A method for high-throughput identification of resistance to wheat scab

Abstract

The present application relates to the technical field of plant disease detection, in particular to a method for high-throughput identification of resistance to wheat scab, comprising the following steps: (1) preparing a Fusarium graminearum spore suspension; (2) selecting wheat ears at the flowering stage and immersing them in the Fusarium graminearum spore suspension to obtain inoculated ear samples; (3) randomly dividing the inoculated ear samples into an initial inoculum calibration group and a culture detection group, extracting DNA from the initial inoculum calibration group to obtain initial inoculum calibration DNA samples; (4) culturing the inoculated ear samples of the culture detection group in an incubator; after the end of the culture, washing the ear samples and extracting DNA to obtain end-point detection DNA samples; (5) performing real-time fluorescent quantitative PCR detection on the initial inoculum calibration DNA samples and the end-point detection DNA samples, respectively, to characterize the expansion of Fusarium graminearum in wheat ear tissue in terms of the relative proliferation of the pathogenic bacteria and evaluate the resistance. The method provided by the present application is suitable for batch screening of resistance to scab in wheat germplasm resources.

Description

Technical Field

[0001] This invention relates to the field of plant disease detection technology, specifically a high-throughput method for identifying wheat scab resistance. Background Technology

[0002] Fusarium head blight (FHB) is an important disease of wheat caused by pathogenic fungi such as Fusarium graminearum and Fusarium asiatica. It mainly occurs during the heading and flowering stages of wheat and is prone to spread under hot, humid, and rainy conditions. FHB not only causes necrosis of the wheat ear tissue, hinders grain filling, and reduces yield, but also leads to the accumulation of fungal toxins such as deoxynivalenol in the grains, seriously affecting the quality and safety of wheat.

[0003] Currently, wheat scab resistance identification mainly employs methods such as field inoculation identification, natural disease identification, and indoor inoculation identification. Field inoculation identification typically involves artificial inoculation of the test materials during the wheat heading and flowering stage. After the disease develops to a suitable investigation period, the scab resistance of wheat materials is evaluated by investigating indicators such as the percentage of diseased spikelets, the percentage of diseased spikelets, or the disease index. This method is widely used in wheat scab resistance breeding and germplasm resource screening. However, field identification usually involves multiple stages, including material planting, timely inoculation, disease development, and maturity investigation, resulting in a long identification cycle. It is also susceptible to environmental factors such as temperature, humidity, rainfall, and field management conditions. Furthermore, manual investigation requires counting the number of diseased spikelets on each plant or spike, which is labor-intensive. The results are easily influenced by the investigator's experience, visual judgment, and differences in investigation scale, exhibiting a degree of subjectivity and failing to meet the needs of rapid, objective, and standardized evaluation for large-scale breeding populations.

[0004] To improve identification efficiency, some studies have used real-time quantitative PCR to detect pathogen-specific genes, characterizing the degree of Fusarium graminearum infection by measuring the relative content of pathogens in wheat tissue at the culture endpoint. However, under artificial inoculation conditions, the number of initial spores received by different wheat spike samples during actual inoculation is not entirely consistent. These differences may stem from factors such as spike type, spike length, tissue surface condition, and the degree of contact between the spore suspension and the spike. Therefore, even if different materials have similar actual pathogen proliferation capabilities, the pathogen content detected at the culture endpoint may vary significantly due to different initial inoculation amounts. Conversely, samples with higher initial inoculation amounts may still show higher pathogen content even if subsequent pathogen proliferation is inhibited to some extent. If wheat scab resistance is evaluated solely based on the pathogen content at the culture endpoint, differences in detection results caused by variations in initial inoculation amounts may be mistakenly interpreted as differences in pathogen proliferation capabilities between materials, leading to biased resistance evaluation results.

[0005] Therefore, how to correct for differences in initial inoculation amounts among different samples based on rapid quantitative detection of pathogen content, so as to more accurately reflect the actual relative proliferation of Fusarium graminearum in wheat tissues, is an urgent problem to be solved in the rapid and accurate identification of wheat scab resistance. Summary of the Invention

[0006] In view of the problems existing in the prior art, the present invention provides a high-throughput method for identifying wheat scab resistance.

[0007] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0008] A high-throughput method for identifying wheat scab resistance includes the following steps:

[0009] (1) Preparation of Fusarium graminearum spore suspension;

[0010] (2) Select wheat ears at the flowering stage, immerse the wheat ears in a suspension of Fusarium graminearum spores, remove them and hang them vertically on a sterile rack to drain the liquid, and obtain inoculated ear samples;

[0011] (3) The inoculated spikelet samples were randomly divided into an initial inoculation amount correction group and a culture and detection group. DNA was extracted from the initial inoculation amount correction group to obtain the initial inoculation amount correction DNA sample.

[0012] (4) The inoculated ear samples of the culture and detection group were placed in an incubator for culture. After the culture was completed, the ear samples were washed, and then the liquid on the surface of the ear was removed to obtain the washed ear samples. DNA was extracted to obtain the DNA sample for endpoint detection.

[0013] (5) The initial inoculation amount correction DNA sample and the endpoint detection DNA sample were subjected to real-time fluorescence quantitative PCR detection. The initial relative content and endpoint relative content of pathogens were calculated based on the target pathogen gene and the plant internal reference gene. The relative proliferation of pathogens was calculated. The relative proliferation of pathogens was used to characterize the extent of Fusarium graminearum expansion in wheat ear tissue and to evaluate resistance.

[0014] Initial relative content:

[0015] ;

[0016] Relative content at endpoint:

[0017] ;

[0018] Relative proliferation of pathogens:

[0019] ;

[0020] in, and The copy numbers of the OSP24 and Actin genes in the DNA sample were corrected based on the initial inoculum amount, respectively. and The endpoints are the copy numbers of the OSP24 and Actin genes in the DNA sample, respectively.

[0021] Furthermore, the concentration of the Fusarium graminearum spore suspension was 5 × 10⁻⁶. 5 per mL.

[0022] Furthermore, in step (2), the wheat ears are immersed in the Fusarium graminearum spore suspension for 30-60 seconds.

[0023] Furthermore, in step (4), the culture conditions for the inoculated spikelet samples of the culture and detection group are 25℃ and 95% relative humidity for 48h.

[0024] Furthermore, in step (4), after removing the awns from the ear of wheat, place it in a sterile centrifuge tube, add sterile PBS buffer, shake and wash for 30 seconds, discard the washing solution, and repeat the washing 1-3 times.

[0025] This invention provides a method for high-throughput identification of wheat scab resistance, and its application in plant disease detection.

[0026] The beneficial effects of adopting the above technical solution include:

[0027] This invention provides a high-throughput method for identifying wheat scab resistance. By setting an initial inoculum size to calibrate the sample, the relative pathogen content at the initial inoculation stage and the end of the culture are detected separately, and the relative pathogen proliferation is calculated. This avoids misjudgments caused by differences in the initial inoculum size when evaluating solely based on the pathogen content at the end of the culture. Simultaneously, this invention uses PBS buffer to wash the scion samples after culture to remove free spores and residual bacterial solution from the surface of the scion, reducing interference from exogenous spores on the quantitative results of pathogen detection and improving the accuracy and repeatability of the test results.

[0028] This invention combines the relative proliferation of pathogens with the detection of resistance-related molecular markers Fhb1, Fhb2, Fhb5, and QFhs.crc-2DL. The results of molecular marker detection are used to assist in the verification of resistance phenotype evaluation results, demonstrating high consistency and reliability between the two methods. The wheat scab resistance identification method provided by this invention is highly quantitative, has good repeatability, low subjective error, and is suitable for batch screening of wheat germplasm resources. Attached Figure Description

[0029] Figure 1 This is a flowchart of the high-throughput method for identifying wheat scab resistance according to the present invention. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] See attached document Figure 1 This invention provides a high-throughput method for identifying wheat scab resistance.

[0032] Fusarium graminearum (CGMCC 3.4521) was purchased from the China General Microbiological Culture Collection Center.

[0033] Example 1

[0034] (1) Preparation of Fusarium graminearum spore suspension

[0035] Select Fusarium graminearum mycelium blocks and inoculate them onto PDA medium. Incubate at 25-28℃ for 3-5 days until the colonies have fully grown. Then, select a small amount of mycelium and inoculate it onto mung bean soup medium. Incubate on a shaker at 28℃ for 3-5 days until conidia are produced. Adjust the spore concentration to 5 × 10⁻⁶ using sterile water. 5 A suspension of Fusarium graminearum spores was prepared by spores / mL.

[0036] (2) Wheat ear inoculation

[0037] Wheat ears at the flowering stage were selected, with Sumai No. 3, Wangshuibai, and Yangmai 158 chosen as experimental varieties. Ten to fifteen ears of each variety were randomly selected, retaining 3-5 cm of the stem below the rachis during harvesting. The harvested ears were grouped according to ear length and fresh weight, discarding those that were significantly too small or too large. The wheat ears were then completely immersed in a Fusarium graminearum spore suspension for 30-60 seconds, gently agitating the ears during immersion to ensure full contact between the spore suspension and the ear tissue. After removal, the ears were vertically suspended on a sterile rack and drained for 2-3 minutes to obtain inoculated ear samples.

[0038] (3) Preparation of initial inoculum amount calibration samples

[0039] The inoculated spikelets of each variety were randomly divided into an initial inoculation amount correction group and a culture and testing group. Three spikelets were randomly selected as initial inoculation amount correction samples, denoted as T0, and the remaining spikelets were used as culture and testing samples, denoted as T1.

[0040] The T0 samples were placed on sterile filter paper to absorb surface liquid. 100 mg of ear tissue was weighed and ground into powder in liquid nitrogen to obtain initial tissue samples. DNA was extracted from these initial tissue samples using a uniform sample weight, lysis system, and elution volume to obtain initial inoculum correction DNA samples. Three T0 samples were prepared for each variety and tested separately. The average value was calculated based on the test results of each T0 sample and used as the initial inoculum correction value for the corresponding variety.

[0041] (4) Cultivation and cleaning of inoculated spikelets

[0042] The inoculated ear samples for the culture and testing group were placed in an incubator and cultured for 48 hours at 25℃ and 95% relative humidity. During culture, a certain distance was maintained between the ear samples to avoid cross-contamination. After culture, the awns were removed from the ear samples, which were then placed in sterile centrifuge tubes. Sterile PBS buffer was added, with the volume of PBS buffer not exceeding the base of the ear sample. The samples were gently shaken and washed for 30 seconds, then the washing solution was discarded. This washing process was repeated twice. Subsequently, the liquid on the surface of the ear was absorbed using sterile filter paper to obtain the washed ear samples. After washing, 100 mg of ear tissue was weighed, ground in liquid nitrogen, and total DNA was extracted to obtain the DNA sample for endpoint detection.

[0043] (5) Quantitative detection of relative biomass of pathogens and resistance analysis

[0044] Using the OSP24 gene of Fusarium graminearum as the quantitative target for pathogen detection and the Actin gene of wheat as the plant internal reference gene, standard curves for the OSP24 gene and Actin gene were established respectively. The initial inoculum-corrected DNA samples and the endpoint detection DNA samples were subjected to real-time quantitative PCR detection. The Ct values ​​were converted to the corresponding gene copy numbers according to their respective standard curves. Three replicate wells were set for each detection.

[0045] The initial relative content is calculated according to the following formula:

[0046] ;

[0047] The relative content at the endpoint is calculated using the following formula:

[0048] ;

[0049] The relative proliferation of pathogens is calculated using the following formula:

[0050] ;

[0051] in, and The copy numbers of the OSP24 and Actin genes in the DNA sample were corrected based on the initial inoculum amount, respectively. and The endpoints are the copy numbers of the OSP24 and Actin genes in the DNA sample, respectively.

[0052] (7) Detection of tightly linked molecular markers for resistance genes

[0053] Eight markers closely linked to Fhb1, Fhb2, Fhb4, Fhb5, and QFhs.crc-2DL were used to detect the PCR sites in four different wheat varieties. For Fhb1, Sumai 3 was used as a positive control, with TaHRC-STS as the flanking marker. For Fhb2, Fhb4, and Fhb5, Wangshuibai was used as a positive control, with WMC398, WGRB969, GWM513, GWM149, MAG9482, and WMC752 as flanking markers, respectively. Yangmai 158 was used as a positive control for the QFhs.crc-2DL site. Primers were synthesized by Beijing Bomaide Biotechnology Co., Ltd. The PCR reaction volume was 12 μL, containing 10 μL of 1.1×T3 Super PCR Mix (Beijing Qingke Biotechnology Co., Ltd.) and forward and reverse primers (both at a concentration of 10 μmol·L⁻¹). -1 0.5 μL each of template DNA (200 ng / μL) -1 1 μL. PCR amplification program: 98℃ pre-denaturation for 2 min, 98℃ denaturation for 10 s, 55℃-61℃ annealing for 10 s, 72℃ extension for 10 s, for a total of 35 cycles; 72℃ extension for 2 min, storage at 4℃. Fhb1 PCR products were detected by agarose gel electrophoresis, and PCR products of other genes were detected by 8% non-denaturing polyacrylamide gel electrophoresis, observed by silver staining.

[0054] Example 2

[0055] The difference between Example 2 and Example 1 is that after the inoculated ear sample culture is completed, step (4) cleaning is not performed, and the liquid on the surface of the ear is directly removed. All other steps are the same.

[0056] Example 3

[0057] The difference between Example 3 and Example 1 is that in step (4), the inoculated spikelet sample is washed once with sterile PBS buffer for 30 seconds, while the other steps are the same.

[0058] Example 4

[0059] The difference between Example 4 and Example 1 is that in step (4), the inoculated spikelet samples are washed three times with sterile PBS buffer for 30 seconds, while the other steps are the same.

[0060] Experimental results

[0061] 1. The endpoint DNA extracted in Examples 1-4 was tested to evaluate the effect of different washing cycles on the removal of residual non-infectious spore DNA on the ear surface. The results are shown in Table 1.

[0062] The results showed that in Example 2, due to the lack of washing, a large number of ungerminated and infecting spores remained on the surface, leading to the detection of... The data is artificially inflated and has poor intragroup repeatability. >20%). Washing the scion samples twice effectively removed spores physically adhering to the surface, resulting in the best repeatability.

[0063] Table 1. Effects of different washing treatments on the endpoint pathogen detection amount and coefficient of variation.

[0064]

[0065] 2. The effect of initial inoculation amount correction on the results of wheat scab resistance identification is shown in Table 2.

[0066] The initial spore attachment amount of *Wangshuibai* was significantly higher than that of *Sumai 3*. If only the endpoint is considered... The value of *Pseudomonas aeruginosa* is easily misjudged as "medium-resistant"; however, the relative proliferation rate using this invention... After initial correction, the difference in initial slurry application was successfully deducted, truly reflecting that Wangshui Bai and Sumai No. 3 belong to the same high-resistance type, which greatly improved the accuracy of high-throughput screening.

[0067] Table 2 Results of wheat scab resistance identification after initial inoculum amount correction

[0068]

[0069] 3. Results of high-throughput resistance identification of different wheat varieties

[0070] The results are shown in Table 3, showing the relative proliferation of material 1 under test. The value was 0.095±0.011, which was relatively low, indicating high resistance. It also showed positive molecular markers for both Fhb1 and Fhb5. The relative proliferation of test material 4... The value was 0.112±0.014, also indicating high resistance, and the QFhs.crc-2DL positive molecular marker was detected; the relative proliferation of test material 2 was... The value was 0.241±0.023, indicating moderate resistance, and the Fhb2 positive molecular marker was detected; the relative proliferation of test material 3 was... The highest value was 0.782 ± 0.052, indicating susceptibility to disease, and no resistance-related molecular markers were detected. These results demonstrate that this invention, by determining the relative proliferation of *Fusarium graminearum* in wheat spike tissue, [achieves its intended purpose]. This method can distinguish the level of Fusarium head blight resistance in different wheat materials. Further verification was performed using resistance-related molecular markers Fhb1, Fhb2, Fhb5, and QFhs.crc-2DL. The results of molecular marker detection showed high consistency with the resistance phenotype evaluation results. Therefore, the wheat Fusarium head blight resistance identification method provided by this invention has good accuracy, reliability, and application value.

[0071] Table 3. Fusarium head blight resistance identification of each tested material

[0072]

[0073] Note: "--" indicates not detected.

[0074] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A high-throughput method for identifying wheat scab resistance, characterized in that, Includes the following steps: (1) Preparation of Fusarium graminearum spore suspension; (2) Select wheat ears at the flowering stage, immerse the wheat ears in the Fusarium graminearum spore suspension, drain the liquid, and obtain inoculated ear samples; (3) The inoculated spikelet samples were randomly divided into an initial inoculation amount correction group and a culture detection group. DNA was extracted from the initial inoculation amount correction group to obtain the initial inoculation amount correction DNA sample. (4) The inoculated ear samples of the culture and detection group were placed in an incubator for culture. After the culture was completed, the ear samples were washed, and then the liquid on the surface of the ear was removed to obtain the washed ear samples. DNA was extracted to obtain the DNA sample for endpoint detection. (5) The initial inoculation amount correction DNA sample and the endpoint detection DNA sample were respectively subjected to real-time fluorescence quantitative PCR detection. The initial relative content and endpoint relative content of pathogens were calculated based on the target pathogen gene and the plant internal reference gene. The relative proliferation of pathogens was calculated. The relative proliferation of pathogens was used to characterize the extent of Fusarium graminearum expansion in wheat ear tissue and to evaluate resistance. Initial relative content: ； Relative content at endpoint: ； Relative proliferation of pathogens: ； in, and The copy numbers of the OSP24 and Actin genes in the DNA sample were corrected based on the initial inoculum amount, respectively. and The endpoints are the copy numbers of the OSP24 and Actin genes in the DNA sample, respectively.

2. The method for high-throughput identification of wheat scab resistance according to claim 1, characterized in that, The concentration of the *Fusarium graminearum* spore suspension was 5 × 10⁻⁶. 5 per mL.

3. The method for high-throughput identification of wheat scab resistance according to claim 1, characterized in that, Step (2) The wheat ears are immersed in the Fusarium graminearum spore suspension for 30-60 seconds.

4. The method for high-throughput identification of wheat scab resistance according to claim 1, characterized in that, In step (4), the culture conditions for the inoculated spikelet samples of the culture and detection group are 25℃ and 95% relative humidity for 48 h.

5. The method for high-throughput identification of wheat scab resistance according to claim 1, characterized in that, In step (4), after removing the awns from the ear of wheat, place it in a sterile centrifuge tube, add sterile PBS buffer, shake and wash for 30 seconds, discard the washing solution, and repeat the washing 1-3 times.

6. The application of the high-throughput method for identifying wheat scab resistance according to any one of claims 1-5 in plant disease detection.

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