Method for evaluating vomitoxin synthesis capability of fusarium graminearum induced by different layers of wheat grains and application of method
Through stratified co-cultivation and multi-dimensional index evaluation, the pericarp and seed coat layers of wheat grains were identified as key to vomitoxin induction, solving the problems of unstable evaluation and insufficient stratified assessment in existing technologies, realizing an efficient and precise breeding method, and ensuring food safety.
Patent Information
- Application Number
- CN202510841113.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-23
- Publication Date
- 2025-10-31
AI Technical Summary
Existing methods for evaluating the induction capacity of vomitoxin in wheat grains are greatly affected by environmental factors and human operational errors, resulting in unstable results that fail to reflect the intrinsic characteristics of the grains. Furthermore, they lack detailed assessments of different stratified regions, which affects breeding outcomes.
Wheat grains were divided into five layers (P1-P6) and co-cultured with Fusarium graminearum spore liquid. The expression level of the TRI gene was measured by real-time quantitative PCR, and the spore germination rate and mycelial growth rate were observed to comprehensively evaluate the induction ability of different layers.
The study achieved high repeatability and stability under laboratory conditions, revealing that the pericarp and seed coat are key to inducing vomitoxin, providing precise breeding targets, and improving breeding efficiency and food safety.
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Abstract
Description
Technical Field
[0001] This invention belongs to the fields of plant disease resistance identification and evaluation and food safety technology, specifically involving a method for evaluating the ability of different stratifications of wheat grains to induce Fusarium graminearum to synthesize vomitoxin and its application. Background Technology
[0002] Fusarium head blight (FHB) is a devastating global disease of wheat caused by the Fusarium graminearum species complex (FGSC). This disease not only causes significant yield losses, but the fungal toxins produced after the pathogen infects the grains, particularly deoxynivalenol (DON, commonly known as vomitoxin), pose a serious threat to human and animal health. Fusarium graminearum has a long infection window, lasting from flowering to late grain development, and even remaining active during storage, resulting in toxin contamination throughout the entire food chain, "from farm to table." Therefore, effectively controlling Fusarium head blight and its associated toxin contamination is a formidable challenge to ensuring food security and safety.
[0003] Accurately assessing the ability of wheat grains to induce the synthesis of DON toxin by Fusarium graminearum (i.e., the grain's "toxin-inducing capacity") is crucial for understanding toxin accumulation mechanisms and screening and breeding wheat varieties resistant to (or tolerant of) toxin accumulation. Currently, the mainstream and considered "gold standard" method for evaluating the DON toxin-inducing capacity of wheat grains is the field single-floret drip inoculation method. This method involves artificially injecting a suspension of Fusarium graminearum spores into a specific location on the ear (usually the floret of the fifth spikelet at the tip) during the wheat flowering stage, followed by creating favorable conditions for disease development through measures such as heat preservation and moisture retention. After harvesting, wheat grains infected with Fusarium head blight are chemically analyzed to determine their DON toxin content, thus reflecting the toxin-inducing capacity of the variety or material.
[0004] However, this traditional field identification method has significant and insurmountable inherent defects: (1) High environmental dependence: The severity of disease and the final amount of toxin accumulation after inoculation are easily affected by environmental conditions (such as temperature, humidity, light, and rainfall) during and after inoculation. Adverse weather (such as drought, high temperature, and rainstorm) often leads to disease failure or uneven severity. (2) Human operation error: The standardization of inoculation operation (such as the position of the spikelet injected, the concentration of spore solution, and the control of moisturizing conditions) directly affects the success rate and consistency of inoculation, and there are large differences between different operators or different batches. (3) Fluctuation and low repeatability of results: The superposition of the above environmental and human factors leads to significant fluctuations in the severity of disease and the amount of toxin accumulation between years, locations, and even between different replicates in the same year, resulting in poor reproducibility and repeatability of experimental data. (4) Difficulty in truly reflecting the intrinsic characteristics of grains: Environmental interference makes it impossible for experimental results to stably and reliably reflect the inherent, genetically controlled ability of grains to induce or inhibit DON synthesis, affecting the accurate evaluation of the toxin resistance potential of breeding materials.
[0005] These limitations severely restrict the accuracy, reliability, and efficiency of evaluating grain toxin induction capacity based on field inoculation methods, becoming a bottleneck for in-depth analysis of toxin accumulation mechanisms and accelerating toxin-resistant wheat breeding. Therefore, it is urgent to establish an in vitro evaluation system that is unaffected by environmental interference, highly standardized, and reproducible, to overcome the inherent defects of field trials and provide stable and reliable technical support for research on toxin biosynthesis mechanisms and resistance breeding.
[0006] Furthermore, existing evaluation methods (both field-based and in vitro methods) generally treat the entire grain as a homogeneous whole for toxin determination. However, different anatomical structures of wheat grains (such as the cortex / bran, aleurone layer, endosperm, and embryo) can exhibit significant differences in physical structure, chemical composition, and response to pathogen infection. The lack of sophisticated methods for assessing the ability of different grain strata (such as the cortex and endosperm) to specifically induce or inhibit DON synthesis limits our understanding of the spatial distribution patterns of toxin accumulation within the grain and hinders the possibility of precision breeding targeting resistance in specific tissues. Summary of the Invention
[0007] To address the shortcomings of existing technologies, this invention provides a method and its application for evaluating the ability of different layers of wheat grains to induce Fusarium graminearum to synthesize vomitoxin. This method can evaluate the induction ability of different layers of wheat grains for vomitoxin, not only clarifying the main layers in which Fusarium graminearum induces vomitoxin synthesis in wheat grains, but also revealing the spatial specificity of wheat-pathogen interactions. This provides a new perspective for disease-resistant breeding and allows for the targeted selection of new wheat varieties that possess both resistance to Fusarium head blight and low toxin accumulation characteristics. This has important practical value for ensuring food safety and achieving green wheat production.
[0008] This invention is achieved through the following technical solution:
[0009] A method for evaluating the ability of different stratifications of wheat grains to induce Fusarium graminearum to synthesize vomitoxin includes the following steps:
[0010] Step 1) Divide the whole wheat grains into five different layers of wheat flour P1, P2, P3, P4 and P5, and mix P1-P5 together to make whole wheat flour P6.
[0011] Step 2) The wheat flour P1-P6 obtained in Step 1) were co-cultured with Fusarium graminearum spore liquid and the vomitoxin production of Fusarium graminearum under different stratified culture conditions was determined.
[0012] Step 3) Collect the mycelia co-cultured in Step 2) and extract RNA. Use real-time quantitative PCR to determine the expression level of TRI, a key gene involved in vomitoxin synthesis in the mycelia.
[0013] Step 4) Observe the effect of different layers of wheat flour on the germination rate of Fusarium graminearum spores;
[0014] Step 5) Determine the growth rate of Fusarium graminearum on water agar medium containing wheat flour of different layers;
[0015] Step 6) By measuring the vomitoxin yield obtained in Step 2), the TRI gene expression level obtained in Step 3), the spore germination rate obtained in Step 4), and the mycelial growth rate obtained in Step 5), the induction capacity of different layers of wheat grains on vomitoxin synthesis was comprehensively evaluated.
[0016] Preferably, in step 1), P1 is the pericarp and seed coat, accounting for 0-7% of the wheat grain weight; P2 is the aleurone layer and sub-aleurone layer, accounting for 8%-30% of the wheat grain weight; P3 is the outer endosperm, accounting for 31%-50% of the wheat grain weight; P4 is the meso-endosperm, accounting for 51%-70% of the wheat grain weight; and P5 is the inner endosperm, accounting for 71%-100% of the wheat grain weight.
[0017] Preferably, the preparation method of the Fusarium graminearum spore solution in step 2) is as follows: The strain is inoculated onto potato dextrose agar plates and cultured at 28°C for 3 days. Then, 3-5 mycelial blocks are taken from the edge of the colony using a sterile punch and transferred to 150 mL of sterilized mung bean soup medium. The medium is cultured at 28°C and 160 rpm in a constant temperature shaker for 120 h. The spores are collected, and the spore concentration is adjusted to 1×10⁻⁶ using sterile distilled water. 5 per mL.
[0018] Preferably, the specific steps of step 2) are as follows: Weigh equal masses of wheat flour P1-P6 with different layers, add Fusarium graminearum spore liquid to each, mix thoroughly, and incubate in a constant temperature shaker at 28℃ and 160rpm for 3 days. Then, transfer to a constant temperature incubator at 25℃ for 7 days, dry at 70℃, and determine the vomitoxin yield using LC-MS.
[0019] Preferably, the specific steps of step 3) are as follows: collect the mycelia that have been co-cultured for 3 days in step 2), press out the water and freeze them in liquid nitrogen, extract mycelial RNA by TRIzol reagent method, amplify the three key genes TRI1, TRI5 and TRI10 involved in the synthesis of vomitoxin by real-time fluorescence quantitative PCR, and calculate the relative expression levels.
[0020] Preferably, the specific steps of step 4) are as follows: equal mass of wheat flour P1-P6 with different layers are placed in a spore suspension of Fusarium graminearum, and samples are taken after 3 hours. The spore morphology and germination status are observed using a laser confocal microscope.
[0021] Preferably, the specific steps of step 5) are as follows: equal mass of wheat flour of different layers P1-P6 are thoroughly mixed with water agar medium to prepare wheat flour-water agar medium culture plates; a 1 cm diameter mycelial cake is taken from the edge of the colony using a sterile punch and placed at the center of the plate; the colony morphology and growth rate under different culture conditions are observed to determine the effect of different layers of wheat flour on colony growth.
[0022] The above-mentioned method for evaluating the ability of different wheat grain stratification to induce Fusarium graminearum to synthesize vomitoxin is applied in wheat breeding.
[0023] Preferably, the wheat breeding involves selecting new wheat varieties that possess both resistance to Fusarium head blight and low vomitoxin accumulation.
[0024] The beneficial effects of this invention are as follows:
[0025] (1) The standardized in vitro culture system established in this invention completely eliminates dependence on field environmental conditions. By precisely controlling key parameters such as temperature, humidity, culture medium composition, and pathogen inoculation amount in the laboratory, the significant interference caused by weather changes, field microenvironment, and human error in the traditional single-flower drip inoculation method in the field is completely eliminated. This method has high repeatability and reproducibility, ensuring the stability and comparability of evaluation results between different batches and different laboratories, and providing a solid and reliable technical platform for accurately assessing the toxin-inducing ability of the grain itself.
[0026] (2) The core innovation of this invention lies in the first systematic evaluation of the ability of the fine anatomical structure of wheat grains (P1-P5 layers) to regulate the growth and metabolism of Fusarium graminearum. This method successfully divided the grains into five layers according to anatomical function (P1: pericarp and seed coat; P2: aleurone layer and sub-aleurone layer; P3: outer endosperm; P4: meso-endosperm; P5: inner endosperm) and whole wheat flour (P6), and conducted in vitro co-culture experiments on each layer. This method overcomes the limitation of traditional methods that treat the grain as a homogeneous whole, and for the first time reveals the tissue specificity of the wheat-Fusarium graminearum interaction in a spatial dimension.
[0027] (3) Using the method of this invention, the pericarp and seed coat layers of wheat grains (P1) were clearly discovered and confirmed for the first time as key tissue layers for inducing the synthesis of vomitoxin by Fusarium graminearum. Specifically, this was manifested in the following ways: ① Highest toxin production: Fusarium graminearum co-cultured with the P1 layer showed significantly higher DON synthesis levels than other layers (P2-P5) and whole wheat flour (P6). ② Strongest gene induction: The P1 layer significantly upregulated the expression level of the key gene (TRI gene) for DON toxin synthesis in Fusarium graminearum. ③ Promotion of cell growth: The P1 layer also significantly promoted the biomass accumulation and mycelial growth rate of Fusarium graminearum. This discovery overturned previous understanding, indicating for the first time that the outermost tissue of wheat grains (P1) is rich in key substances (or signals) that can specifically induce pathogenic toxin production and promote its growth, providing a new entry point and key experimental evidence for in-depth analysis of the molecular mechanism of toxin synthesis.
[0028] (4) The method of this invention integrates multi-dimensional evaluation indicators, including quantitative analysis of DON toxin content, expression analysis of key virulence genes (TRI genes), determination of pathogen spore germination rate, and observation of mycelial growth rate. This multi-parameter simultaneous detection strategy can comprehensively, objectively, and accurately evaluate the integrated regulatory effect (induction or inhibition) of each grain stratification on the growth, metabolism, and pathogenicity of Fusarium graminearum, resulting in a more systematic and reliable evaluation.
[0029] (5) The method of this invention has a clear process and standardized operation steps. It does not rely on field seasons and complex inoculation techniques and can be carried out efficiently and in batches in the laboratory. It is not only suitable for comparing the grain toxin induction ability of different wheat varieties (lines), but also facilitates the screening of specific compounds that can induce DON toxin synthesis.
[0030] (6) The core discovery of this invention—the pericarp / seed coat layer (P1)—is a key layer for inducing toxin production and has significant breeding application value: ① Precise breeding target: By clarifying the key target tissue layer (P1) for improvement, breeders can be guided to specifically screen and create wheat germplasm resources enriched with anti-induction (or inhibitory) substances in the pericarp / seed coat. ② Dual breeding objectives: It helps to cultivate breakthrough new wheat varieties that possess both Fusarium head blight resistance (reducing incidence) and low toxin accumulation (especially inhibiting the induction effect of the key layer), reducing the dual risks of Fusarium head blight and toxin contamination from the source. ③ Improved breeding efficiency: Combined with the in vitro stratified evaluation method of this invention, breeding materials with low toxin induction ability can be screened efficiently and accurately in early generations, significantly shortening the breeding cycle and improving selection efficiency.
[0031] (7) By cultivating and promoting low-toxin-accumulation wheat varieties screened based on this invention, the pollution level of DON toxin in grains can be effectively reduced, ensuring food and feed safety and reducing the harm of toxins to human and animal health. At the same time, this method and its application help reduce the use of chemical pesticides, which meets the requirements of sustainable agricultural development and has important practical significance and social value for achieving green and safe wheat production. Attached Figure Description
[0032] Figure 1 The yield of vomitoxin in wheat grains of Yangmai 25(A) and YDXX-18(B) after co-culturing with Fusarium graminearum at different stratifications;
[0033] Figure 2 Determination of the ability of different stratifications of wheat grains of Yangmai 25 and YDXX-18 to induce expression of Fusarium graminearum TRI gene: A is the relative expression level of TRI1 gene; B is the relative expression level of TRI5 gene; C is the relative expression level of TRI10 gene.
[0034] Figure 3 The effect of different grain stratification of wheat varieties Yangmai 25 and YDXX-18 on the spore germination rate of Fusarium graminearum;
[0035] Figure 4 The effect of different grain stratification of wheat grains of Yangmai 25 and YDXX-18 on the growth morphology of Fusarium graminearum colonies;
[0036] Figure 5Dynamic determination of Fusarium graminearum colony growth in different strata of wheat grains of Yangmai 25(A) and YDXX-18(B). Detailed Implementation
[0037] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.
[0038] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the experimental methods without specific conditions are all conventional methods in the art.
[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0040] Example 1
[0041] A method for evaluating the ability of different wheat grain stratifications to induce Fusarium graminearum to synthesize vomitoxin, the specific steps of which are as follows:
[0042] (1) Selecting materials
[0043] In this embodiment, a group of wheat lines with excellent agronomic traits and moderate susceptibility to Fusarium head blight were selected as experimental varieties, namely YDXX-18 and Yangmai 25.
[0044] (2) Prepare layered wheat flour
[0045] 50g of plump, uniformly sized wheat grains were selected and milled using a small-scale layered mill to separate the grains into five layers: P1, P2, P3, P4, and P5, as well as P6 (whole wheat flour composed of the above five layers). The wheat flour was divided into 6 groups to investigate the ability of wheat flour with different layers to induce Fusarium graminearum to synthesize vomitoxin.
[0046] Among them, P1-P5 represent different layers of wheat grains, with each layer representing a certain percentage of the grain portion, as detailed below:
[0047] P1 (pericarp and seed coat): This is the outermost protective structure of wheat grains, accounting for only 0-7% of the total grain weight, and plays a role in protecting the internal tissues.
[0048] P2 (aleurone layer and sub-aleurone layer): Located inside the pericarp and seed coat, outside the outer endosperm, accounting for 8% to 30% of the grain weight, and containing more protein and dietary fiber.
[0049] P3 (outer endosperm): Located within the aleurone layer and sub-aleurone layer, and outside the meso-endosperm, it accounts for 31% to 50% of the grain weight and its main components are starch and protein, etc.
[0050] P4 (Mesoendosperm): Located inside the outer endosperm and outside the inner endosperm, it accounts for 51% to 70% of the grain weight and is an important part of wheat grains for storing nutrients.
[0051] P5 (endosperm): This is the innermost part of the wheat grain, accounting for 71% to 100% of the grain's weight. It mainly contains nutrients such as starch and some proteins.
[0052] (3) Preparation of Fusarium graminearum spore liquid
[0053] The activated *Fusarium graminearum* strain (kindly provided by Professor Jiang Cong of Northwest A&F University) was inoculated onto potato dextrose agar (PDA) plates and cultured at 28°C for 3 days. Subsequently, 3–5 mycelial fragments were collected from the colony edge using a sterile punch and transferred to 150 mL of sterilized mung bean soup medium. The medium was then cultured at 28°C and 160 rpm in a constant-temperature shaker for 120 h. Spores were collected, and the spore concentration was adjusted to 1 × 10⁻⁶ using sterile distilled water. 5 per mL.
[0054] (4) Determination of vomitoxin production
[0055] Weigh out 0.5g of each of the six wheat flour samples (Yangmai 25 and YDXX-18) and place them in sterile conical flasks. Add 50mL of the spore solution prepared above, mix well, and incubate at 28℃ and 160rpm in a constant temperature shaker for 3 days. Then, transfer them to a constant temperature incubator at 25℃ for 7 days of static incubation and dry them in a 70℃ oven.
[0056] The co-cultured mixture was transferred to a 10 mL centrifuge tube, and 4 mL of extraction buffer (49.5% acetonitrile + 1% formic acid + 49.5% double-distilled water) was added. The mixture was thoroughly mixed in a shaker and placed in a constant temperature shaker at 28℃ and 160 rpm for 30 min. After centrifugation at 3400 rpm for 5 min, 2 mL of the supernatant was transferred to a new 10 mL centrifuge tube, a salt packet was added, and the mixture was rapidly shaken for 1 min. The tube was then centrifuged at 3400 rpm for 5 min. 300 μL of the supernatant was transferred to a glass test tube and purged with nitrogen until the solvent was completely evaporated. 500 μL of extraction buffer was added to dissolve the supernatant. The mixture was filtered through an organic filter membrane into a sample vial, and the vomitoxin content was detected by liquid chromatography-mass spectrometry (LC-MS).
[0057] The peak area data of the two test powder samples were detected by a triple quadrupole liquid chromatography-mass spectrometry (TSQ-Vantage, Thermo Fisher SCIENTIFIC) instrument, and the absolute content of vomitoxin in the test powder samples was calculated.
[0058] The chromatographic conditions were as follows: the column used was an ACQUITY UPLC HSS T3 (2.1×100mm, 1.8μm); the injection volume was 10μL; the flow rate was 0.35mL / min; the column temperature was 40℃; and gradient elution was performed using a mobile phase consisting of an aqueous phase and an organic phase (B).
[0059] The mass spectrometry conditions are as follows: positive ion mode is used, and the elution time of vomitoxin is generally 2.88 min.
[0060] First, the vomitoxin gradient standard samples were measured using the instrument to obtain a vomitoxin assay standard curve. After detecting the peak area of the test powder samples, the concentration of vomitoxin in the test powder samples was calculated according to the standard curve. Based on the amount of test powder used (0.5g in this example) and the total mass of the powder samples, the absolute content of vomitoxin in each test powder sample was calculated. The formula for calculating the absolute content of vomitoxin is as follows:
[0061]
[0062] In the formula: ISTD Area represents the peak area detected by the triple quadrupole liquid chromatography-mass spectrometry (LC-MS) instrument.
[0063] Experimental results are as follows Figure 1 As shown, when co-cultured with wheat grains P1 (pericarp and seed coat), the DON toxin production of Fusarium graminearum was significantly higher than that of the whole wheat flour group (P6) and other grain stratification groups (P2-P5). This indicates that the pericarp and seed coat layers of wheat grains are the main strata that induce the synthesis of DON toxin by Fusarium graminearum, and that a large number of metabolites that induce the synthesis of DON toxin by Fusarium graminearum may be enriched in these strata.
[0064] (5) Measurement of TRI gene expression
[0065] Six wheat flour samples (0.5g each) from Yangmai 25 and YDXX-18 were weighed and placed in sterile conical flasks. 50mL of the prepared spore solution was added, and the mixture was thoroughly mixed. The samples were then incubated at 28℃ and 160rpm for 3 days. Mycelia were collected, the water was pressed out, and the samples were placed in 2mL centrifuge tubes. Grinding beads were added, and the tubes were frozen in liquid nitrogen. The samples were then ground for 2 minutes. 1mL of TRIzol extraction solution was added, and the mixture was thoroughly shaken and mixed. 400μL of chloroform was added, and the mixture was thoroughly mixed. The mixture was allowed to stand at room temperature for 3 minutes, then ground at 4℃ and 12000rpm. Centrifuge for 10 min; transfer 450 μL of supernatant to a 1.5 mL enzyme-free centrifuge tube, add isopropanol at a 1:1 ratio, mix by inverting 5-10 times, and incubate at -20°C for 30 min; centrifuge at 12000 rpm for 10 min at 4°C and discard the supernatant; add 1 mL of 75% ethanol, centrifuge at 12000 rpm for 5 min at 4°C, discard the supernatant, and dry in a clean bench; finally, add 100 μL of DEPC water to dissolve for 5 min; reverse transcribe cDNA using a reverse transcription kit (Novizan, Nanjing).
[0066] The expression level of the TRI gene in *Fusarium graminearum* co-cultured with the aforementioned wheat flour was detected using real-time quantitative PCR. The reaction system contained 2×ChamQ Universal SYBR qPCR Master Mix, specific primers, template DNA, and ddH2O. The thermal cycling conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 5 s; 60℃ annealing for 30 s, for 40 cycles. The fluorescence signal was collected after each cycle, and the Ct value was obtained using Bio-Rad CFX Maestro software to calculate the expression level of the target gene.
[0067] The relative expression levels of TRI1, TRI5, and TRI10 are as follows: Figure 2 As shown, when co-cultured with wheat grain P1 (pericarp and seed coat), Fusarium graminearum TRI1 ( Figure 2 (A), TRI5 Figure 2 (B), TRI10 Figure 2 The expression level of C) was significantly higher than that of the whole wheat flour group (P6) and other grain stratification groups (P2-P5), indicating that the stratification of Fusarium graminearum TRI gene expression induced by wheat grains was the pericarp and seed coat stratification (P1).
[0068] (6) Determination of the germination rate of Fusarium graminearum spores
[0069] Six wheat flour samples (0.5g each) from Yangmai 25 and YDXX-18 were weighed and placed in sterile conical flasks. 50mL of the spore suspension prepared above was added, mixed evenly, and cultured in a shaker at 28℃ and 160rpm for 3h. The spore state was observed using a laser confocal microscope.
[0070] Experimental results are as follows Figure 3 As shown, there were no significant differences in the germination position and morphology of spores co-cultured with different wheat grains in different layers, indicating that different layers of wheat grains do not affect the germination of Fusarium graminearum spores.
[0071] (7) Determination of the growth rate of Fusarium graminearum
[0072] Six wheat flour samples (0.5g each) from Yangmai 25 and YDXX-18 were weighed and thoroughly mixed with 45mL of water agar (WA) medium to prepare wheat flour-WA medium culture plates. The medium was divided into three equal parts. After complete solidification, a Fusarium graminearum block PH-1 of the same size and growth state was inoculated at the center of the medium (a 1cm diameter mycelial cake was taken from the edge of the colony using a sterile punch and placed at the center of the plate). The colony morphology and mycelial growth rate of Fusarium graminearum on different layered wheat flour mediums were observed (3-5 days).
[0073] Experimental results are as follows Figure 4 , 5 As shown, after co-culturing Fusarium graminearum with wheat grains in different layers, dense aerial hyphae were formed in both cases, and the colonies were regularly round. Figure 4 Furthermore, the diameter decreases from the outside to the inside (P1→P5) as the layers deepen. Figure 5 This morphological characteristic indicates that the nutrients required for the growth of the pathogen are concentrated in the outer tissues of the wheat grain (such as P1).
[0074] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. The scope of protection of the present invention is determined by the scope claimed in the claims. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.
Claims
1. A method for evaluating the ability of different stratifications of wheat grains to induce Fusarium graminearum to synthesize vomitoxin, characterized in that, Includes the following steps: Step 1) Divide the whole wheat grains into five different layers of wheat flour P1, P2, P3, P4 and P5, and mix P1-P5 together to make whole wheat flour P6. Step 2) The wheat flour P1-P6 obtained in Step 1) were co-cultured with Fusarium graminearum spore liquid and the vomitoxin production of Fusarium graminearum under different stratified culture conditions was determined. Step 3) Collect the mycelia co-cultured in Step 2) and extract RNA. Use real-time quantitative PCR to determine the expression level of TRI, a key gene involved in vomitoxin synthesis in the mycelia. Step 4) Observe the effect of different layers of wheat flour on the germination rate of Fusarium graminearum spores; Step 5) Determine the growth rate of Fusarium graminearum on water agar medium containing wheat flour with different layers; Step 6) Evaluate the ability of different layers of wheat grain to induce vomitoxin synthesis by measuring the vomitoxin yield obtained in Step 2), the TRI gene expression level obtained in Step 3), the spore germination rate obtained in Step 4), and the mycelial growth rate obtained in Step 5.
2. The method for evaluating the ability of different stratifications of wheat grains to induce Fusarium graminearum to synthesize vomitoxin, as described in claim 1, is characterized in that... Step 1) P1 is the pericarp and seed coat, accounting for 0-7% of the wheat grain weight; P2 is the aleurone layer and sub-aleurone layer, accounting for 8%-30% of the wheat grain weight; P3 is the outer endosperm, accounting for 31%-50% of the wheat grain weight; P4 is the meso-endosperm, accounting for 51%-70% of the wheat grain weight; and P5 is the inner endosperm, accounting for 71%-100% of the wheat grain weight.
3. The method for evaluating the ability of different stratifications of wheat grains to induce Fusarium graminearum to synthesize vomitoxin, as described in claim 1, is characterized in that... Step 2) describes the preparation method of Fusarium graminearum spore suspension as follows: The strain is inoculated onto potato dextrose agar plates and cultured at 28°C for 3 days. Then, 3-5 mycelial blocks are taken from the edge of the colony using a sterile punch and transferred to 150 mL of sterilized mung bean soup medium. The medium is cultured at 28°C and 160 rpm in a constant temperature shaker for 120 h. Spores are collected and the spore concentration is adjusted to 1×10⁻⁶ using sterile distilled water. 5 per mL.
4. The method for evaluating the ability of different stratifications of wheat grains to induce Fusarium graminearum to synthesize vomitoxin, as described in claim 1, is characterized in that... The specific steps of step 2) are as follows: Weigh equal masses of wheat flour P1-P6 with different layers, add Fusarium graminearum spore liquid to each, mix thoroughly, and incubate in a constant temperature shaker at 28℃ and 160rpm for 3 days. Then, transfer to a constant temperature incubator at 25℃ for 7 days, dry at 70℃, and determine the vomitoxin yield using LC-MS.
5. The method for evaluating the ability of different stratifications of wheat grains to induce Fusarium graminearum to synthesize vomitoxin, as described in claim 1, is characterized in that... The specific steps of step 3) are as follows: collect the mycelia that have been co-cultured for 3 days in step 2), press out the water and freeze them in liquid nitrogen, extract mycelial RNA by TRIzol reagent method, amplify the three key genes TRI1, TRI5 and TRI10 involved in the synthesis of vomitoxin by real-time fluorescence quantitative PCR, and calculate the relative expression levels.
6. The method for evaluating the ability of different stratifications of wheat grains to induce Fusarium graminearum to synthesize vomitoxin, as described in claim 1, is characterized in that... The specific steps of step 4) are as follows: Wheat flour of equal mass and different layers P1-P6 are placed in the spore suspension of Fusarium graminearum. After 3 hours, samples are taken and the spore morphology and germination status are observed using a laser confocal microscope.
7. The method for evaluating the ability of different stratifications of wheat grains to induce Fusarium graminearum to synthesize vomitoxin, as described in claim 1, is characterized in that... The specific steps of step 5) are as follows: Mix equal masses of wheat flour of different layers P1-P6 thoroughly with water agar medium to prepare wheat flour-water agar medium culture plates; use a sterile punch to take a 1cm diameter mycelial cake from the edge of the colony and place it at the center of the plate; observe the colony morphology and growth rate under different culture conditions to determine the effect of different layers of wheat flour on colony growth.
8. The application of the method for evaluating the ability of different stratifications of wheat grains to induce Fusarium graminearum to synthesize vomitoxin in wheat breeding, as described in any one of claims 1-7.
9. The application according to claim 8, characterized in that, The wheat breeding program aims to select new wheat varieties that possess both resistance to Fusarium head blight and low vomitoxin accumulation.