Application of selenium-containing composite initiator as inhibitor for generating deoxynivalenol in corn

Abstract

The invention relates to an application of a selenium-containing composite initiator as an inhibitor for generating deoxynivalenol in corn, which comprises the following steps: coating corn seeds with the selenium-containing composite initiator to form the corn seeds containing the selenium-containing composite initiator; the selenium-containing composite initiator enhances the resistance of corn to fusarium graminearum and inhibits the synthesis of deoxynivalenol by reprogramming seed metabolism and activating jasmonic acid (JA)-mediated defensive reaction in the seedling stage, and converts phenylpropyl metabolism of seedlings from flavonoid biosynthesis to antifungal phenol biosynthesis. Therefore, seed vigor is synchronized with innate immunity, and an effective strategy is provided for corn crop protection.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, in particular to use of a selenium-containing composite initiator as a deoxynivalenol production inhibitor in corn. Background Art

[0002] Fusarium graminearum (F. graminearum) causes stalk and ear rot in corn and produces deoxynivalenol (DON), a contaminant threat to corn. Under favorable temperature and humidity conditions, these fungi can multiply and produce toxins, seriously contaminating corn crops and processed products. Furthermore, this toxin can also be produced during corn storage if it is not adequately dried or stored improperly.

[0003] At present, some studies have begun to focus on how to inhibit the synthesis of deoxynivalenol, thereby inhibiting the growth and toxin production of Fusarium graminearum. For example, CN118923671A discloses the use of fungal inhibitors composed of resveratrol, which can significantly inhibit the growth of Fusarium graminearum hyphae, destroy the cell membrane of Fusarium graminearum, inhibit the expression of toxin-producing genes of Fusarium graminearum and inhibit the production of deoxynivalenol by Fusarium graminearum; CN119498308A discloses the use of plant hormone methyl jasmonate as an inhibitor of Fusarium graminearum growth and DON synthesis, providing a plant-derived inhibitor for the formulation of a safe, pollution-free and green strategy for the prevention and control of wheat fusarium fusarium; CN117898307A discloses a biocontrol bacterium Burkholderia pyrrolate, which is used to inhibit the production of deoxynivalenol toxin by wheat fusarium fusarium.

[0004] Although some progress has been made, the relevant technologies focus more on the growth period after the crops have seedled, resulting in limited absorption capacity of the crops for the corresponding inhibitors, and the inhibitory effect still needs to be further improved. Summary of the Invention

[0005] In response to the deficiencies in the prior art, the present invention provides a use of a selenium-containing composite initiator as an inhibitor of deoxynivalenol (DON) production in corn. The present invention forms corn seeds containing the selenium composite initiator by coating the selenium composite initiator with corn seeds. The selenium composite initiator can enhance the vitality of corn crops at the seed stage and activate the innate immune capacity, thereby better inhibiting the production of deoxynivalenol in corn, further improving the overall resistance of corn crops, being more conducive to their growth and improving their quality.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a use of a selenium-containing composite initiator as an inhibitor of deoxynivalenol production in corn, wherein the selenium-containing composite initiator is coated on corn seeds to form corn seeds containing the selenium composite initiator, and the corn seeds containing the selenium composite initiator are planted to inhibit deoxynivalenol in corn.

[0008] The corn seeds containing the selenium composite initiator of the present invention are prepared by a method comprising the following steps:

[0009] (1) providing a first multi-inlet vortex mixer having four channels, introducing a selenium source as a first liquid flow into the first channel of the first multi-inlet vortex mixer, introducing a stabilizer as a second liquid flow into the second channel, introducing water as a third liquid flow into the third channel, and introducing a reducing agent as a fourth liquid flow into the fourth channel, wherein the first liquid flow, the second liquid flow, the third liquid flow, and the fourth liquid flow are instantaneously collided and mixed in the first multi-inlet vortex mixer to form a selenium-containing composite suspension;

[0010] (2) providing a second multi-inlet vortex mixer connected in series with the first multi-inlet vortex mixer in step (1), introducing the selenium-containing composite suspension obtained in step (1) into the first channel of the second multi-inlet vortex mixer as the first liquid flow, introducing the first ligand into the second channel as the second liquid flow, introducing the second ligand into the third channel as the third liquid flow, and introducing water into the fourth channel as the fourth liquid flow, the first liquid flow, the second liquid flow, the third liquid flow and the fourth liquid flow collide and mix instantaneously in the second multi-inlet vortex mixer to form a selenium-containing suspension for coating;

[0011] (3) The sterilized corn seeds are first immersed in the selenium-containing coating suspension obtained in step (2), and the immersed seeds are then immersed in a crosslinking agent solution, and after drying, corn seeds containing the selenium composite initiator are formed.

[0012] The "selenium-containing composite initiator" in the present invention refers to a selenium-containing composite initiator formed by compounding selenium elements with components such as stabilizers, and then undergoing an instantaneous collision mixing preparation process in a first multi-inlet vortex mixer and a second multi-inlet vortex mixer connected in series. After coating the seeds, the selenium-containing composite initiator can be used as a corn seed vitality initiator, so that the corn seeds can show excellent effects in inhibiting the production of deoxynivalenol in corn during the later planting process, thereby effectively inhibiting the production of corn toxins.

[0013] A series of plant physiology studies demonstrated that coating corn seeds with the selenium-containing composite initiator described in this invention reprogrammed seed metabolism and activated jasmonic acid (JA)-mediated defense responses during the seedling stage, enhancing corn resistance to Fusarium graminearum and inhibiting deoxynivalenol synthesis. Simultaneously, it shifted seedling phenylpropanoid metabolism from flavonoid biosynthesis to antifungal phenolic biosynthesis, thereby establishing a chemical barrier against fungal attack. This metabolic reprogramming significantly reduced the stalk rot severity index (DSI) and effectively inhibited deoxynivalenol biosynthesis.

[0014] Preferably, in step (1) of preparing the corn seeds containing the selenium composite initiator, the selenium source is any one of sodium selenite, sodium selenate or selenium dioxide, or a combination of at least two of them.

[0015] Preferably, the reducing agent is any one of cysteine, hydrazine hydrate, ascorbic acid, sodium sulfite, sodium thiosulfate or glutathione, or a combination of at least two thereof.

[0016] Preferably, the stabilizer is any one of polysaccharides, organic compounds, polymers or polypeptides, or a combination of at least two of them, preferably a combination of polysaccharides and polypeptides.

[0017] In the present invention, the polysaccharide compound may be, for example, at least one of chitosan, chitosan oligosaccharides, carboxymethyl chitosan, sodium alginate, codonopsis polysaccharide, wolfberry polysaccharide, dandelion polysaccharide, tea polysaccharide, konjac polysaccharide or lentinan; the organic compound may be, for example, at least one of melatonin, sodium lauryl sulfate, cetyltrimethylammonium bromide, resveratrol, Tween (20, 40, 60, 80), proanthocyanidin, chlorogenic acid or epigallocatechin gallate; the polymer may be, for example, at least one of polyethylene glycol, polyvinyl alcohol or polyvinyl pyrrolidone; the polypeptide may be, for example, at least one of soybean peptide, peanut peptide, corn peptide, pea peptide or rice peptide.

[0018] The stabilizer in the present invention is preferably a combination of polysaccharides and polypeptides. By combining the polysaccharides and polypeptides, the selenium-containing composite suspension obtained in step (1) can be made more stable, thereby maximizing the activity of the selenium composite initiator when coating corn seeds, thereby more effectively achieving the inhibitory effect on deoxynivalenol in corn.

[0019] When the stabilizer of the present invention is a combination of polysaccharide and polypeptide, the mass ratio between the polysaccharide and the polypeptide can be, for example, (1-5): (1-5), wherein the first and second "1-5" can be selected from 1, 2, 3, 4, 5.

[0020] Preferably, the molar ratio of the selenium element in the selenium source to the reducing agent is 1:(2-6), for example, 1:2, 1:3, 1:4, 1:5 or 1:6.

[0021] Preferably, the mass ratio of selenium in the selenium source to the stabilizer is 1:(5-50), for example 1:5, 1:8, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:42, 1:45 or 1:50.

[0022] Preferably, in step (1) of preparing the corn seeds containing the selenium composite initiator, the flow rates of the first liquid flow and the second liquid flow are each independently 4 to 8 mL / min, for example, 4 mL / min, 5 mL / min, 6 mL / min, 7 mL / min or 8 mL / min, and the flow rates of the third liquid flow and the fourth liquid flow are each independently 25 to 50 mL / min, for example, 25 mL / min, 30 mL / min, 35 mL / min, 40 mL / min or 50 mL / min.

[0023] Preferably, in the selenium-containing composite suspension obtained in step (1), the average particle size of selenium is 20 to 80 nm, for example, 20 nm, 40 nm, 60 nm or 80 nm.

[0024] Preferably, in step (2) of preparing the corn seeds containing the selenium composite initiator, the first ligand is any one of carboxymethyl chitosan, sodium alginate, methyl cellulose or cellulose nanocrystals, or a combination of at least two thereof.

[0025] Preferably, the second ligand is any one of pectin, xanthan gum, gum arabic or gelatin, or a combination of at least two of them.

[0026] In the present invention, the mass ratio of the selenium-containing composite suspension obtained in step (1) to the first ligand and the second ligand is not particularly limited, and is based on the ability to achieve good coating of corn seeds. For example, the mass ratio of selenium element to the first ligand and the second ligand in the selenium-containing composite suspension can be 1:(15-30):(15-30), wherein the first and second "15-30" can be selected from 15, 18, 20, 25 or 30.

[0027] Preferably, in step (2) of preparing the corn seeds containing the selenium composite initiator, the flow rates of the first liquid flow and the second liquid flow are each independently 5 to 10 mL / min, for example, 5 mL / min, 6 mL / min, 8 mL / min, 9 mL / min or 10 mL / min, and the flow rates of the third liquid flow and the fourth liquid flow are each independently 30 to 60 mL / min, for example, 30 mL / min, 40 mL / min, 45 mL / min, 50 mL / min or 60 mL / min.

[0028] Preferably, in step (2) of preparing the corn seeds containing the selenium composite initiator, the selenium concentration in the selenium-containing coating suspension formed is 40-60 mg / L, for example, 40 mg / L, 50 mg / L or 60 mg / L.

[0029] In the present invention, by regulating the selenium concentration in the selenium-containing coating suspension within an appropriate range, the production of deoxynivalenol in corn can be better inhibited, and the growth of corn crops can be promoted to the greatest extent.

[0030] Preferably, in step (3) of preparing the corn seeds containing the selenium composite initiator, the immersion time is 2 to 12 hours, for example, 2 hours, 5 hours, 8 hours, 10 hours or 12 hours.

[0031] Preferably, the soaking time is 3 to 6 hours, such as 3 hours, 4 hours, 5 hours or 6 hours.

[0032] Preferably, the cross-linking agent is any one of zinc sulfate, copper sulfate, calcium chloride or manganese sulfate, or a combination of at least two thereof.

[0033] Preferably, the mass concentration of the cross-linking agent is 1-5%, for example, 1%, 2%, 3%, 4% or 5%.

[0034] Compared with the prior art, the present invention has at least the following beneficial effects:

[0035] In the present invention, corn seeds containing the selenium composite initiator are coated with a selenium composite initiator, wherein the selenium composite initiator can enhance the resistance of corn to Fusarium graminearum and inhibit the synthesis of deoxynivalenol by reprogramming seed metabolism and activating jasmonic acid (JA)-mediated defense responses in the seedling stage, while at the same time shifting the phenylpropanoid metabolism of the seedlings from flavonoid biosynthesis to antifungal phenolic biosynthesis, thereby synchronizing seed vigor with innate immunity, providing an effective strategy for corn crop protection, and achieving effective inhibition of corn toxins. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1The figure is a schematic diagram of the process of preparing corn seeds containing selenium composite initiator in the present invention.

[0037] Figure 2 The apparent phenotypes of the corn seedlings corresponding to the control group (CK), reference group (FgCK), Example 1, Examples 3-4, Examples 6-8 and Comparative Example 1 are shown.

[0038] Figure 3 The disease severity index (DSI) of the corn seedlings corresponding to the reference group (FgCK), Example 1, Examples 3-4, Examples 6-8 and Comparative Example 1 is shown.

[0039] Figure 4 The jasmonic acid (JA) content in the corn seedlings corresponding to the reference group (FgCK), Example 1, Examples 3-4, Examples 6-8 and Comparative Example 1 is shown.

[0040] Figure 5 The total flavonoids content in the corn seedlings corresponding to the reference group (FgCK), Example 1, Examples 3-4, Examples 6-8 and Comparative Example 1 is shown.

[0041] Figure 6 The total phenolic content in the corn seedlings corresponding to the reference group (FgCK), Example 1, Examples 3-4, Examples 6-8 and Comparative Example 1 is shown.

[0042] Figure 7 The correlation and interaction between jasmonic acid, total flavonoids and total phenols in the corn seedlings corresponding to Example 1 are shown. DETAILED DESCRIPTION

[0043] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. It should be understood by those skilled in the art that the specific embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0044] Preparation Example 1

[0045] This preparation example provides a corn seed containing a selenium composite initiator, and the preparation process is as follows: Figure 1 As shown, step (1) and step (2) are carried out in a first multi-inlet vortex mixer and a second multi-inlet vortex mixer connected in series, and the preparation process specifically includes the following steps:

[0046] (1) introducing a sodium selenite solution (selenium source) into the first channel of a first multi-inlet vortex mixer as a first liquid flow, introducing a stabilizer composed of chitosan oligosaccharide and soybean peptide in a mass ratio of 1:2 into the second channel as a second liquid flow, introducing water into the third channel as a third liquid flow, and introducing ascorbic acid (reducing agent) into the fourth channel as a fourth liquid flow;

[0047] The first liquid stream, the second liquid stream, the third liquid stream and the fourth liquid stream are instantaneously collided and mixed in a first multi-inlet vortex mixer, the mass ratio of selenium to stabilizer in the sodium selenite solution is 1:30, the molar ratio of selenium to ascorbic acid in the sodium selenite solution is 1:5, the flow rates of the first liquid stream and the second liquid stream are both 6 mL / min, and the flow rates of the third liquid stream and the fourth liquid stream are both 30 mL / min, to form a selenium-containing composite suspension, wherein the average particle size of the selenium is 35±5 nm;

[0048] (2) introducing the selenium-containing composite suspension obtained in step (1) into the first channel of the second multi-inlet vortex mixer, introducing the carboxymethyl chitosan solution (ligand 1) into the second channel, introducing the pectin solution (ligand 2) into the third channel, and introducing water into the fourth channel;

[0049] The first liquid stream, the second liquid stream, the third liquid stream and the fourth liquid stream are instantaneously collided and mixed in a second multi-inlet vortex mixer, the mass ratio of selenium to carboxymethyl chitosan and pectin in the selenium-containing composite suspension is 1:20:20, the flow rates of the first liquid stream and the second liquid stream are both 8 mL / min, and the flow rates of the third liquid stream and the fourth liquid stream are both 50 mL / min, to form a selenium-containing coating suspension with a selenium concentration of 50 mg / L;

[0050] (3) After the corn seeds are surface-sterilized with 2% H2O2, they are rinsed with distilled water to ensure that the residual disinfectant is completely removed. The seeds are first immersed in the selenium-containing coating suspension obtained in step (2) for 8 hours, then immersed in calcium chloride with a mass concentration of 2% for 5 hours, and vacuum-dried at 35°C for 48 hours to form corn seeds containing a selenium composite initiator.

[0051] Preparation Example 2

[0052] This preparation example provides a corn seed containing a selenium composite initiator, and the preparation process is as follows: Figure 1 As shown, step (1) and step (2) are carried out in a first multi-inlet vortex mixer and a second multi-inlet vortex mixer connected in series, and the preparation process specifically includes the following steps:

[0053] (1) A selenium dioxide solution (selenium source) was introduced into the first channel of the first multi-inlet vortex mixer as a first liquid flow, a stabilizer composed of lentinan and peanut peptide in a mass ratio of 1:1 was introduced into the second channel as a second liquid flow, water was introduced into the third channel as a third liquid flow, and a cysteine ​​solution (reducing agent) was introduced into the fourth channel as a fourth liquid flow;

[0054] The first liquid stream, the second liquid stream, the third liquid stream, and the fourth liquid stream are instantaneously collided and mixed in a first multi-inlet vortex mixer, the mass ratio of selenium to stabilizer in the selenium dioxide solution is 1:10, the molar ratio of selenium element to cysteine ​​in the selenium dioxide solution is 1:3, the flow rates of the first liquid stream and the second liquid stream are both 4 mL / min, and the flow rates of the third liquid stream and the fourth liquid stream are both 40 mL / min, to form a selenium-containing composite suspension, wherein the average particle size of the selenium is 42±5 nm;

[0055] (2) introducing the selenium-containing composite suspension obtained in step (1) into the first channel of a second multi-inlet vortex mixer, introducing a sodium alginate solution (ligand 1) into the second channel, introducing a gum arabic solution (ligand 2) into the third channel, and introducing water into the fourth channel;

[0056] The first liquid stream, the second liquid stream, the third liquid stream, and the fourth liquid stream are instantaneously collided and mixed in a second multi-inlet vortex mixer, the mass ratio of selenium to sodium alginate and gum arabic in the selenium-containing composite suspension is 1:25:15, the flow rates of the first liquid stream and the second liquid stream are both 6 mL / min, and the flow rates of the third liquid stream and the fourth liquid stream are both 45 mL / min, forming a selenium-containing coating suspension with a selenium concentration of 45 mg / L;

[0057] (3) After the corn seeds are surface-sterilized with 2% H2O2, they are rinsed with distilled water to ensure that the residual disinfectant is completely removed. The seeds are first immersed in the selenium-containing coating suspension obtained in step (2) for 10 hours, then immersed in zinc sulfate with a mass concentration of 3% for 6 hours, and vacuum-dried at 40°C for 42 hours to form corn seeds containing a selenium composite initiator.

[0058] Preparation Example 3

[0059] Compared with Preparation Example 1, the stabilizer combination in step (1) was adjusted to a single stabilizer, chitosan oligosaccharide, and the mass ratio of selenium to chitosan oligosaccharide in the sodium selenite solution was 1:30. Other conditions were exactly the same as Preparation Example 1.

[0060] Preparation Example 4

[0061] Compared with Preparation Example 1, the stabilizer combination in step (1) was adjusted to a single stabilizer, soybean peptide, and the mass ratio of selenium to soybean peptide in the sodium selenite solution was 1:30. The other steps were exactly the same as Preparation Example 1.

[0062] Preparation Example 5 to Preparation Example 8

[0063] Compared with Preparation Example 1, the amount of water introduced into the fourth channel in step (2) was changed so that step (2) formed a selenium-containing coating suspension with a selenium concentration of 5 mg / L (Preparation Example 5), 25 mg / L (Preparation Example 6), 75 mg / L (Preparation Example 7) and 100 mg / L (Preparation Example 8), respectively. The rest was exactly the same as Preparation Example 1.

[0064] Preparation Example 9

[0065] Compared with Preparation Example 1, the stabilizer in step (1) was replaced by Tween-20 and soybean peptide in a mass ratio of 1:2, and the mass ratio of selenium in the sodium selenite solution to the stabilizer was maintained at 1:30. The other steps were exactly the same as Preparation Example 1.

[0066] Preparation Example 10

[0067] Compared with Preparation Example 1, the stabilizer in step (1) was replaced by a combination of chitosan oligosaccharide and polyethylene glycol in a mass ratio of 1:2, and the mass ratio of selenium in the sodium selenite solution to the stabilizer was maintained at 1:30. Other steps were exactly the same as Preparation Example 1.

[0068] Comparative Preparation Example 1

[0069] This comparative preparation example provides corn seeds impregnated with inorganic selenium, and the preparation method thereof comprises the following steps:

[0070] (1) After surface disinfection of corn seeds with 2% H2O2, rinse with distilled water to ensure complete removal of residual disinfectant;

[0071] (2) The corn seeds sterilized in step (1) are immersed in a sodium selenite solution having a selenium concentration of 50 mg / L, taken out after 15 hours, and vacuum-dried at 35° C. for 48 hours to obtain the inorganic selenium-impregnated corn seeds.

[0072] Comparative Preparation Example 2

[0073] This comparative preparation example provides a method for preparing corn seeds coated with selenium complexes by a stirring method, wherein the preparation method comprises the following steps:

[0074] (1) A sodium selenite solution and a stabilizer composed of chitosan oligosaccharide and soybean peptide in a mass ratio of 1:2 were added to a first beaker, and after stirring for 30 minutes, an aqueous solution of ascorbic acid was added dropwise to the beaker; the mass ratio of selenium in the sodium selenite solution to the stabilizer was 1:30; the molar ratio of selenium in the sodium selenite solution to ascorbic acid was 1:5, and the ascorbic acid was added at a rate of 30 mL / min, to finally form a selenium-containing suspension;

[0075] (2) Add carboxymethyl chitosan solution and pectin to a second beaker, stir for 30 minutes, then add the selenium-containing suspension of step (1), and continue stirring for 300 minutes to form a selenium-containing coating suspension with a selenium concentration of 50 mg / L, wherein the mass ratio of selenium to carboxymethyl chitosan and pectin is 1:20:20;

[0076] (3) After the corn seeds are surface-sterilized with 2% H2O2, they are rinsed with distilled water to ensure that the residual disinfectant is completely removed. The seeds are first immersed in the selenium-containing coating suspension obtained in step (2) for 8 hours, then immersed in calcium chloride with a mass concentration of 2% for 5 hours, and vacuum-dried at 35°C for 48 hours to form corn seeds coated with selenium complexes prepared by a stirring method.

[0077] Test Case

[0078] For the corn seeds provided in the above-mentioned Preparation Examples 1 to 10 and Comparative Preparation Examples 1 to 2, after rinsing with distilled water to remove residual selenium adhering to the surface, corn seeds that have not been treated with any selenium were used as a control and were uniformly planted in a controlled environment greenhouse facility. The specific growth parameters were: lighting conditions of 14h light / 10h dark; humidity was controlled at 75%, and growth temperature was 24°C.

[0079] 1. Preparation of Fusarium graminearum Spore Suspension

[0080] The Fusarium graminearum strain was inoculated onto a PDA plate and cultured in a dark incubator (25°C) for at least 10 days. Several 5×5 mm agar blocks were aseptically cut and placed into sterile CMC medium. The culture was incubated in an incubator at 200 rpm and 28°C for 3 days. The spore suspension in the CMC medium was filtered through two layers of gauze and then centrifuged at 3800×g for 5 minutes. The supernatant was discarded, and the concentrated conidia were resuspended and washed twice with sterile water before being diluted to 1.0×10 6 / ml, add 0.001% Tween-20 to the diluent and set aside.

[0081] 2. Fusarium graminearum fungal inoculation and disease severity index (DSI) calculation

[0082] 2.1 After culturing corn seeds that had not been treated with any selenium, corn seeds of Preparation Examples 1 to 10, and Comparative Preparation Examples 1 to 2 for 12 days, a 1 mm deep wound was made at the base of the stem of the corresponding corn seedling using a 1 mL syringe under lighting conditions of 24° C. and 14 h light / 10 h dark. 20 μL of water was added to the wound, and 20 μL of spore suspension was applied to the wound, thereby forming: a reference group (FgCK, i.e., inoculated with Fusarium graminearum fungi but not treated with any selenium), Examples 1 to 10, and Comparative Examples 1 to 2, and the relative humidity was maintained at 75%. Three days after inoculation, the phenotype of each corn seedling was first observed. At the same time, corn seedlings that had not been treated with any selenium and not inoculated with Fusarium graminearum fungi were cultured in the same manner as a control group (CK).

[0083] Figure 2 The apparent phenotypes of the corn seedlings corresponding to the control group (CK), reference group (FgCK), Example 1, Examples 3-4, Examples 6-8 and Comparative Example 1 are shown.

[0084] pass Figure 2 It can be seen that the control group performed normally because it was not inoculated with the Fusarium graminearum fungus; while the reference group, which was inoculated with the Fusarium graminearum fungus but not treated with any selenium, showed yellow-brown lesions on its leaves, most leaves were necrotic, and the stems were seriously damaged. The leaves of Example 1 showed serious stress effects and reduced chlorophyll content, while the leaves of Example 1 had almost no lesions, the stems were harder and thicker, and the degree of rot was lower, with the best overall performance. Examples 3 to 4 and Examples 6 to 8 all showed a certain degree of stem rot, softening and leaf fading, which were slightly worse than Example 1.

[0085] 2.2 The disease severity index (DSI) of each maize seedling was evaluated and statistically analyzed. The calculation of the disease severity index (DSI) refers to the article Sun, Y., Ruan, X., Ma, L., Wang, F., Gao, X. Rapid Screening and Evaluation of Maize Seedling Resistance to Stalk Rot Caused by Fusarium spp. Bio Protoc (2018), 8(10), e2859. DOI: 10.21769 / BioProtoc.2859.

[0086] Figure 3 The disease severity index (DSI) of the corn seedlings corresponding to the reference group (FgCK), Example 1, Examples 3-4, Examples 6-8 and Comparative Example 1 is shown.

[0087] pass Figure 3 It can be seen that compared with the reference group and comparative example 1, the disease severity index of Examples 1, Examples 3 to 4, and Examples 6 to 8 of the present invention has decreased. This shows that, compared with no selenium treatment or selenium treatment but inorganic selenium impregnation treatment of comparative example 1, the method of coating corn seeds with the selenium composite initiator used in the present invention can more effectively reduce the disease severity index, which means that the present invention better achieves the inhibitory effect on deoxynivalenol.

[0088] pass Figure 3 It can also be seen that Example 1 has the lowest disease severity index, and Examples 3 and 4 are inferior to Example 1. This shows that when the stabilizer is adjusted to a single type, it will reduce the inhibitory effect on deoxynivalenol to a certain extent. It also proves that when a combination of polysaccharides and polypeptides is used as a stabilizer, it can better play the role of a deoxynivalenol production inhibitor.

[0089] The disease severity index of Examples 6 to 8 is slightly higher than that of Examples 1 and Examples 3 to 4, which shows that the selection of selenium concentration in the present invention has an important influence on the disease severity index of corn seedlings. When the selenium concentration of Example 6 is 25 mg / L or the selenium concentration of Example 7 is 75 mg / L, the inhibitory effect on Fusarium graminearum fungi is not as good as that of Example 1 when the selenium concentration is 50 mg / L. When the selenium concentration is further increased, that is, a concentration of 100 mg / L is used in Example 8, the disease severity index is lower than that of Examples 6 and 7, but still higher than that of Examples 1 and Examples 3 to 4. This fully illustrates that the present invention can obtain a more excellent deoxynivalenol inhibitory effect by regulating the selenium concentration within an appropriate range (40 to 60 mg / L).

[0090] 3. Extraction and Analysis of Deoxynivalenol

[0091] Accurately weigh 5 g of straw sample into a 50 mL centrifuge tube. Deoxynivalenol was extracted with 20 mL of acetonitrile / water / acetic acid solution (70:29:1, v / v / v). The sample was vortexed for 30 minutes and then centrifuged. 0.5 mL of the supernatant was mixed with 0.5 mL of water and centrifuged at 12,000 rpm / min for 10 minutes at 4°C. The supernatant was filtered through a 0.2 μm polytetrafluoroethylene membrane, and the filtrate was collected in a sample injection vial. Deoxynivalenol content was analyzed using a Shimadzu 8045 mass spectrometer.

[0092] The deoxynivalenol (DON) levels of corn seedlings treated with the control group (CK), reference group (FgCK), Examples 1 to 10, and Comparative Examples 1 to 2 were analyzed and determined. The results are shown in Table 1.

[0093] Table 1

[0094] DON (μg / g) CK / FWf 0.032 Example 1 0.015 Example 2 0.016 Example 3 0.018 Example 4 0.019 Example 5 0.022 Example 6 0.022 Example 7 0.023 Example 8 0.021 Example 9 0.023 Example 10 0.022 Comparative Example 1 0.028 Comparative Example 2 0.026

[0095] As can be seen from Table 1, compared to FgCK and Comparative Examples 1-2, the levels of deoxynivalenol in Examples 1-10 provided by the present invention were significantly reduced, indicating that the selenium composite initiator provided by the present invention can significantly alleviate the impact of Fusarium graminearum infection on corn crop growth and effectively reduce the level of deoxynivalenol. This also shows that when Comparative Example 1 uses sodium selenite as a selenium source to directly impregnate corn seeds, its effect in reducing the content of deoxynivalenol is significantly inferior to that of the present invention; when Comparative Example 2 uses only stirring preparation without using the instantaneous nanoprecipitation method, its inhibitory effect on deoxynivalenol is also inferior to that of the present invention.

[0096] The deoxynivalenol content in Examples 1 and 2 is the lowest, indicating that they have the best inhibitory effect on deoxynivalenol, while the deoxynivalenol content in Examples 3-4 is slightly higher than that in Example 1, which shows that when a single type of stabilizer is used, the inhibition of deoxynivalenol will be slightly reduced compared to the combination of two stabilizers; Examples 9-10 respectively use a combination of polypeptides or polysaccharides with other types of stabilizers. By comparing with Example 1, it can be seen that even though Examples 9-10 also use a combination of stabilizers, when the polysaccharide or polypeptide in Example 1 is replaced by other types of stabilizers, the inhibitory effect of deoxynivalenol is still inferior to that of Example 1, which further confirms that the stabilizer after the combination of polysaccharides and polypeptides has a better effect in reducing the level of deoxynivalenol.

[0097] Comparing Example 1 with Examples 5-8, it can be seen that when the selenium concentration is lower than the concentration of 40-60 mg / L used in the present application (Examples 5-6), or when the concentration is higher than the concentration used in the present application (Examples 7-8), the inhibitory effect on deoxynivalenol decreases, falling short of that of Example 1. In particular, for Example 8, when the selenium concentration is twice that of the present invention, the increased selenium concentration actually results in a lower inhibitory effect on deoxynivalenol than that of Example 1. This fully demonstrates that, in the present invention, by regulating the selenium concentration within the appropriate range (40-60 mg / L), a more excellent inhibitory effect on deoxynivalenol can be achieved.

[0098] 4. Analysis of phytohormone JA, total flavonoids, and total phenolic compounds in corn plants

[0099] For metabolite extraction, 100 mg of sample was homogenized in 1 mL of extraction buffer containing 80% methanol and 0.1% formic acid in ultrapure water. The homogenate was sonicated for 30 minutes to enhance extraction efficiency and then centrifuged at 12,000 rpm for 5 minutes. To ensure maximum metabolite recovery, the extraction process was repeated twice, and all supernatants were combined. The combined supernatants were dried with nitrogen and then diluted to 100 μL with 60% ethanol. Prior to analysis, the sample was cleaned up with 100 mg of C18 material and filtered through a 0.22 μm nylon syringe filter to remove particulate matter.

[0100] JA analysis was performed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) on an Agilent G6465B triple quadrupole system equipped with a reversed-phase C18 column (EclipsePlus C18, 2.1 × 50 mm, 1.8 μm). Total flavonoids and total phenols were assayed using the LHT-2-G Plant Flavonoid Test Kit and the TP-2-G Plant Total Phenol Test Kit, respectively, from Suzhou Keming Company.

[0101] Figures 4-6 The jasmonic acid JA ( Figure 4 )、Total flavonoids( Figure 5 ) and total phenols ( Figure 6 ) content.

[0102] pass Figures 4-6 It can be seen that compared with the reference group and comparative example 1, the jasmonic acid JA and total phenol content in the corn seedlings of Example 1, Examples 3-4 and Examples 6-8 of the present invention are higher, and the total flavonoid content is reduced, which reflects that the antibacterial substances in the corn seedlings of Example 1, Examples 3-4 and Examples 6-8 are accumulated, and this accumulation is mainly caused by the redirection of downstream phenylpropanoid metabolites after the activation of the JA signaling pathway.

[0103] In addition, the disease severity index (DSI) and correlation analysis of DON with jasmonic acid JA, total flavonoids and total phenolic content were performed on the corn seedlings treated in Example 1. Figure 7 shown.

[0104] Figure 7The correlation and interaction between JA, total flavonoids, and total phenols are shown. The correlation coefficient matrix shows that under Fusarium graminearum infection, JA and total phenols showed an extremely significant positive correlation (0.79***), while total flavonoids and total phenols also showed a significant negative correlation (0.72***). The network diagram on the left further reveals the complexity of the relationship between DON, DSI, and the three: the orange line (total flavonoids) and the green / white line (total phenols) form a mutually exclusive connection, which fully verifies that the seeds coated with selenium complex initiators activate JA signals and antagonize the balance between flavonoid and phenolic metabolism, thereby promoting the synthesis of antibacterial phenolic substances while inhibiting flavonoid accumulation, thereby improving resistance to Fusarium graminearum and inhibiting toxin accumulation.

[0105] according to Figure 7 The content shown, combined with the present invention provides Figures 4-6 , which also fully illustrates that each embodiment provided by the present invention can effectively achieve the improvement of the resistance of Fusarium graminearum and the inhibition of toxin accumulation.

[0106] In summary, the present invention uses a selenium composite initiator to coat corn seeds to form corn seeds containing a selenium composite initiator, wherein the selenium composite initiator can enhance the resistance of corn to Fusarium graminearum and inhibit the synthesis of deoxynivalenol by reprogramming seed metabolism and activating jasmonic acid (JA)-mediated defense responses in the seedling stage, while at the same time shifting the phenylpropanoid metabolism of the seedlings from flavonoid biosynthesis to antifungal phenolic biosynthesis, thereby synchronizing seed viability with innate immunity, providing an effective strategy for corn crop protection and effectively inhibiting the production of corn toxins.

Claims

1. A use of a selenium-containing composite initiator as an inhibitor of deoxynivalenol production in corn, characterized in that: coating corn seeds with a selenium-containing composite initiator to form corn seeds containing the selenium composite initiator; The corn seeds containing the selenium composite initiator are prepared by a method comprising the following steps: (1) providing a first multi-inlet vortex mixer having four channels, introducing a selenium source as a first liquid flow into the first channel of the first multi-inlet vortex mixer, introducing a stabilizer as a second liquid flow into the second channel, introducing water as a third liquid flow into the third channel, and introducing a reducing agent as a fourth liquid flow into the fourth channel, wherein the first liquid flow, the second liquid flow, the third liquid flow, and the fourth liquid flow are instantaneously collided and mixed in the first multi-inlet vortex mixer to form a selenium-containing composite suspension; (2) providing a second multi-inlet vortex mixer connected in series with the first multi-inlet vortex mixer in step (1), introducing the selenium-containing composite suspension obtained in step (1) into the first channel of the second multi-inlet vortex mixer as the first liquid flow, introducing the first ligand into the second channel as the second liquid flow, introducing the second ligand into the third channel as the third liquid flow, and introducing water into the fourth channel as the fourth liquid flow, the first liquid flow, the second liquid flow, the third liquid flow and the fourth liquid flow collide and mix instantaneously in the second multi-inlet vortex mixer to form a selenium-containing suspension for coating; (3) The sterilized corn seeds are first immersed in the selenium-containing coating suspension obtained in step (2), and the immersed seeds are then immersed in a crosslinking agent solution, and after drying, corn seeds containing the selenium composite initiator are formed.

2. The use according to claim 1, characterized in that In step (1) of preparing the corn seeds containing the selenium composite initiator, the selenium source is any one of sodium selenite, sodium selenate or selenium dioxide, or a combination of at least two thereof; Preferably, the reducing agent is any one or a combination of at least two of cysteine, hydrazine hydrate, ascorbic acid, sodium sulfite, sodium thiosulfate or glutathione; Preferably, the stabilizer is any one of polysaccharides, organic compounds, polymers or polypeptides, or a combination of at least two of them. More preferably, the stabilizer is a combination of polysaccharides and polypeptides.

3. The use according to claim 1 or 2, characterized in that The molar ratio of selenium element in the selenium source to the reducing agent is 1:(2-6); Preferably, the mass ratio of selenium in the selenium source to the mass ratio of the stabilizer is 1:(5-50).

4. The use according to any one of claims 1 to 3, characterized in that In the step (1) of preparing the corn seeds containing the selenium composite initiator, the flow rates of the first liquid flow and the second liquid flow are each independently 4 to 8 mL / min, and the flow rates of the third liquid flow and the fourth liquid flow are each independently 25 to 50 mL / min; Preferably, in the selenium-containing composite suspension obtained in step (1), the average particle size of selenium is 20 to 80 nm.

5. The use according to any one of claims 1 to 4, characterized in that In step (2) of preparing the corn seeds containing the selenium composite initiator, the first ligand is any one of carboxymethyl chitosan, sodium alginate, methyl cellulose or cellulose nanocrystals, or a combination of at least two thereof; Preferably, the second ligand is any one of pectin, xanthan gum, gum arabic or gelatin, or a combination of at least two of them.

6. The use according to any one of claims 1 to 5, characterized in that In step (2) of preparing the corn seeds containing the selenium composite initiator, the flow rates of the first liquid flow and the second liquid flow are independently 5-10 mL / min, and the flow rates of the third liquid flow and the fourth liquid flow are independently 30-60 mL / min.

7. The use according to any one of claims 1 to 6, characterized in that In the step (2) of preparing the corn seeds containing the selenium composite initiator, the selenium concentration in the formed selenium coating suspension is 40-60 mg / L.

8. The use according to any one of claims 1 to 7, characterized in that In step (3) of preparing the corn seeds containing the selenium composite initiator, the soaking time is 2 to 12 hours; Preferably, the soaking time is 3 to 6 hours.

9. The use according to any one of claims 1 to 8, characterized in that In step (3) of preparing the corn seeds containing the selenium composite initiator, the cross-linking agent is any one of zinc sulfate, copper sulfate, calcium chloride or manganese sulfate, or a combination of at least two thereof; Preferably, the mass concentration of the cross-linking agent is 1-5%.

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