Method for inducing and identifying type of modified mycotoxin in corn

Abstract

The invention relates to the technical field of fungaltoxin induction methods, in particular to a method for inducing and identifying the type of modified fungaltoxin in corn. According to the induction identification method disclosed by the invention, the modified fungaltoxin can be accurately induced and generated in the corn for subsequent species identification. The induction method disclosed by the invention is simple and rapid to operate, flexible in inoculation part, capable of accurately identifying the toxin type, high in inoculation efficiency and capable of overcoming the defects of complex mycotoxin types and mutual interference of a natural induction method.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of mycotoxin induction methods, and in particular to a method for inducing and identifying modified mycotoxin types in corn. BACKGROUND

[0002] Corn is extremely susceptible to fungal infection and contamination with mycotoxins during growth, which is the most important factor affecting the quality and safety of corn and seriously affects consumer safety. Mycotoxins, like other heterologous substances, can be metabolized by living plants to form modified mycotoxins. Modified mycotoxins can be interconverted with free forms during human and animal digestion, increasing the uncertainty of toxin contamination levels and causing huge economic losses and health risks to animals and humans. Therefore, how to accurately identify the types of modified mycotoxins in corn and monitor and scientifically evaluate the contamination levels of mycotoxins in corn during production has become a primary problem to protect human and animal health.

[0003] Currently, in the research on the identification of the types of modified mycotoxins naturally formed during crop growth, the ways to obtain modified mycotoxins are mainly divided into natural production, inoculation of toxin-producing strains and inoculation of toxins. (1) Natural production: for example, Streit, E. et al. identified the types of modified mycotoxins naturally produced in Australian feed and grains. Palacios, S. A. et al. analyzed the contamination levels of modified vomitoxin naturally occurring in Argentine wheat. Disadvantages: under natural conditions, the types of fungi are complex and affect each other during the formation of modified toxins, resulting in inaccurate identification of types. (2) Inoculation of toxin-producing strains: Kurt E. Richardson et al. inoculated Fusarium graminearum on rice to identify the types of mycotoxins. Disadvantages: the growth of the strain is affected by climate conditions, and if the temperature and humidity during growth are not suitable, the test may fail and no toxins may be produced. (3) Mycotoxin induction: Nathanail prepared a solution of labeled T2 and injected 10 μL of the mycotoxin solution into each spike at the flowering stage of wheat. After each treatment, the spikes were covered with small transparent plastic bags for 24 hours, and 2 mL of double-distilled water was sprayed using a handheld sprayer. High relative humidity was ensured to promote the diffusion of mycotoxins into plant cells. Modified T2 toxins were induced by repeated inoculation of mycotoxins and their metabolic types were identified. The mycotoxin induction technique has the advantages of clear toxin types and direct induction of modified toxins metabolized by plants. Disadvantages: the current application of this technology is mainly concentrated on wheat and barley. 13 Nathanail prepared a solution of labeled T2 and injected 10 μL of the mycotoxin solution into each spike at the flowering stage of wheat. After each treatment, the spikes were covered with small transparent plastic bags for 24 hours, and 2 mL of double-distilled water was sprayed using a handheld sprayer. High relative humidity was ensured to promote the diffusion of mycotoxins into plant cells. Modified T2 toxins were induced by repeated inoculation of mycotoxins and their metabolic types were identified. The mycotoxin induction technique has the advantages of clear toxin types and direct induction of modified toxins metabolized by plants. Disadvantages: the current application of this technology is mainly concentrated on wheat and barley.

[0004] Compared with the production process of the maize and the cereal crops, the period of contaminating the mycotoxin is quite different, and the external phenotypes of the maize and the cereal crops are quite different, for example, there are gaps between the grains of the cereal crops, and the gaps are exposed, and the surface of the ear of the maize is covered by the leaf. Therefore, the existing technology for identifying the type of the modified mycotoxin induced by the mycotoxin cannot be directly used for the maize, and a new mycotoxin inoculation technology needs to be established according to the period of the maize infected by the mycotoxin and the structure characteristics of the plant, so as to induce and identify the type of the modified mycotoxin in the maize and the proportion between different types. SUMMARY

[0005] In order to solve the above technical problems, the present application provides a method for inducing and identifying the type of the modified mycotoxin in the maize, comprising the following steps: 20-30 days after the maize is in bloom, the leaf covering the maize is cut along the longitudinal direction to form a cut with a width of 4-5 cm and a length of 8-10 cm, and the leaf covering the maize is not cut off; the mycotoxin is injected into the root of the gap between the grains of the maize in an area of 6-10 cm 2 , and the inoculation amount of the mycotoxin is 20 μg or more, and then the leaf covering the maize is covered on the ear of the maize again and fixed, and the inoculation is continuously performed for more than 5 days.

[0006] The cereal crops are usually inoculated with the mycotoxin at the flowering stage, but the maize is infected by the fungus and then produces the toxin at the late stage of the ear maturation, so the present application selects to inoculate the mycotoxin 20-30 days after the maize is in bloom (the grain filling period of the maize), which is consistent with the period of naturally producing the toxin.

[0007] In addition, there is a gap between each grain of the ear of the cereal crops, and the toxin is easy to be inoculated. However, the surface of the ear of the maize is covered by the leaf, and the condition of the grain cannot be directly observed. Therefore, the present application cuts a certain area of the leaf when inoculating the mycotoxin, but in order to avoid the loss of the light and the water of the grain of the maize, avoid the grain of the maize from maturing too fast under the light condition, and at the same time, avoid the infection of the fungus, and at the same time, obtain the modified mycotoxin with a higher concentration, the present application controls the size of the cut, the inoculation area, and covers the cut leaf on the ear of the maize after the inoculation, so as to ensure that the growth state of the inoculation part and the other parts is consistent, and does not affect the normal growth state of the maize plant, so that the modified mycotoxin is successfully induced and obtained.

[0008] The inoculation success rate can be improved by continuously inoculating for more than 5 days (once a day).

[0009] Preferably, the mycotoxin is zearalenone (ZEN).

[0010] Preferably, the inoculation amount of the mycotoxin is 40-160 μg.

[0011] Preferably, the concentration of the inoculated zearalenone solution is 100 mg / L or more.

[0012] More preferably, the concentration of the inoculated zearalenone solution is 200 mg / L or more, and more preferably 200 mg / L to 800 mg / L.

[0013] The zearalenone solution with the above concentration can induce the production of seven modified mycotoxins at the same time.

[0014] Preferably, the volume of the zearalenone solution inoculated per day is 200 μL.

[0015] Preferably, the induction method further comprises: collecting the corn kernels containing the modified mycotoxins 8 to 12 days after the inoculation is stopped.

[0016] The concentration of the modified mycotoxins is high in the above period.

[0017] Preferably, the modified mycotoxins include at least one of α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), zearalanone (ZAN), α-zearalenol-4-glucoside (α-ZEL-Glu), β-zearalenol-4-glucoside (β-ZEL-Glu), zearalenone-4-O-β-D-glucopyranoside (ZEN-Glu), and zearalenone 4-sulfate ammonium salt (ZEN-4-Sul).

[0018] Preferably, the modified mycotoxins include α-zearalenol, β-zearalenol, zearalanone, α-zearalenol-4-glucoside, β-zearalenol-4-glucoside, zearalenone-4-O-β-D-glucopyranoside, and zearalenone 4-sulfate ammonium salt.

[0019] Preferably, the mycotoxin is inoculated by using a flat-head syringe.

[0020] The flat-head syringe can avoid the sharp-head syringe being too sharp to prick the corn kernels.

[0021] Preferably, the fixing is sealed by using adhesive tape.

[0022] Preferably, the variety of the corn is Xianyu 335.

[0023] Further, the application provides the use of the induction method in the research of modified mycotoxins in corn.

[0024] Preferably, the induction method is used to research the types of modified mycotoxins in corn.

[0025] Compared with the prior art, the application has the following beneficial effects: The induction and identification method of the application can accurately induce the production of modified mycotoxins in corn for subsequent type identification. The induction method of the application is simple, fast, flexible in inoculation site, can accurately identify the type of toxin, has high inoculation efficiency, and makes up for the defects of the natural induction method that the types of mycotoxins are complex and interfere with each other. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a type and content detection result diagram of the modified mycotoxin obtained by the induction method of Example 1.

[0027] Figure 2 is a type and content detection result diagram of the modified mycotoxin obtained by different inoculation areas.

[0028] Figure 3 is a type and content detection result diagram of the modified mycotoxin obtained by inoculating different concentrations of mycotoxins. DETAILED DESCRIPTION

[0029] In order to make the objectives, technical solutions and advantages of the application clearer, the technical solutions in the application will be clearly and completely described below. Obviously, the described examples are part of the examples of the application, rather than all the examples. Based on the examples in the application, all other examples obtained by those skilled in the art without creative labor fall within the scope of protection of the application. In the examples provided in the specification, the specific techniques or conditions are not specified, and the techniques or conditions are described in the literature in the art, or according to the product manual. The reagents or instruments used are not specified by the manufacturer, and are conventional products that can be purchased from a regular channel. The corn variety used in the following examples is Xianyu 335.

[0030] Example 1 The present embodiment provides an induction method of modified mycotoxins in corn, and the steps are as follows: Select 24 corn plants with a growth state of 25 days after corn heading, and divide them into a treatment group and a control group, each with 12 plants. The treatment method of the treatment group is as follows: cut the corn husk along the longitudinal direction to form a cut with a width of 5 cm and a length of 10 cm, and keep the husk on the corn ear without cutting; take 200 μL of 200 mg / L zearalenone solution and inject it into the cut with a flat syringe, and the injection depth is 6 cm 2Roots in the gaps of corn kernels were injected within the area and marked; then the corn husks were re-covered on the corn ears and sealed and fixed with adhesive tape, and inoculated continuously for 5 days, once a day. Distilled water was used instead of zearalenone solution to inoculate in the same way as the control group. After the last day of inoculation, corn kernels of the treatment and control groups were collected at intervals of 5 days, 10 days, 15 days, and 20 days.

[0031] Further, the embodiment analyzes the types and contents of modified mycotoxins in corn kernels by liquid chromatography tandem mass spectrometry. The steps of liquid chromatography tandem mass spectrometry analysis are as follows: The sample was crushed by a hammer-type cyclone mill, and after passing through a 20-mesh sieve, it was mixed and used. 5 g (accurate to 0.01 g) of the crushed sample was weighed into a 50 mL centrifuge tube, 100 microliters of internal standard working solution (0.1 μg / mL [ 13 C 18 ] - ZEN) was added, and then 20 mL of 80% acetonitrile water (v:v=80:20) solution was added. After oscillation extraction for 40 minutes, 6 g of anhydrous magnesium sulfate and 1.5 g of anhydrous sodium acetate were added, and vortexed for 1 minute. Centrifugation was performed at 4000 rpm / min for 5 min, and the supernatant was removed for purification. 10 mL of the above-mentioned sample to be purified was removed into a 15 mL centrifuge tube, and 50.0 mg of C 18 was added per mL, oscillated for 1 min, centrifuged, and 4 mL of the supernatant was accurately removed into a glass test tube, dried under nitrogen at 40℃, 1.0 mL of 5% acetonitrile water solution was added, mixed, 0.22 micrometer filter membrane was used for filtration, and liquid chromatography tandem mass spectrometry determination was performed.

[0032] Liquid chromatography conditions: chromatographic column Kinetex C 18 column (100×2.1 mm, 2.6 microns); column temperature: 35℃; injection amount: 2 microliters; flow rate: 0.4 mL / min; mobile phase and gradient elution conditions are shown in Table 1. Mobile phase A: 0.1% (v / v) formic acid aqueous solution; mobile phase B: acetonitrile.

[0033] Table 1 Gradient elution conditions

[0034] Mass spectrometry reference conditions ionization mode: electrospray ionization (ESI); spray voltage: 4500V; atomization temperature: 550℃; curtain gas: 35L / h; atomization gas: 50.0 mL / min, auxiliary gas: 50.0 mL / min, and the gas flow was adjusted before use to make the mass spectrometry sensitivity meet the detection requirements; scanning mode: negative ion scanning; detection mode: multiple reaction monitoring, and the parameters are shown in Table 2.

[0035] Table 2 Multiple reaction monitoring parameters

[0036] Note: For quantitative ions.

[0037] The standard working solution of different mycotoxins was injected respectively, and the standard working curve was drawn with the peak area as the vertical coordinate and the mass concentration of the standard working solution as the horizontal coordinate. The sample was quantified by the standard working curve.

[0038] The detection results are shown in Table 1. Figure 1 As shown in Table 1, after stopping inoculation, 7 modified mycotoxins were produced at an interval of 5 days, which were α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), zearalanone (ZAN), α-zearalenol-4-glucoside (α-ZEL-4-glucoside, α-ZEL-Glu), β-zearalenol-4-glucoside (β-ZEL-4-glucoside, β-ZEL-Glu), zearalenone-4-O-β-D-glucopyranoside (ZEN-Glu), and zearalenone 4-sulfate ammonium salt (ZEN-4-Sul). The concentrations of each compound reached a maximum at an interval of 10 days, and then showed a downward trend as the interval further extended.

[0039] Example 2 This example is based on Example 1, and the influence of each parameter change on the induction result is investigated by single factor variable experiment.

[0040] 1. Screening of mycotoxin inoculation period Mycotoxins were inoculated on the corn ears according to the method of Example 1 at 15 days, 25 days and 40 days after the corn ears emerged, respectively. The test results showed that the corn at 15 days after the corn ears emerged did not form kernels after stopping inoculation for 5 days, and the sample could not be collected. The corn at 25 days after the corn ears emerged formed corn kernels that could be completely separated after stopping inoculation for 5 days. The corn at 40 days after the corn ears emerged completed kernel filling after stopping inoculation for 5 days, and the endosperm changed from paste to wax, the kernel hardness increased, and the transport efficiency of mycotoxins in the corn kernels decreased, which was not conducive to the induction of modified mycotoxins.

[0041] 2. Screening of corn leaf cutting size The corn husk was cut into a 3 cm wide and 6 cm long cut (cut 1) and a 5 cm wide and 10 cm long cut (cut 2), respectively, and inoculated with mycotoxins according to the method of Example 1. It was found that the corn husk gradually dehydrated and shriveled after inoculation with mycotoxins, and the cut 1 husk shriveled to expose the inoculation site, causing the corn kernels to be exposed to light, increasing the hardness of the kernels and premature maturation. Although the cut 2 husk also shriveled, it still covered the inoculation site after shrinking, ensuring normal growth of the site.

[0042] 3. Screening of inoculation area On the basis of the method of Example 1, only the inoculation area was changed to 3 cm 2 , 8 cm 2 and 15 cm 2 , and the results are shown in Figure 2 . It was found that it was difficult to inject 200 μL of zearalenone solution into the gap between the corn kernels in the range of 3 cm 2 , and the sample size was too small when sampling, which could not complete the subsequent species identification and concentration analysis. In the range of 8 cm 2 and 15 cm 2 , the sample size collected in the range of 15 cm 2 was greater than that in the range of 8 cm 2 . It was found during subsequent analysis that as the sample size increased, the analyte in the sample was diluted, and the content of modified mycotoxins in the sample decreased, so 6 cm 2 -10 cm 2 was selected as the inoculation range.

[0043] 4. Screening of mycotoxin inoculation concentration On the basis of the method of Example 1, only the mycotoxin inoculation concentration was adjusted to 50 mg / L, 100 mg / L and 200 mg / L, respectively. The samples were collected 5 days after inoculation, and the content of modified mycotoxins in the corn kernels was determined.

[0044] The results are shown in Figure 3 . It was found by analysis that when the concentration was 50 mg / L, no zearalenone and modified mycotoxins were detected in the sample due to the low inoculation concentration. Zearalenone and 4 kinds of modified mycotoxins were detected in the sample inoculated with 100 mg / L. When the inoculation concentration was 200 mg / L, zearalenone and 7 kinds of modified mycotoxins were present in the corn kernels collected 5 days after inoculation, so 100 mg / L was the lowest inoculation concentration, and more than 200 mg / L was better.

[0045] It should be pointed out finally that the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit the same; and although the present application has been described in detail with reference to the foregoing embodiments, it should be appreciated by those skilled in the art that the technical solutions recorded in the foregoing embodiments can be modified, or some technical features thereof can be replaced equivalently; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method of inducing identification of modified mycotoxin types in corn, characterized by, comprising: After 20~30 days of corn tasseling, the corn husk is cut along the longitudinal direction to form a 4~5 cm wide and 8~10 cm long incision, and the corn husk is not cut off on the corn ear; the mycotoxin is injected into the root gap between the corn kernels in an area of 6~10 cm 2 The inoculation amount of the mycotoxin is 20 μg or more, and then the corn husk is covered on the corn ear part and fixed, and the inoculation is continuously performed for 5 days or more.

2. The induction method of claim 1, wherein, the mycotoxin is zearalenone.

3. The induction method of claim 2, wherein, the inoculum of mycotoxin is 40-160 μg.

4. The induction method of claim 1, wherein, further comprising: the corn kernels containing the modified mycotoxin are harvested 8-12 days after inoculation is stopped.

5. The induction method of claim 1, wherein, the modified mycotoxin includes at least one of α-zearalanol, β-zearalanol, zearalanone, α-zearalanol-4-glucoside, β-zearalanol-4-glucoside, zearalenone-4-glucoside, and zearalenone-4-sulfate ammonium salt.

6. The induction method of claim 5, wherein, the modified mycotoxin includes α-zearalanol, β-zearalanol, zearalanone, α-zearalanol-4-glucoside, β-zearalanol-4-glucoside, zearalenone-4-glucoside, and zearalenone-4-sulfate ammonium salt.

7. The induction method of claim 1, wherein, the mycotoxin is inoculated using a flat-ended syringe.

8. The induction method of claim 1, wherein, the securing is performed using a tape seal.

9. Use of the induction method of any one of claims 1-8 to study modified mycotoxins in corn.

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