacter (Enterobacter Hormanehei) described in the first aspect.

In a third aspect, there is provided the use of the enterobacteria of the first aspect or the microbial inoculum of the second aspect in selenium metabolism.

The fourth aspect provides the use of the enterobacter of the first aspect or the microbial inoculum of the second aspect in plant selenium-enriched or selenium-enriched plant cultivation.

A fifth aspect provides the use of an enterobacterium according to the first aspect or a bacterial agent according to the second aspect for modulating the diversity of endogenous microorganisms in a plant.

A sixth aspect provides a method of cultivating a plant rich in selenium or selenium-enriched plants, the method comprising: contacting the enterobacter of the first aspect or the microbial inoculum of the second aspect with a plant to be selenium enriched or a plant to be selenium enriched.

A seventh aspect provides a selenium-enriched microbial fertilizer comprising the enterobacteria of the first aspect or the microbial inoculum of the second aspect, and optionally an inorganic and/or organic selenium fertilizer.

Through the technical scheme of the invention, the following beneficial effects can be obtained:

(1) compared with the existing selenium-rich mode, the selenium-rich microbial inoculum provided by the invention has the advantages that the selenium content in plants can be greatly increased, the environmental pollution risk of selenium salt is eliminated, and the defects of decreased benefit, serious non-point source pollution and the like of the traditional fertilizer are avoided through the absorption promotion effect of high-selenium-rich microorganisms on inorganic selenium salt. In addition, the microbial fermentation technology can be used for rapidly realizing mass preparation and industrial production of the microbial fertilizer, and the method has the advantages of low fermentation production cost, short period and high yield, and is suitable for large-area application demonstration and industrial popularization.

(2) The selenium-enriched strain provided by the invention can tolerate selenite and/or selenate with high concentration, so that the applicable window of the strain to the selenium salt environment is increased.

(3) The selenium-rich strain provided by the invention can be colonized in plants, particularly in rhizosphere and leaves of the plants, thereby providing a foundation for cultivating the plants rich in selenium and selenium-rich plants.

(4) The selenium-enriched strain provided by the invention can also influence the diversity of endophytic microorganisms of plants, for example, the ratio of Alcaligenes in plant leaves can be increased, so that the selenium-enriched level is further increased.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Biological preservation

The Enterobacter (Enterobacter hormaechei) provided by the invention is preserved in the China general microbiological culture Collection center of the culture Collection management Committee of microorganisms at 4 months and 7 days in 2022, the preservation number is CGMCC No.24655, the preservation address is No. 3 of the Beijing university Hokko No. 1 of the sunward district, China academy of sciences (abbreviated as CGMCC), and the short term is NS-3.

Drawings

In order that the present disclosure may be more readily and clearly understood, the following detailed description of the present disclosure is provided in connection with specific embodiments thereof and the accompanying drawings, in which,

FIG. 1 shows 6 endophytic bacteria in the presence of 10mM Se⁴⁺The growth conditions of (1);

FIG. 2 shows sodium selenite tolerance of 6 endophytic bacteria;

FIG. 3 shows 6 endophytic bacteria in 20mM Se⁴⁺The reduction rate of (2);

FIG. 4 is a fluorescent microscope picture of NS3-GFP that was successfully constructed;

FIG. 5A is a photograph of the colonization of the root of Cardamine cordifolia by strain NS 3-GFP;

FIG. 5B is a gel electrophoresis of GFP fragments amplified from roots, stems and leaves of Cardamine violifolia;

FIG. 6 shows the plant Selenocyanine SeCys of Cardamine violifolia₂(fig. 6A), methylselenocysteine mesecs (fig. 6B), nano-selenium SeNPs (fig. 6C), and total selenium (fig. 6D) content;

FIG. 7 shows the effect of Enterobacter NS-3 on the beta-diversity of microorganisms present in the rhizosphere soil (FIG. 7A), roots (FIG. 7B), and leaves (FIG. 7C) of Cardamine violifolia;

FIG. 8 shows the effect of Enterobacter NS-3 on microbial communities in Cardamine violifolia rhizosphere soil (FIG. 8A), roots (FIG. 8B), and leaves (FIG. 8C).

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

In a first aspect, the invention provides an Enterobacter (Enterobacter hormichei) with the preservation number of CGMCC No. 24655.

The enterobacter CGMCC No.24655(NS-3) belongs to the enterobacter, belongs to Proteobacteria, belongs to Enterobacteriaceae, is a gram-negative bacillus, is separated from cardamine violifolia, and is a plant endophyte.

Specifically, the method comprises the steps of firstly separating and purifying cardamine violifolia to obtain 6 strains of endophytes, then carrying out stress culture on the 6 strains of endophytes in an selenite environment, evaluating the tolerance of selenite with different concentrations and the reduction capability of the selenite, obtaining an endophyte which can survive in a higher selenite environment and has higher nitrite reduction capability, and further comparing through a 16s sequence (shown as SEQ ID NO: 1), determining that the strain is Enterobacter hopcalis (Enterobacter hormonechi) and is numbered as NS-3.

SEQ ID NO:1：

According to the invention, the method for separating endophyte from cardamine violifolia specifically comprises the following steps: sterilizing Cardamine violifolia, mashing, grinding, coating the diluted solution on a flat plate, and separating and purifying to obtain endophytic bacteria. More specifically, sterilizing roots, stems and leaves of cardamine violifolia; after the surface of the material is disinfected, the material is fully ground, the material is diluted in a gradient mode twice, the diluted material is coated on a solid flat plate, the culture is carried out, endophytic bacteria with different forms are counted, the purified endophytic bacteria are obtained, and the obtained endophytic bacteria are stored in a refrigerator with the temperature of 80 ℃ below zero by using a glycerol tube with the concentration of 40% for later use.

Wherein, the solid plate can be LB culture medium, which contains yeast powder 8-12g/L, peptone 8-12g/L, sodium chloride 4-6g/L, agar powder 1.2-2g/L, pH 6.5.5-7.5.

According to the present invention, the enterobacteria provided by the present invention can produce a large amount of viable enterobacteria by liquid culture, and the method of culture is not particularly limited as long as the enterobacteria can be proliferated, and for example, viable enterobacteria can be inoculated into a fermentation medium and cultured under aerobic conditions to obtain a fermentation broth.

Preferably, the preparation process of the viable bacteria can further comprise activating and purifying the preserved NS-3 on a solid plate, then inoculating the NS-3 into a seed culture medium for culture to obtain a seed solution, finally inoculating the seed solution into a fermentation culture medium, and obtaining a fermentation liquid after fermentation culture.

Wherein the seed culture medium can be LB liquid culture medium, for example, can contain 8-12g/L yeast powder, 8-12g/L peptone, 4-6g/L, pH 6.5 sodium chloride 6.5-7.5. Preferably, the activated and purified NS-3 is inoculated into a seed culture medium and cultured to obtain a seed solution. In the present invention, the conditions for the seed culture preferably include: the rotation speed is 150-250rpm, the temperature is 25-40 ℃, and the time is 8-10 hours.

Wherein the fermentation medium can be LB liquid medium, for example, it can contain yeast powder 8-12g/L, peptone 8-12g/L, sodium chloride 4-6g/L, pH 6.5-7.5. Preferably, the seed culture is inoculated in the fermentation medium in an amount of 0.5-1.5% by volume, and preferably cultured at 25-40 ℃ and 250rpm of 150 ℃ for 15-25 hours.

In the present invention, the viable bacteria of enterobacter in the fermentation liquid can be further separated, and the method for separating is not particularly limited as long as the bacteria can be enriched from the culture liquid, and for example, the separation can be achieved by a centrifugation and/or filtration method, and the conditions for centrifugation and filtration can be known conditions, and the present invention is not described herein again.

According to the present invention, in order to facilitate the study of the properties and characteristics of this strain, the Enterobacter CGMCC No.24655 further contains a tracking plasmid, for example, a vector into which a fluorescent protein (GFP) is to be inserted. The vector may be a broad-host vector, such as pUC19, among others.

Wherein, in case the strain contains a tracking plasmid, the fermentation medium may further contain an antibiotic compatible with the tracking plasmid, for example, carbenicillin (Cb), and the content thereof may be 350-450. mu.g/mL, for example, 400. mu.g/mL.

In a second aspect, the present invention provides a bacterial agent comprising an enterobacterium (enterobacterium homanehei) as described above.

According to the present invention, the form of the microbial inoculum may not be particularly limited, and may be, for example, a liquid microbial inoculum, a semi-liquid microbial inoculum, a concentrated microbial inoculum, a compressed microbial inoculum, or a solid microbial inoculum. The solid microbial inoculum can be a dry microbial inoculum, for example, a freeze-dried microbial inoculum, a spray-dried microbial inoculum, and the like.

Preferably, the microbial inoculum contains viable cells of said enterobacteria. The number of the live bacteria in the microbial inoculum can be selected in a wide range as long as the requirements of relevant standards are met, and preferably, the content of the live bacteria in the microbial inoculum is more than 2 hundred million cfu/g of the microbial inoculum.

According to the present invention, the microbial inoculum can be prepared according to a conventional method in the field, and according to a specific embodiment of the present invention, the method for preparing the microbial inoculum comprises: the enterobacteria are fermented and cultured in a fermentation medium, and the viable count is 10⁸More than cfu/mL to obtain a liquid microbial inoculum; concentrating the liquid microbial inoculum, for example, by solid-liquid separation to obtain a semi-liquid microbial inoculum; then adding a freeze-drying protective agent into the semi-liquid microbial inoculum, and adjusting the concentration of viable bacteria to 10¹⁰More than cfu/mL, and drying the obtained mixed material to obtain the dry microbial inoculum.

According to the present invention, the solid-liquid separation method can be performed by means of techniques conventional in the art, for example, centrifugation, filtration, etc., as long as the activity of enterobacteria is not significantly affected. According to a preferred embodiment of the present invention, the centrifugation, for example, can be performed in a refrigerated centrifuge at the speed of 5000-12000rpm for 5-20min to obtain the bacterial pellet.

According to the present invention, the lyoprotectant may be any one of various cryoprotectants that are conventional in the art, for example, at least one of skim milk powder, maltodextrin, trehalose, dextran, glycerin, and the like.

According to the present invention, preferably, before adding the lyoprotectant to the semi-liquid microbial inoculum, the method further comprises washing the semi-liquid microbial inoculum with a buffer. The buffer may be a buffer conventionally used in the art for washing the cells, and may be, for example, physiological saline or PBS buffer.

The drying method is not particularly limited, and may be, for example, freeze-drying, oven-drying, air-drying or spray-drying.

The strain provided by the invention can be colonized in roots and leaves of cardamine violifolia, so that the selenium absorption of plants is promoted, selenium salt is converted into nano-selenium endophytic bacteria, the selenium enrichment of the plants is assisted, the environmental pollution risk of the selenium salt is eliminated, and the defects of diminishing benefits, serious non-point source pollution and the like of the traditional fertilizer are overcome. In addition, the microbial fermentation technology can be used for rapidly realizing mass preparation and industrial production of the microbial fertilizer, and the method has the advantages of low fermentation production cost, short period and high yield, and is suitable for large-area application demonstration and industrial popularization.

According to the present invention, as described above, the strain provided by the present invention can promote the absorption and transformation of selenium by plants. On the other hand, the strain provided by the invention can be colonized on roots and leaves of plants so as to be converted into endophytes of the plants, thereby providing convenient conditions for the cultivation of selenium-enriched plants.

As shown in the embodiment of the invention, the microbial inoculum provided by the invention can also regulate the diversity of microorganisms in plants.

Preferably, the endogenous microorganism is an endogenous microorganism in a plant leaf.

Preferably, the enterobacteria are capable of increasing the occupancy of the alcaligenes.

According to the present invention, the plant mentioned in the above aspects is not particularly limited, and preferably, the plant is a plant selected from the group consisting of cruciferae, liliaceae, solanaceae, actinidiaceae, and theaceae, and the like, and more preferably, a plant of genus capsella of cruciferae (e.g., cardamine violifolia), a plant of genus brassica of brassicaceae (e.g., cauliflower, chinese cabbage), a plant of genus allium of liliaceae (e.g., garlic), a plant of genus capsicum of solanaceae (e.g., capsicum), a plant of genus actinidia of actinidiaceae (e.g., kiwi), and a plant of genus camellia of theaceae (tea plant).

Specifically, the strain or microbial inoculum provided by the invention can be applied to the cultivation soil of a plant, so as to realize contact with the plant. Further, the liquid containing the strain or the microbial inoculum of the present invention may be sprayed on the stem and leaf of the plant to contact the plant, and the former is preferable in the present invention.

According to the invention, the selenium-enriched microbial fertilizer can also contain acceptable auxiliary agents in the fertilizer.

In addition, according to the present invention, the strain or microbial inoculum can also be used in combination with other conventional fertilizers, including but not limited to inorganic and/or organic selenium fertilizers, phosphate fertilizers, nitrogen fertilizers, potassium fertilizers, and the like.

According to the invention, the selenium-rich microbial fertilizer can be prepared into different types of fertilizers such as granular fertilizer, liquid fertilizer and the like according to different application modes.

Examples

The methods and materials in the following examples are all conventional methods, unless otherwise specified.

Example 1

This example demonstrates the selection of selenium-enriched strains

1.6 strains of endophytic bacteria are obtained by separating and purifying cardamine violifolia, and are numbered as NS-1, NS-3, NS-15, NS-18, NS-19 and NS-G2.

2. Selenite selenium tolerance assay: activating the separated 6 cardamine hirsute endophytic bacteria to 10mM Na₂SeO₃Culturing in solid LB culture medium at 37 deg.C for 12-24 h.

FIG. 1 shows that these 6 strains contained 10mM Na₂SeO₃Growth of solid LB medium. As can be seen from FIG. 1, at 10mM Na₂SeO₃These 6 strains of bacteria were able to grow in LB medium and were single red colonies.

FIG. 2 shows the growth of various cardamine hirsute endophytic bacteria at different sodium selenite concentrations (0, 20, 40, 80, 100, 140, 180, 200, 240, 300, 400mM) and measured for spectrophotometric values at 600 nm. As can be seen in FIG. 2, the endophyte NS-1 was resistant to sodium selenite concentrations of 180 mM; the concentration of the sodium selenite which is tolerated by the endophyte NS-3 is 200 mM; the concentration of the sodium selenite which is tolerated by the endophyte NS-15 is 20 mM; the concentration of the sodium selenite which is tolerated by the endophyte NS-18 is 100 mM; the concentration of the resistant sodium selenite of the endophyte NS-19 is 20 mM; endophyte NS-G2 tolerated sodium selenite concentration of 180 mM.

3. And (3) selenite reduction rate determination: single clones were picked up in 5mL of liquid LB medium and inoculated with 100. mu.L of each of 20mM Na₂SeO₃The liquid LB fermentation medium of (1) was subjected to shaking culture at 37 ℃ and 200rpm for about 24 hours. Collecting 1mL of the prepared fermentation broth, placing in 1.5mL centrifuge tube, numbering, centrifuging at 12000rpm for 5min, discarding supernatant, washing with normal saline for 3 times, and adding 20mL of 1M Na₂And (3) mixing the S solution, fully reacting for 1h, centrifuging at 12000rpm for 2min again, taking the supernatant, measuring the light absorption value at 500nm, and calculating the reduction efficiency of 6 endophytic bacteria by measuring the amount of elemental selenium generated in the supernatant, wherein the result is shown in figure 3.

As can be seen from FIG. 3, the endophyte NS-3 can release 62.07% of 20mM Na within 24h₂SeO₃The reduction to red elemental selenium is the highest of 6 strains.

Example 2

This example illustrates the colonization ability of 6 endophytic bacteria in Cardamine cordifolia.

Genetic operation is carried out on green fluorescent protein GFP by using a broad-host vector pUC19 to enable the GFP to become recombinant plasmids, the recombinant plasmids are respectively transferred into 6 endophytic bacteria by an electrical transformation method, and through a back grafting experiment, only the strain NS3-GFP can be colonized in cardamine violifolia, and the other 5 strains cannot be colonized. The constructed engineered bacterium NS3-GFP capable of expressing green fluorescent protein can observe green fluorescence under a fluorescence microscope as shown in FIG. 4.

In addition, in order to widely apply the strain NS-3 to actual production, the NS-3 is further applied to cauliflower, garlic, hot pepper, Chinese cabbage, kiwi fruit and tea tree, the selenium enrichment effect of the NS-3 is explored, and the result shows good selenium enrichment effect.

Example 3

This example is presented to illustrate the verification of NS-3 selenium-rich function

1. Preparation of NS3-GFP selenium-rich microbial inoculum

The strain NS3-GFP was activated onto an LB plate containing Cb 400. mu.g/mL, and cultured at 37 ℃ for 12-16 hours. Picking the monoclonal to a liquid LB culture medium containing Cb 400 mu g/mL, carrying out shaking culture for 10-12h at 37 ℃ at 200rpm, transferring the monoclonal to a 10mL liquid LB culture medium containing Cb 400 mu g/mL at the inoculation amount of 1%, carrying out shaking culture for 18h at 37 ℃ at 200 rpm. Taking the shaken bacterial liquid in a 50mL centrifuge tube, centrifuging at 4 deg.C and 6000rpm for 10min, and resuspending with sterile water to obtain 10 concentration bacterial liquid⁸cfu/mL (i.e., OD600 ═ 0.8), and bottled.

2. Fungicide NS3-GFP selenium-rich functional verification

1) Adding NS3-GFP bacterial liquid into the potting soil when cardamine violifolia grows to 5 cm. The bacterial liquid preparation method is as follows, strain NS3-GFP is activated to LB plate containing Cb 400 mug/mL, and cultured for 12-16h at 37 ℃. The single clone is picked up to be cultured in liquid LB culture medium containing Cb 400 mug/mL for 10-12h at 37 ℃ under 200rpm with shaking, and is respectively transferred in 10mL liquid LB culture medium containing Cb 400 mug/mL with 1 percent of inoculum size for culturing for 18h at 37 ℃ under 200rpm with shaking. Taking the shaken bacterial liquid in a 50mL centrifuge tube, centrifuging at 4 deg.C and 6000rpm for 10min, and resuspending with sterile water to obtain 10 concentration bacterial liquid⁸cfu/mL (namely OD600 ═ 0.8), after bacterial liquid is prepared, the bacterial liquid is beaten around the root of cardamine violifolia by a needle tube, and 300mL of the bacterial liquid is watered. Adding strain NS3-GFP bacterial liquid into cardamine violifolia potted plant without adverse reaction, starting to apply sodium selenite, and according to experimental grouping conditions, adding the sodium selenite into the cardamine violifolia potted plantAdding sodium selenite with selenium content of 0, 50, 100, 200mg/kg into soil, and applying twice, once every 20 days.

2) After a microbial inoculum NS3-GFP is inoculated into cardamine violifolia potted plants, roots are taken and sliced to observe the colonization condition of the strain NS3-GFP in cardamine violifolia under a laser confocal microscope, and the roots of cardamine violifolia are observed by a laser confocal microscope, and the result is shown in figure 5A, that the strain with green fluorescence mark emits green fluorescence, which indicates that endophyte is well colonized on the roots of plants, then, in order to further explore the colonization situation of the endophyte NS-3 in the cardamine violifolia, total DNA of the cardamine violifolia root and the cardamine violifolia is respectively extracted, primers are designed to amplify GFP fragments, and as a result, as shown in FIG. 5B, the total DNA of the cardamine violifolia potting soil, the roots and the cardamine violifolia added with the fungicide NS3-GFP amplifies GFP fragments which are shared with the strain NS3-GFP and have the size of about 500bp, and the result proves that the endophyte can colonize at the root, the stem and the leaf of the cardamine violifolia.

3) As shown in FIGS. 6A (6B) (6C) (6D), two months after adding bacterial agent NS3-GFP into cardamine violifolia potting soil, cardamine violacea plant selenocysteine SeCys₂The contents of the methyl selenocysteine MeSeCys, the nano-selenium SeNPs and the total selenium are obviously increased, and when 200mg/kg of selenium fertilizer is applied, the bacterial agent NS3-GFP is used for treating the SeCys of the cardamine violifolia leaves₂The highest content is 2243.765mg/kg, which is 56.78% higher than that of the non-inoculated group; when the selenium fertilizer is applied to 50mg/kg, the MeSeCys content of cardamine violifolia leaves treated by the fungicide NS3-GFP is obviously increased, and is 87.82% higher than that of a non-inoculated group; when 200mg/kg of selenium fertilizer is applied, the highest content of SeNPs of cardamine violifolia leaves treated by the fungicide NS3-GFP is 1851.625mg/kg, which is 41.04 percent higher than that of a non-inoculated group; when 200mg/kg of selenium fertilizer is applied, the highest total selenium content of cardamine violifolia leaves treated by the fungicide NS3-GFP reaches 4180.465mg/kg, the selenium content is increased by 48.78% compared with a control group without applying the selenium fertilizer, and the selenium content is increased by 99.8% compared with a control group without applying the selenium fertilizer or adding the fungicide NS3-GFP, so that the selenium content of cardamine violifolia plants can be effectively increased by adding the fungicide NS-3 and applying the selenium fertilizer from an external source, and the plants are rich in selenium.

Example 4

This example illustrates the effect of Enterobacter NS-3 on the diversity of Endomethacin-producing microorganisms

After the cardamine violifolia is harvested, roots, stems and leaves are collected, the total DNA of endophytes in the leaves is extracted by a Fast DNA TM Spin kit for Soil kit, and the prepared DNA sample is subjected to sequencing analysis. Sequencing results show that adding enterobacter NS-3 does not affect beta-diversity of cardamine violifolia (fig. 7A) (fig. 7B) (fig. 7C), and taking adding no fungicide NS-3 as a blank control group, the effect of adding fungicide NS-3 to cardamine violifolia rhizosphere soil, roots and leaf endophytes in cardamine violifolia potted plants is evaluated, as shown in fig. 8A, the percentage content of microbial flora of cardamine violifolia rhizosphere soil is not affected by enterobacter NS-3, as shown in fig. 8B, after 50 and 200mg/kg selenium treatment, enterobacter is increased obviously, and is increased by 4.3% and 7.7% respectively, which indicates that enterobacter NS-3 can be colonized in cardamine violifolia. As shown in FIG. 8C, after adding Enterobacter NS-3, Alcaligenes was increased in Cardamine violifolia leaves.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Sequence listing

<110> northwest university

<120> enterobacter and microbial inoculum and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1407

<212> DNA

<213> Enterobacter hollandii (Enterobacter hormecei)

<400> 1

tgcagtcgaa cggtaacagg aagcagcttg ctgctttgct gacgagtggc ggacgggtga 60

gtaatgtctg ggaaactgcc tgatggaggg ggataactac tggaaacggt agctaatacc 120

gcataacgtc gcaagaccaa agagggggac cttcgggcct cttgccatcg gatgtgccca 180

gatgggatta gctagtaggt ggggtaacgg ctcacctagg cgacgatccc tagctggtct 240

gagaggatga ccagccacac tggaactgag acacggtcca gactcctacg ggaggcagca 300

gtggggaata ttgcacaatg ggcgcaagcc tgatgcagcc atgccgcgtg tatgaagaag 360

gccttcgggt tgtaaagtac tttcagcggg gaggaaggcg ataaggttaa taaccttgtc 420

gattgacgtt acccgcagaa gaagcaccgg ctaactccgt gccagcagcc gcggtaatac 480

ggagggtgca agcgttaatc ggaattactg ggcgtaaagc gcacgcaggc ggtctgtcaa 540

gtcggatgtg aaatccccgg gctcaacctg ggaactgcat tcgaaactgg caggctagag 600

tcttgtagag gggggtagaa ttccaggtgt agcggtgaaa tgcgtagaga tctggaggaa 660

taccggtggc gaaggcggcc ccctggacaa agactgacgc tcaggtgcga aagcgtgggg 720

agcaaacagg attagatacc ctggtagtcc acgccgtaaa cgatgtcgac ttggaggttg 780

tgcccttgag gcgtggcttc cggagctaac gcgttaagtc gaccgcctgg ggagtacggc 840

cgcaaggtta aaactcaaat gaattgacgg gggcccgcac aagcggtgga gcatgtggtt 900

taattcgatg caacgcgaag aaccttacct actcttgaca tccagagaac ttagcagaga 960

tgctttggtg ccttcgggaa ctctgagaca ggtgctgcat ggctgtcgtc agctcgtgtt 1020

gtgaaatgtt gggttaagtc ccgcaacgag cgcaaccctt atcctttgtt gccagcggtt 1080

aggccgggaa ctcaaaggag actgccagtg ataaactgga ggaaggtggg gatgacgtca 1140

agtcatcatg gcccttacga gtagggctac acacgtgcta caatggcgca tacaaagaga 1200

agcgacctcg cgagagcaag cggacctcat aaagtgcgtc gtagtccgga ttggagtctg 1260

caactcgact ccatgaagtc ggaatcgcta gtaatcgtgg atcagaatgc cacggtgaat 1320

acgttcccgg gccttgtaca caccgcccgt cacaccatgg gagtgggttg caaaagaagt 1380

aggtagctta accttcggga gggcgct 1407

Claims

1. An Enterobacter (Enterobacter hormecei) with the preservation number of CGMCC No. 24655.

2. An agent for bacteria comprising the Enterobacter (Enterobacter hormaechei) according to claim 1.

3. Use of an enterobacterium according to claim 1 or a bacterial agent according to claim 2 in selenium metabolism.

4. Use according to claim 3, wherein the selenium metabolism is the conversion of selenate and/or selenite to elemental selenium.

5. The use of the enterobacter of claim 1 or the microbial inoculum of claim 2 in the cultivation of selenium-enriched plants or selenium-enriched plants.

6. Use of the enterobacterium of claim 1 or the microbial agent of claim 2 for modulating the diversity of endogenous microorganisms in a plant.

7. Use according to claim 5 or 6, wherein the plant is a crucifer, preferably a cardamine species.

8. Use according to claim 6, wherein the endogenous microorganism is an endogenous microorganism in a plant leaf; and/or

Said enterobacteria are capable of increasing the occupancy of Alcaligenes.

9. A method for cultivating selenium-rich plants or selenium-rich plants is characterized by comprising the following steps: contacting the enterobacter of claim 1 or the microbial inoculum of claim 2 with a plant to be selenium enriched or a plant to be selenium enriched cultivated.

10. A selenium-enriched microbial fertilizer, characterized in that it contains enterobacter of claim 1 or the microbial inoculum of claim 2, and optionally inorganic and/or organic selenium fertilizer.