Plant essential oil compound liquid for inhibiting growth and toxin production of fusarium graminearum and application of plant essential oil compound liquid

Abstract

The invention discloses plant essential oil compound liquid for inhibiting growth and toxin production of fusarium graminearum and application of the plant essential oil compound liquid. The plant essential oil compound liquid provided by the invention is formed by compounding carvacrol and an aldehyde compound, wherein the aldehyde compound is one or two of cinnamyl aldehyde and citral; the concentration of the carvacrol is 4-40 [mu] g / mL, the concentration of the aldehyde compound is 8-85 [mu] g / mL, and the volume ratio of the carvacrol to the aldehyde compound is 1: (1-5). The plant essential oil compound liquid can inhibit growth and toxin production of fusarium graminearum, can inhibit generation of main pathogenic bacteria of wheat, reduces the use amount of chemical pesticides, improves the quality of cereals such as wheat and the like, and provides an important thought for prevention and control of wheat stem rot.

Description

Technical Field

[0001] The invention relates to the technical field of food preservation and fresh-keeping, and in particular to a plant essential oil compound liquid for inhibiting the growth and toxin production of Fusarium graminearum and an application thereof. Background Art

[0002] Fusarium graminearum is a pathogenic fungus that causes wheat and barley fusarium head blight and stem base rot. The epidemic of stem base rot not only causes a reduction in grain production, but also leads to serious contamination of fusarium toxins, threatening food and feed safety. Fusarium graminearum produces fusarium toxins that are toxic to humans and livestock in the field and during grain storage. Among them, deoxynivalenol (DON), also known as vomitoxin, is commonly found in moldy cereal feed raw materials such as wheat and corn. It has become one of the most common mycotoxins in feed and feed raw materials in my country, and the detection rate of DON is as high as 90%. DON can bind to ribosomes to inhibit protein synthesis, causing health problems such as digestive tract damage, anorexia, immune dysfunction, and miscarriage in humans and livestock. At the same time, DON, as a key virulence factor, is necessary for the extensive infection of Fusarium in crops such as wheat. Therefore, controlling the growth and toxin production of Fusarium graminearum from the source has important theoretical significance and practical value for improving grain quality, reducing the economic losses caused by DON pollution to the aquaculture industry, and ensuring food safety.

[0003] The defects of traditional mildew prevention methods: The main method to control Fusarium graminearum and DON pollution is chemical fungicides, which not only cause environmental safety problems, but also increase wheat resistance if used for a long time; and ultraviolet light irradiation can also produce drug-resistant mutants of Fusarium graminearum. Currently, researchers are trying to develop different new green mildew inhibitors to reduce the negative effects of traditional mildew prevention methods (chemical fungicides, ultraviolet light radiation).

[0004] Essential oils (EOs) are secondary metabolites with aromatic odors extracted from plants. Studies have shown that essential oils have good antibacterial effects on a variety of pathogenic fungi, can inhibit the growth of pathogenic fungi and the production of mycotoxins, and are important raw materials for natural antibacterial agents. Active substances in essential oils such as carvacrol, cinnamaldehyde and citral have been widely used in the fields of food and agriculture. Single-component essential oils can play a certain role in antibacterial, preservation and freshness preservation, but single-component essential oils have the disadvantages of narrow antibacterial spectrum and easy to produce drug resistance. In addition, due to the characteristics of biodegradability and volatility of essential oils, adding low-dose single-component essential oils may not achieve the expected prevention and control effect, and high doses will increase the cost of use. In recent years, studies have found that different essential oils are compounded in a certain proportion to treat Aspergillus flavus, Aspergillus ochreus and Aspergillus niger, which are common in the storage of grain and oil, can not only achieve the expected mildew prevention effect, but also play a synergistic role. At present, there is no report or application of compound essential oils for the prevention and control of wheat stem rot. Summary of the invention

[0005] The object of the present invention is to overcome the deficiencies of the prior art and provide a plant essential oil compound liquid for inhibiting the growth and toxin production of Fusarium graminearum. The present invention selects 2 or 3 plant essential oils with excellent growth inhibition effects on Fusarium graminearum from 7 different plant essential oils for compounding, and develops a plant essential oil compound liquid for inhibiting the growth and toxin production of Fusarium graminearum. It is found that the prevention and control effect of the compounding of 2 plant essential oils (carvacrol and cinnamaldehyde) is better than that of the compounding of 3 plant essential oils (carvacrol, cinnamaldehyde and citral). The plant essential oil compound liquid of the present invention can inhibit the growth and toxin production of Fusarium graminearum, can inhibit the occurrence of major pathogenic bacteria of wheat, reduce the use of chemical pesticides, improve the quality of cereals such as wheat, and provide an important idea for the prevention and control of wheat stem base rot.

[0006] To achieve the above purpose, the technical solution designed by the present invention is as follows:

[0007] The invention provides an application of plant essential oil in inhibiting the growth and toxin production of Fusarium graminearum. The plant essential oil is selected from carvacrol, cinnamaldehyde and citral.

[0008] The present invention also provides a plant essential oil compound liquid for inhibiting the growth and toxin production of Fusarium graminearum, wherein the compound liquid is compounded with carvacrol and aldehyde compounds, wherein the aldehyde compounds are one or both of cinnamaldehyde and citral;

[0009] The concentration of carvacrol is 4-40 μg / mL, the concentration of the aldehyde compound is 8-85 μg / mL, and the volume ratio of carvacrol to the aldehyde compound is 1:1-5.

[0010] Furthermore, when the aldehyde compound is cinnamaldehyde, the compound solution is compounded from carvacrol and cinnamaldehyde, wherein the concentration of carvacrol is 4 to 40 μg / mL, the concentration of cinnamaldehyde is 8 to 75 μg / mL, and the volume ratio of carvacrol to cinnamaldehyde is 1:1 to 3;

[0011] Alternatively, when the aldehyde compound is citral, the compound solution is compounded from carvacrol and citral, wherein the concentration of carvacrol is 4 to 40 μg / mL, and the concentration of citral is 10 to 85 μg / mL;

[0012] The volume ratio of carvacrol to citral is 1:1-3.

[0013] Furthermore, the compound solution is compounded by carvacrol with a concentration of 4.83 to 29 μg / mL and cinnamaldehyde with a concentration of 35.72 to 53.58 μg / mL, and the volume ratio of carvacrol to cinnamaldehyde is 1:1;

[0014] Alternatively, the compound liquid is compounded by carvacrol with a concentration of 4.83 to 29 μg / mL and citral with a concentration of 10.15 to 60.92 μg / mL, and the volume ratio of carvacrol to citral is 1:1.

[0015] Furthermore, the compound solution is compounded by carvacrol with a concentration of 29 μg / mL and cinnamaldehyde with a concentration of 53.58 μg / mL, and the volume ratio of carvacrol to cinnamaldehyde is 1:1.

[0016] Furthermore, the compound liquid is compounded with carvacrol at a concentration of 29 μg / mL and citral at a concentration of 20.31 μg / mL, and the volume ratio of carvacrol to citral is 1:1.

[0017] Furthermore, when the aldehyde compound is a combination of cinnamaldehyde and citral;

[0018] The concentration of carvacrol is 4-40 μg / mL; the concentration of cinnamaldehyde is 8-75 μg / mL, and the concentration of citral is 10-85 μg / mL;

[0019] The volume ratio of carvacrol, cinnamaldehyde and citral is 1:1-2:1-2.

[0020] Furthermore, the concentration of carvacrol is 9.67 μg / mL; the concentration of cinnamaldehyde is 17.86 μg / mL, and the concentration of citral is 40.62 μg / mL;

[0021] The volume ratio of carvacrol, cinnamaldehyde and citral is 1:1:1.

[0022] The present invention also provides an antibacterial agent, which comprises the plant essential oil compound liquid.

[0023] The present invention also provides an application of an antibacterial agent in inhibiting the germination of Fusarium graminearum spores.

[0024] The invention also provides an application of the antibacterial agent in preventing and treating wheat stem base rot.

[0025] The present invention also provides an application of the plant essential oil compound liquid as an additive in preparing feed.

[0026] The present invention also provides a feed, wherein the feed contains the plant essential oil compound liquid, wherein the addition amount of the plant essential oil compound liquid is 200-500 mg / kg.

[0027] Principle of the present invention:

[0028] 1. The present invention is based on a relatively new concept of wheat stem rot prevention and control, and is proposed and completed using a well-known principle in the field of microbiology: plant essential oils have antibacterial and antiseptic effects, inhibit toxin production, and the rational combination of plant essential oils with different components can play a synergistic role. Carvacrol, cinnamaldehyde and citral, plant essential oils with excellent antibacterial effects, are compounded to obtain a plant essential oil compound liquid with the best antibacterial effect.

[0029] 2. Fusarium graminearum produces DON toxins that are toxic to humans and livestock in the field and during grain storage, and can cause the occurrence of stem base rot in wheat crops. The plant essential oil compound liquid of the present invention can inhibit the production of Fusarium graminearum, thereby controlling the generation of DON toxins and the occurrence of stem base rot in wheat crops. The present invention not only avoids and controls the generation of drug resistance of Fusarium graminearum in wheat stem base rot, but also reduces the cost of prevention and control of stem base rot in wheat crops.

[0030] Beneficial effects of the present invention:

[0031] 1. The inhibitory effect of 7 single-component plant essential oils on the growth of Fusarium graminearum hyphae is better than that of the traditional mildew inhibitor sodium diacetate. The single-component plant essential oils carvacrol, cinnamaldehyde and citral can destroy the hyphae structure of Fusarium graminearum, inhibit DON biosynthesis and the expression of key elements of DON synthesis. The present invention uses carvacrol and aldehyde compounds (cinnamaldehyde and citral) to compound to obtain a plant essential oil compound liquid, which avoids and controls the production of drug resistance of Fusarium graminearum of wheat stem base rot, while reducing the cost of prevention and control of wheat crop stem base rot; and the prevention and control effect of the compound of 2 plant essential oils (carvacrol and cinnamaldehyde) is better than that of the compound of 3 plant essential oils (carvacrol, cinnamaldehyde and citral).

[0032] 2. The three plant essential oils of the present invention, carvacrol, cinnamaldehyde and citral, can be used as natural active ingredients. The plant essential oil compound liquid prepared by them can be applied to the prevention and control of wheat stem base rot, providing a new solution for the prevention and control of feed source DON pollution.

[0033] 3. The plant essential oil compound liquid developed by the present invention can be used as a feed additive to reduce the pollution caused by Fusarium graminearum and its toxins during grain storage, and provide a new strategy for preventing feed mildew and controlling DON in feed. Specifically, adding the plant essential oil compound liquid to feed can inhibit the growth of Fusarium graminearum and the synthesis of DON in feed, effectively reduce feed source DON pollution, and improve the quality of grains such as wheat. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a diagram showing the antibacterial effect of different plant essential oils on Fusarium graminearum on plates;

[0035] Figure 2 This is the result diagram of the effects of carvacrol, cinnamaldehyde and citral on the morphological structure of Fusarium graminearum hyphae;

[0036] Figure 3 This is a graph showing the inhibitory effects of carvacrol, cinnamaldehyde and citral on the spore germination of Fusarium graminearum;

[0037] In the figure, Figure A is EC 50 Figure A is a graph showing the conidia morphology after treatment with different doses of carvacrol, cinnamaldehyde, citral or sodium diacetate for 48 hours; Figure B is a graph showing the spore germination rate and spore germination inhibition rate of Fusarium graminearum in the groups treated with carvacrol, cinnamaldehyde, citral or sodium diacetate;

[0038] Figure 4 This is a graph showing the inhibitory effects of carvacrol, cinnamaldehyde and citral on the synthesis of DON toxin from Fusarium graminearum;

[0039] Figure 5 This is a graph showing the effects of carvacrol, cinnamaldehyde and citral on the expression of key gene components of DON toxin synthesis in Fusarium graminearum;

[0040] Compared with the control group, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001;

[0041] Figure 6 This is the result diagram of the inhibitory effect of the antibacterial agent on the spore germination of Fusarium graminearum;

[0042] In the figure, Figure A is a conidia morphology diagram after 48 hours of treatment; Figure B is a diagram showing the effect of antibacterial agent treatment on the spore germination rate and spore germination inhibition rate of Fusarium graminearum;

[0043] Figure 7 This is a diagram showing the effect of single-component plant essential oil or antibacterial agent in preventing Fusarium graminearum from infecting wheat seedlings;

[0044] In the figure, Figure A is a result diagram showing that a single component plant essential oil or antibacterial agent inhibits Fusarium graminearum from infecting wheat seedlings;

[0045] Figure B shows the effect of single-component plant essential oil or antibacterial agent on the DON toxin content in wheat infected with Fusarium graminearum; compared with the control group, * indicates P < 0.05, ** indicates P < 0.01, *** Indicates P < 0.001; compared with the PH-1 group, # indicates P < 0.05, ## Indicates P<0.01. DETAILED DESCRIPTION

[0046] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0047] Example 1 Inhibitory effects of different plant essential oils on the mycelial growth of Fusarium graminearum

[0048] 1 Test method

[0049] The mycelium growth rate method was used to determine the inhibitory effects of seven plant essential oils, including citral, cinnamaldehyde, eugenol, geraniol, menthol, carvacrol, and linalool, on the mycelial growth of Fusarium graminearum. At the same time, the traditional mildew inhibitor sodium diacetate was selected as a positive control.

[0050] (1) The PH-1 strain of Fusarium graminearum was cultured on sodium carboxymethyl cellulose (CMC) medium for 5 days, conidia were collected, and the conidia suspension was adjusted to 1×10 5 CFU / mL.

[0051] (2) Use tweezers to place a sterile circular filter paper (6 mm in diameter) on a prepared PDA plate (9 cm in diameter) containing different concentrations of plant essential oils, and transfer 3 μL of conidia suspension (1×10 5 CFU / mL) onto circular filter paper. The concentrations of plant essential oils or sodium diacetate in the culture medium of each group are shown in Table 1.

[0052] (3) After inoculation, the plates were placed in a dark incubator at 28°C for 3 days. The colony diameters of each treatment were measured using the cross method, and the inhibition rates of different plant essential oils on mycelial growth were calculated.

[0053]

[0054] Table 1 Concentration of plant essential oil or sodium diacetate in culture medium

[0055]

[0056]

[0057] 2 Test results

[0058] From Table 2 and Figure 1 It can be seen that the EC of seven plant essential oils, including citral, cinnamaldehyde, eugenol, geraniol, menthol, carvacrol, and linalool, against Fusarium graminearum PH-1 50 The EC values ​​of the traditional mildew inhibitor sodium diacetate against Fusarium graminearum PH-1 were determined by the same method. 50 It is 1130.00μg / mL.

[0059] It can be seen that the seven kinds of plant essential oils have an inhibitory effect on the mycelial growth of Fusarium graminearum and have an inhibitory effect on the EC of the pathogen. 50 The above results showed that carvacrol, cinnamaldehyde and citral in the seven plant essential oils had excellent antibacterial effects on Fusarium graminearum PH-1.

[0060] Table 2 Results of toxicity test of different plant essential oils or sodium diacetate against Fusarium graminearum

[0061]

[0062] Example 2 Effects of carvacrol, cinnamaldehyde and citral on the mycelial morphology of Fusarium graminearum

[0063] 1 Test method

[0064] (1) Inoculate Fusarium graminearum PH-1 into the EC 50 The cells were cultured on PDA plates containing carvacrol (38.66 μg / mL), cinnamaldehyde (71.44 μg / mL) and citral (81.23 μg / mL) at 28°C in a dark incubator for 6 days.

[0065] (2) Use a sterile scalpel to cut 6 to 10 bacterial blocks (<3 mm) with hyphae at the edge of the colony. At the same time, use the traditional mildew inhibitor sodium diacetate as a positive control.

[0066] (3) The cut bacterial blocks were quickly transferred to 2.5% glutaraldehyde solution for fixation, dehydrated, infiltrated, embedded, and prepared into ultrathin sections. The sections were then sliced ​​using a scanning electron microscope (JSM-6390 / LV, NTC, Japan) and a Tecnai G microscope (Germany, Canada).2 The morphology and structure of mycelium were observed using a 20TWIN transmission electron microscope (FEI, Hillsboro, USA).

[0067] 2 Test results

[0068] The scanning electron microscopy results are as follows Figure 2 As shown in the figure, the mycelium of Fusarium graminearum in the control group was full, with regular tubular shape and smooth tube wall. Each mycelium grew evenly, and the width and size were basically the same. The mycelium after carvacrol or citral treatment aggregated, and the mycelium surface was entangled by a fibrous mesh structure. The mycelium after cinnamaldehyde treatment shrank. The mycelium treated with sodium diacetate increased in density and shrank.

[0069] Transmission electron microscopy results Figure 2 As shown in the figure, the surface of mycelial cells in the control group was a regular oval, the organelles inside were arranged closely, and the cell wall was intact and uniform. The organelles of mycelial cells treated with carvacrol or citral were degraded and cavities appeared. The cell wall of mycelial cells treated with cinnamaldehyde or sodium diacetate became thinner and the organelles were blurred.

[0070] The above results show that carvacrol, cinnamaldehyde and citral can all destroy the mycelial morphology of Fusarium graminearum PH-1, which is different from the mode of action of the traditional mildew inhibitor sodium diacetate.

[0071] Example 3 Effects of carvacrol, cinnamaldehyde and citral on conidia germination of Fusarium graminearum

[0072] 1 Test method

[0073] (1) Take 200 μL of fresh spore suspension of Fusarium graminearum PH-1 (1×10 5 CFU / mL), add 100mL containing each EC 50 The culture medium was mixed with carvacrol (38.66 μg / mL), cinnamaldehyde (71.44 μg / mL) and citral (81.23 μg / mL) and sealed, and then cultured at 28°C and 180 r / min in the dark. At the same time, the traditional mildew inhibitor sodium diacetate was selected as a positive control.

[0074] (2) Observe and take photos of the spore germination at 0 h, 2 h, 6 h, 12 h, 24 h, and 48 h, and calculate the spore germination rate and spore germination inhibition rate.

[0075] Spore germination rate (%) = number of germinated spores / (number of germinated spores + number of ungerminated spores) × 100%

[0076] Spore germination inhibition rate (%) = (number of spore germination in the control group - number of spore germination in the treatment group) / number of spore germination in the control group × 100%

[0077] The total number of spores investigated was 200, and germination was considered when the germ tube was longer than half the spore diameter. This was repeated three times.

[0078] 2 Test results

[0079] Depend on Figure 3 A shows that at 0h, the conidia of Fusarium graminearum were sickle-shaped with a thick circle in the middle and sharp ends, and no germination occurred. At 48h, the germ tubes of the conidia in the control group were elongated in many places, germinated into long strips, and extended to both sides to form many branches. Although the germ tubes of the conidia in the carvacrol, cinnamaldehyde or citral treatment groups germinated and slightly elongated, the elongated part was short and granular, and did not fully extend into slender hyphae. Only a few germinated into shorter hyphae. However, the conidia in the sodium diacetate treatment group not only germinated in large quantities, but also a clear reticular structure formed after elongation could be seen.

[0080] Depend on Figure 3 B shows that the spore germination rate of the control group increased linearly over time. At 48h, the spore germination rate of the control group reached 98.17%, while the germination rates of the carvacrol, cinnamaldehyde, and citral treatment groups were 32.67%, 46.17%, and 45.50%, respectively, and the spore inhibition rates were 66.74%, 52.95%, and 53.62%, respectively. The spore germination rate and spore germination inhibition rate of the sodium diacetate treatment group were 56.17% and 42.79%, respectively.

[0081] It can be seen that carvacrol, cinnamaldehyde and citral can inhibit the germination of Fusarium graminearum spores, thereby inhibiting the growth of pathogens, and the inhibition effect is better than that of the traditional mildew inhibitor sodium diacetate.

[0082] Example 4 Effects of carvacrol, cinnamaldehyde and citral on DON toxin production by Fusarium graminearum

[0083] 1 Test method

[0084] (1) Take 0.5 mL of Fusarium graminearum PH-1 (1×10 5 CFU / mL) fresh spore liquid was added into 100 mL of toxin-inducing liquid medium TBI and cultured at 28°C in a shaking incubator at 180 r / min for 1 day.

[0085] (2) Add 100 μL of plant essential oil or 1 mL of sodium diacetate to the culture medium (the final concentration of the drug treatment is preferably the corresponding EC 50 The concentrations were 38.66 μg / mL carvacrol, 71.44 μg / mL cinnamaldehyde, 81.23 μg / mL citral, and 1130.00 μg / mL sodium diacetate). The mixture was incubated at 28°C and 180 r / min in the dark, and repeated 3 times.

[0086] (3) 1.5 mL of bacterial culture was removed on the 3rd, 4th, 5th, 6th, 7th, 8th, 9th and 10th day after treatment, and the supernatant was obtained by centrifugation. The DON toxin in the fermentation supernatant was fully extracted using acetonitrile.

[0087] (4) The DON toxin content was detected by high performance liquid chromatography-tandem mass spectrometry. The high performance liquid chromatography detection conditions were set as follows: BEH-C18 as the chromatographic column, acetonitrile: water (30:70) as the mobile phase, flow rate of 0.3 mL / min, and column temperature of 35°C.

[0088] (5) Mass spectrometry detection conditions are as follows: mass spectrometry uses an electrospray ionization source and a positive ion multiple reaction monitoring (MRM) mode; the spray voltage is 3.2 kV; the ion source temperature is 350 °C; the curtain gas is air and the collision gas is nitrogen. Before use, the flow rates of each gas were adjusted to ensure that the mass spectrometry sensitivity meets the detection requirements. The data were collected in segments. The specific parameters are shown in Table 3.

[0089] Table 3 Mass spectrometry parameters of DON toxin

[0090]

[0091] 2 Test results

[0092] Depend on Figure 4 It can be seen that from the 5th day of the experiment, the content of DON toxin in the supernatant of the control group showed an exponential increase trend, and on the 10th day of the experiment, the content of DON toxin reached 11717.75±634.17μg / L. From the 6th day of the experiment, the content of DON toxin in the supernatant of the sodium diacetate treatment group showed an exponential increase trend, and on the 10th day of the experiment, the content of DON toxin reached 8746.58±1174.09μg / L, indicating that the inhibitory effect of sodium diacetate on the synthesis of DON toxin of Fusarium graminearum was relatively weak. On the 10th day of the experiment, the DON toxin contents in the treatment groups with carvacrol, cinnamaldehyde and citral were 112.30±6.04μg / L, 46.95±6.40μg / L and 195.01±21.84μg / L, respectively, indicating that the three plant essential oils have a strong inhibitory effect on the synthesis of DON toxin of Fusarium graminearum, and are superior to the traditional mildew inhibitor sodium diacetate.

[0093] Example 5 Effects of carvacrol, cinnamaldehyde and citral on the expression of key elements of DON toxin synthesis in Fusarium graminearum

[0094] 1 Test method

[0095] (1) Take 0.5 mL of Fusarium graminearum PH-1 (1×10 5 CFU / mL) fresh spore liquid was added into 100 mL of toxin-inducing liquid medium TBI and cultured at 28°C in a shaking incubator at 180 r / min for 1 day.

[0096] (2) Add 100 μL of plant essential oil or 1 mL of sodium diacetate to the culture medium (the final concentration of the drug treatment is preferably the corresponding EC 50 The concentrations were 38.66 μg / mL carvacrol, 71.44 μg / mL cinnamaldehyde, 81.23 μg / mL citral, and 1130.00 μg / mL sodium diacetate). The mixture was incubated at 28°C and 180 r / min in the dark, and repeated 3 times.

[0097] (3) After 6 days, the mycelium was collected and RNA was extracted. The relative expression levels of FgTRI1, FgTRI4, and FgTRI11, the key elements for DON toxin synthesis, were detected by quantitative PCR.

[0098] 2 Test results

[0099] like Figure 5 It can be seen that carvacrol significantly inhibited the expression of FgTRI1 and FgTRI11, the key elements of DON toxin synthesis, cinnamaldehyde significantly inhibited the expression of FgTRI4 and FgTRI11, and citral significantly inhibited the expression of FgTRI11; while the traditional mildew inhibitor sodium diacetate significantly inhibited the expression of FgTRI4, but significantly increased the expression of FgTRI11.

[0100] The results showed that carvacrol, cinnamaldehyde and citral can inhibit the expression of key elements of DON toxin synthesis, cut off the synthesis pathway of DON toxin in Fusarium graminearum, and thus inhibit the biosynthesis of DON toxin, which is different from the action pathway of traditional mildew inhibitor sodium diacetate.

[0101] Example 6

[0102] Preparation of plant essential oil compound solution 1

[0103] Table 4 shows the component concentrations of the plant essential oil compound liquid. The components of the plant essential oil compound liquid were prepared according to Table 4, and the components were mixed at a volume ratio of 1:1 to obtain the plant essential oil compound liquid. The specific steps are as follows:

[0104] (1) Prepare the above volume ratios with the respective EC 50 1 / 8 times of carvacrol and cinnamaldehyde (i.e., carvacrol concentration is 4.83 μg / mL, cinnamaldehyde concentration is 8.93 μg / mL);

[0105] (2) Carvacrol and cinnamaldehyde are mixed in a ratio of 1:1 to obtain a plant essential oil compound solution 1.

[0106] Table 4 Component concentrations of plant essential oil compound solutions 1 to 42

[0107]

[0108]

[0109] Examples 7 to 48

[0110] Preparation of Plant Essential Oil Compound Liquids 2 to 42

[0111] Table 4 shows the component concentrations of the plant essential oil compound liquids. Prepare the components of the plant essential oil compound liquids according to Table 4, and mix each component in a volume ratio of 1:1 to obtain the plant essential oil compound liquids. The specific steps are the same as those in Example 6.

[0112] Determination of the Inhibitory Effect of the Plant Essential Oil Compound Liquid in Example 49 on the Mycelial Growth of Fusarium graminearum

[0113] 1. Determination of the Inhibitory Effect of Plant Essential Oil Compound Liquids 1 to 16 on the Mycelial Growth of Fusarium graminearum

[0114] 1.1 Test Method

[0115] Evaluate the effect of the combination of carvacrol and cinnamaldehyde according to the fractional inhibitory concentration index (FICI).

[0116]

[0117] Among them, EC 50 (A) 精油联用 is the EC of A when A and B are used in combination 50 , EC 50 (B) 精油联用 is the EC of B when A and B are used in combination 50 . When FICI ≤ 0.5, it is a synergistic effect; when 0.5 < FICI ≤ 1, it is an additive effect; when 1 < FICI ≤ 2, it is an irrelevant effect; when FICI > 2, it is an antagonistic effect.

[0118] Test the antibacterial properties (mycelial growth method) of plant essential oil compound liquids 1 to 16.

[0119] 1.2 Test Results

[0120] The results are shown in Table 5. At the 3rd day, taking the mycelial growth inhibition rate of Fusarium graminearum greater than 50% as the baseline, when the EC 50 combination value of carvacrol and cinnamaldehyde is 1 / 8 of the EC of carvacrol used alone 50 and 1 / 4 times of the EC of cinnamaldehyde used alone 50 (i.e., plant essential oil compound liquid 2), the FICI value of the two is the smallest, showing a synergistic effect.

[0121] During the 3rd to 6th days, the EC 50 combination value of carvacrol and cinnamaldehyde is 1 / 8 of the EC of carvacrol used alone 50 and 1 / 2 times of the EC of cinnamaldehyde used alone 50 (i.e., plant essential oil compound liquid 3), the EC of carvacrol used alone50 1 / 8 of cinnamaldehyde and EC alone 50 3 / 4 times (i.e., plant essential oil compound solution 4), carvacrol alone EC 50 1 / 4 of cinnamaldehyde and EC alone 50 1 / 2 times (i.e. plant essential oil compound solution 7), carvacrol alone EC 50 1 / 4 of cinnamaldehyde and EC alone 50 When the concentration of carvacrol was 3 / 4 times of that of the plant essential oil compound solution 8, the mycelial growth inhibition rate reached 100%, and carvacrol and cinnamaldehyde showed an additive effect on Fusarium graminearum. 50 When 3 / 4 of the EC50 and 3 / 4 of cinnamaldehyde alone were used (i.e., plant essential oil compound solution 16), the mycelial growth inhibition rate reached 100%, and carvacrol and cinnamaldehyde showed no relevant effect on Fusarium graminearum.

[0122] In summary, plant essential oil compound solutions 3, 4, 7, 8 and 16 have the best inhibitory effect on the mycelial growth of Fusarium graminearum.

[0123] Table 5 Inhibitory effect of plant essential oil compound solutions 1 to 16 on the mycelial growth of Fusarium graminearum

[0124]

[0125] Note: The results are expressed as mean ± standard deviation, n = 6. a-i The data in the same column have different shoulder letters (lowercase English letters), indicating significant differences (P<0.05).

[0126] 2. Determination of the inhibitory effect of plant essential oil compound solution 17-32 on the mycelial growth of Fusarium graminearum

[0127] 2.1 Test methods

[0128] The effect of carvacrol and citral mixed was evaluated according to the graded inhibitory concentration index (FICI).

[0129] The antibacterial properties of plant essential oil compound solutions 17-32 were tested (mycelium growth method).

[0130] 2.2 Test results

[0131] The results are shown in Table 6. On the 3rd day, the EC values ​​of carvacrol and citral were 0.1447 W / cm2, 0.133 W / cm2, and 0.127 W / cm2. 50 Combined value is EC for carvacrol alone 50 1 / 4 of citral and EC alone 50 When the concentration of carvacrol and citral was 1 / 4 of that of the plant essential oil blend 22, the FICI values ​​of the two were the smallest, showing a synergistic effect. 50 Combined value is EC for carvacrol alone50 3 / 4 and citral alone EC 50 When the concentration of carvacrol and citral was 1 / 4 times (i.e., plant essential oil compound solution 30), the mycelium growth inhibition rate reached 100% on the 3rd day of the experiment, and with the increase of culture days, the mycelium growth inhibition rate gradually decreased, and the mycelium growth inhibition rate on the 6th day was 59%. Carvacrol and citral showed an additive effect on Fusarium graminearum.

[0132] In summary, only the plant essential oil compound solution 30 achieved a 100% mycelium growth inhibition rate on the third day of the experiment.

[0133] Table 6 Inhibitory effect of plant essential oil compound solutions 17-32 on the mycelial growth of Fusarium graminearum

[0134]

[0135]

[0136] Note: The results are expressed as mean ± standard deviation, n = 6. a-i The data in the same column have different shoulder letters (lowercase English letters), indicating significant differences (P<0.05).

[0137] 3. Determination of the inhibitory effect of plant essential oil compound solution 33-42 on the mycelial growth of Fusarium graminearum

[0138] 3.1 Test methods

[0139] The effect of the mixture of carvacrol, cinnamaldehyde and citral was evaluated according to the graded inhibitory concentration index (FICI) (FICI values ​​of different combinations of plant essential oils were selected ≤ 1, and the plant essential oils showed additive or synergistic effects after compounding).

[0140] The antibacterial properties of plant essential oil compound solutions 33-42 were tested (mycelium growth method).

[0141] 3.2 Test results

[0142] The results are shown in Table 7. Only 1 / 4EC 50 Carvacrol + 1 / 4EC 50 Cinnamaldehyde+1 / 2EC 50 The mycelium growth inhibition rate of citral (i.e., plant essential oil compound liquid 42) on the 3rd day was slightly greater than 50%, while the mycelium growth inhibition rates of other combinations were all less than 50%, indicating that the antibacterial effect of the combination of three plant essential oils was not as good as the antibacterial effect of the above-mentioned two plant essential oils. Therefore, subsequent experimental exploration of the combination of three plant essential oils was no longer considered.

[0143] Table 7 Inhibitory effect of plant essential oil compound liquid 33-42 on the mycelial growth of Fusarium graminearum

[0144]

[0145]

[0146] Note: The results are expressed as mean ± standard deviation, n = 6. a-i The data in the same column have different shoulder letters (lowercase English letters), indicating significant differences (P<0.05).

[0147] Embodiment 50

[0148] Preparation of antibacterial agents 1 to 5

[0149] The antibacterial agent 1 includes a plant essential oil compound solution 3, wherein the concentrations of carvacrol and cinnamaldehyde are 4.83 μg / mL and 35.72 μg / mL respectively (i.e., 1 / 8EC 50 Carvacrol + 1 / 2EC 50 Cinnamaldehyde).

[0150] The antibacterial agent 2 includes a plant essential oil compound solution 4, wherein the concentrations of carvacrol and cinnamaldehyde are 4.83 μg / mL and 53.58 μg / mL respectively (i.e., 1 / 8EC 50 Carvacrol + 3 / 4EC 50 Cinnamaldehyde).

[0151] Antibacterial agent 3 includes plant essential oil compound solution 7, wherein the concentrations of carvacrol and cinnamaldehyde are 9.67 μg / mL and 35.72 μg / mL respectively (i.e., 1 / 4EC 50 Carvacrol + 1 / 2EC 50 Cinnamaldehyde).

[0152] Antibacterial agent 4 includes plant essential oil compound solution 8, wherein the concentrations of carvacrol and cinnamaldehyde are 9.67 μg / mL and 53.58 μg / mL respectively (i.e., 1 / 4EC 50 Carvacrol + 3 / 4EC 50 Cinnamaldehyde).

[0153] The antibacterial agent 5 includes a plant essential oil compound solution 16, wherein the concentrations of carvacrol and cinnamaldehyde are 29 μg / mL and 53.58 μg / mL respectively (i.e., 3 / 4EC 50 Carvacrol + 3 / 4EC 50 Cinnamaldehyde).

[0154] Example 51 Effects of antibacterial agents 1 to 5 on conidia germination of Fusarium graminearum

[0155] 1 Test method

[0156] The effects of the antibacterial agents 1 to 5 of Example 50 on the conidia germination of Fusarium graminearum were determined, and the spore germination rate and the spore germination inhibition rate were calculated.

[0157] 2 Test results

[0158] Depend on Figure 6 A shows that at 48h, 1 / 8EC 50 Carvacrol + 1 / 2EC 50 The germ tubes of conidia in the cinnamaldehyde group (i.e., antibacterial agent 1) were significantly elongated, and some spores germinated and intertwined in a knot-like shape; 1 / 8EC 50 Carvacrol + 3 / 4EC 50 Cinnamaldehyde group (i.e., antibacterial agent 2) and 1 / 4EC 50 Carvacrol + 1 / 2EC 50 In the cinnamaldehyde group (i.e., antibacterial agent 3), conidia showed some germ tube germination and lateral branch growth, more than 70% of conidia did not germinate, and some conidia shrank and became smaller, showing dot-shaped conidia; 1 / 4EC 50 Carvacrol + 3 / 4EC 50 In the cinnamaldehyde group (i.e., antibacterial agent 4), 79% of the conidia were in the ungerminated state; 3 / 4EC 50 Carvacrol + 3 / 4EC 50 Although there were a few germination phenomena of conidia in the cinnamaldehyde group (i.e., antibacterial agent 5), such as linear elongation and lateral branch elongation of conidia, more than 90% of the conidia were dot-shaped and shrunken, indicating that the compound essential oil of carvacrol and cinnamaldehyde can not only inhibit the germination of conidia of Fusarium graminearum, but also destroy the morphology of conidia.

[0159] Depend on Figure 6 B shows that the five combinations of carvacrol and cinnamaldehyde significantly inhibited the germination of conidia of Fusarium graminearum, among which 3 / 4EC 50 Carvacrol + 3 / 4EC 50 The spore germination inhibition rate of the cinnamaldehyde group (i.e., antibacterial agent 5) was close to 100%, and the effect was stable within 48 hours. It can be seen that the combination of carvacrol and cinnamaldehyde showed a strong inhibitory effect on the spore germination of Fusarium graminearum.

[0160] In summary, antibacterial agent 5 had the strongest inhibitory effect on the conidia germination of Fusarium graminearum.

[0161] Example 52 Wheat seedling infection control test

[0162] 1 Test method

[0163] (1) Select wheat seeds with full grains, lay two layers of absorbent paper in a glass culture dish, soak them with sterile distilled water, and place the wheat seeds in the culture dish with the groin facing downward. Culture them in the dark at 25°C and a relative humidity of 75% for 2 to 3 days, and select seedlings with uniform embryo length for pathogen infection.

[0164] (2) In a growth bottle filled with 50 mL of sterile MS medium, single component carvacrol, cinnamaldehyde, citral, sodium diacetate or the five antibacterial agents in Example 50 were added, wherein the concentration of carvacrol was its EC 50 (i.e. 38.66 μg / mL), the concentration of cinnamaldehyde is its EC 50 (i.e. 71.44 μg / mL), the concentration of citral is its EC 50 (i.e. 81.23 μg / mL), the concentration of sodium diacetate is its EC 50 (i.e. 1130 μg / mL).

[0165] (3) Take 5 μL of Fusarium graminearum PH-1 conidia suspension (1×10 5 CFU / mL) was placed in the center of the solidified MS medium and spread evenly using a disposable spreading rod.

[0166] (4) The selected wheat germs were inserted vertically into the culture medium with their groin facing downward (ensuring that the coleoptiles were vertically upward and the top of the buds were above the surface of the culture medium). Each treatment group had 3 replicates, with 15 wheat germs in each replicate. They were placed in a 28°C greenhouse with alternating light and dark (light / dark: 12h / 12h) and cultured. After 10 days of culture, the growth and disease of the wheat seedlings in each culture bottle were observed. The calculation formula for the disease level index and the control effect is as follows:

[0167]

[0168] (5) Wipe the surface moisture of the washed wheat seedlings dry, weigh and record the weight, use acetonitrile to fully extract the DON toxin in the wheat seedlings, and use high performance liquid chromatography to detect the DON toxin content.

[0169] 2 Test results

[0170] Depend on Figure 7 A shows that the wheat in the control group grew very well, with the base of the stem in natural growth color and the upper part in emerald green, and the whole plant was strong and upright. The base of the wheat stem in the group infected with Fusarium graminearum PH-1 was dark brown, the upper part was withered and yellow, and the growth was poor. The infection symptoms at the base of the wheat stem in the single-component carvacrol and cinnamaldehyde treatment groups were significantly alleviated, the upper part of the stem turned green, and the growth was good. The wheat stem base in the citral treatment group still had obvious symptoms of stem base rot, and the upper part of the stem was yellow-green. On the contrary, the symptoms of wheat stem base rot in the traditional mildew inhibitor sodium diacetate group were aggravated, and the whole wheat plant turned black-brown. All five antibacterial agents significantly alleviated the infection symptoms at the base of the wheat stem, with only a little browning at the base of the wheat stem and emerald green above the stem.

[0171] As shown in Table 8, the single components of carvacrol and cinnamaldehyde, as well as the five antibacterial agents, significantly reduced the disease grade index, showing a good control effect. The control effect of the five antibacterial agents was better than that of the single component of plant essential oils, among which 3 / 4EC 50 Carvacrol + 3 / 4EC 50 The combination of cinnamaldehyde (i.e., antifungal agent 5) had the best control effect. On the contrary, the traditional mildew inhibitor sodium diacetate aggravated the disease index of wheat stem base rot.

[0172] Depend on Figure 7 B shows that infection with Fusarium graminearum PH-1 significantly increased the content of DON toxin in wheat. Compared with the PH-1 group, the content of DON toxin in wheat in the sodium diacetate group increased by 6 times, while the single components of carvacrol, cinnamaldehyde and citral could reduce the content of DON toxin in wheat infected with Fusarium graminearum, 1 / 4EC 50 Carvacrol + 3 / 4EC 50 The cinnamaldehyde compound combination (i.e., antibacterial agent 4) has a tendency to reduce the DON toxin content in wheat infected with Fusarium graminearum.

[0173] It can be seen that antibacterial agent 4 has obvious effect in preventing and controlling wheat stem base rot, and has important application value in preventing and controlling the damage caused by Fusarium graminearum infection of wheat.

[0174] Table 8 Effects of single-component plant essential oils or antibacterial agents on disease grade index and control effect of Fusarium graminearum infection on wheat seedlings

[0175]

[0176]

[0177] Note: The results are expressed as mean ± standard deviation, n = 3. a-d The data in the same column have different shoulder letters (lowercase English letters), indicating significant differences (P<0.05).

[0178] Example 53 Preparation of feed

[0179] Adding plant essential oil compound liquid (i.e. feed additive) to ordinary feed, the added amount is 300mg / kg. The probability of mildew of this feed is lower than that of ordinary feed.

[0180] It can be seen from this that adding a compound liquid of plant essential oils to feed can inhibit the growth of Fusarium graminearum and the synthesis of DON in feed, effectively reduce DON contamination in feed sources, and improve the quality of grains such as wheat.

[0181] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all belong to the protection scope of the present invention.

Claims

1. An application of plant essential oil in inhibiting the growth and toxin production of Fusarium graminearum, characterized in that: The plant essential oil is selected from carvacrol, cinnamaldehyde and citral.

2. A plant essential oil compound liquid for inhibiting the growth and toxin production of Fusarium graminearum, characterized in that: The compound liquid is compounded by carvacrol and aldehyde compounds, and the aldehyde compounds are one or both of cinnamaldehyde and citral; The concentration of carvacrol is 4-40 μg / mL, the concentration of the aldehyde compound is 8-85 μg / mL, and the volume ratio of carvacrol to the aldehyde compound is 1:1-5.

3. The plant essential oil compound liquid according to claim 2, characterized in that: When the aldehyde compound is cinnamaldehyde, the compound solution is compounded from carvacrol and cinnamaldehyde, wherein the concentration of carvacrol is 4 to 40 μg / mL, the concentration of cinnamaldehyde is 8 to 75 μg / mL, and the volume ratio of carvacrol to cinnamaldehyde is 1:1 to 3; Alternatively, when the aldehyde compound is citral, the compound solution is compounded from carvacrol and citral, wherein the concentration of carvacrol is 4 to 40 μg / mL, and the concentration of citral is 10 to 85 μg / mL; The volume ratio of carvacrol to citral is 1:1-3.

4. The plant essential oil compound liquid according to claim 3, characterized in that: The compound liquid is prepared by compounding carvacrol with a concentration of 4.83 to 29 μg / mL and cinnamaldehyde with a concentration of 35.72 to 53.58 μg / mL, and the volume ratio of carvacrol to cinnamaldehyde is 1:1; Alternatively, the compound liquid is compounded by carvacrol with a concentration of 4.83 to 29 μg / mL and citral with a concentration of 10.15 to 60.92 μg / mL, and the volume ratio of carvacrol to citral is 1:

1.

5. The plant essential oil compound liquid according to claim 2, characterized in that: When the aldehyde compound is a combination of cinnamaldehyde and citral; The concentration of carvacrol is 4-40 μg / mL; the concentration of cinnamaldehyde is 8-75 μg / mL, and the concentration of citral is 10-85 μg / mL; The volume ratio of carvacrol, cinnamaldehyde and citral is 1:1-2:1-2.

6. A bacteriostatic agent, characterized in that: The antibacterial agent comprises the plant essential oil compound solution according to any one of claims 2 to 5.

7. Use of the antibacterial agent according to claim 6 in inhibiting the germination of Fusarium graminearum spores.

8. Use of the antibacterial agent according to claim 6 in preventing and treating wheat stem base rot.

9. Use of the plant essential oil compound liquid as claimed in claim 2 as an additive in preparing feed.

10. A feed, characterized in that: The feed contains the plant essential oil compound liquid as claimed in any one of claims 2 to 5, wherein the added amount of the plant essential oil compound liquid is 200 to 500 mg / kg.

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