Evaluation method and application of mesosulfuron-methyl herbicide-resistant wheat

Abstract

The invention discloses an evaluation method and application of mesosulfuron-methyl herbicide-resistant wheat, and belongs to the technical field of wheat breeding. The method comprises the following steps: performing endogenous hormone content detection, specific metabolite content detection, wheat plant height, flag leaf form, grain yield and quality index detection on to-be-evaluated wheat sprayed with mesosulfuron-methyl, and comparing a detection result with a non-pesticide-applied control or a known variety; and comprehensively evaluating the tolerance of the wheat to mesosulfuron-methyl. According to the invention, a set of multi-index comprehensive evaluation system is constructed. The method effectively overcomes the defects that a traditional method depends on subjective field observation, evaluation lags behind and indexes are single, achieves objective identification of mesosulfuron-methyl resistance of wheat varieties, and provides reliable technical support for rapid screening of resistant varieties, early evaluation of breeding materials and safe field medication. The invention also provides application of the method in preparation of a detection kit.

Description

Technical Field

[0001] This invention belongs to the field of wheat breeding technology, specifically an evaluation method for wheat resistant to mesosulfuron-methyl herbicide and its application. Background Technology

[0002] Wheat, as one of the most important food crops, has its production level directly affecting food security. Weeds in wheat fields not only compete with wheat for growth resources such as water, fertilizer, and sunlight, but also act as a bridge between pests and diseases, seriously harming wheat yield and quality. Although chemical weeding has become the primary control method in wheat production due to its advantages of high efficiency, time-saving, and labor-saving, the problem of herbicide damage is becoming increasingly prominent with the large-scale and continuous use of herbicides and can no longer be ignored.

[0003] Among numerous chemical herbicides, mesosulfuron-methyl, a systemic sulfonylurea herbicide, exerts its weed-control effect by inhibiting the activity of acetolactate synthase, showing significant efficacy against various noxious grassy weeds in wheat fields, such as jointed goatgrass, wild oat, and bromegrass. However, this herbicide poses varying degrees of phytotoxicity risks to different wheat varieties. Differences in tolerance among varieties and the effects of environmental stress can lead to growth inhibition, leaf deformities, and even yield reduction in sensitive varieties.

[0004] Currently, the methods for evaluating wheat tolerance to mesosulfuron-methyl herbicide mainly rely on field symptom observation or final yield determination. These traditional methods have significant shortcomings: First, field symptom observation is difficult to achieve objective and quantitative assessment, not only because observers have varying levels of experience and subjective judgment standards, but also because visual observation cannot capture subtle changes that are difficult to identify. Relying solely on morphological indicators and yield to judge wheat tolerance to mesosulfuron-methyl herbicide results in inaccurate results and involves a large workload of measurement and statistical work. Second, existing evaluation systems mostly focus on visible morphological indicators, ignoring changes in endogenous physiological indicators of plants in the early stages of mesosulfuron-methyl stress, and there is still a lack of comprehensive evaluation methods that can integrate multi-dimensional and multi-period indicators. Summary of the Invention

[0005] To overcome the aforementioned deficiencies of existing technologies, this invention for the first time reveals and applies the correlation between changes in the content of specific metabolites and endogenous hormones such as gibberellin, indoleacetic acid, and abscisic acid and the tolerance of wheat to mesosulfuron-methyl, using these as one of the early and sensitive evaluation indicators. Combined with physiological phenotype and other detection indicators, this invention aims to provide an early and systematic comprehensive evaluation method for wheat resistant to mesosulfuron-methyl herbicide.

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

[0007] An evaluation method for wheat resistant to mesosulfuron-methyl herbicide includes the following steps performed sequentially:

[0008] S1. Stress treatment: Apply mesosulfuron-methyl to the wheat to be evaluated during the greening stage;

[0009] S2. Physicochemical index detection: On days 10-15 after application of mesosulfuron-methyl, the content of endogenous hormones and metabolites were detected.

[0010] S3. Phenotypic index detection: Phenotypic index detection shall be carried out on days 18 to 24 after application;

[0011] S4. Yield and quality index testing: Harvest the wheat to be evaluated and conduct yield and quality index testing;

[0012] S5. Comprehensive evaluation: Take the test results of the wheat to be evaluated and compare them with the control group of wheat that has not been treated or wheat known to be resistant to mesosulfuron-methyl herbicide to evaluate the tolerance of the wheat to be evaluated to mesosulfuron-methyl herbicide;

[0013] The endogenous hormones include gibberellin, indoleacetic acid, and abscisic acid;

[0014] The metabolites include creatine phosphate disodium salt and cystine.

[0015] The inventors unexpectedly discovered a correlation between changes in the levels of endogenous hormones gibberellin, indoleacetic acid, and abscisic acid, as well as metabolites creatine phosphate disodium salt and cystine, and wheat tolerance to mesosulfuron-methyl, providing clues for the early diagnosis of mesosulfuron-methyl herbicide damage.

[0016] Preferably, the physicochemical indicators are tested on the 14th day after the application of mesosulfuron-methyl;

[0017] Phenotypic markers were measured on day 21 after administration of mesosulfuron-methyl;

[0018] One method involved spraying 30 g / L mesosulfuron-methyl dispersible oil suspension twice, after the wheat turned green and before the jointing stage, with a 15-day interval between the two applications.

[0019] Furthermore, the physicochemical indicators were tested on wheat leaves. Specifically, wheat leaves were taken, wrapped in aluminum foil, and quickly placed in liquid nitrogen. Dry ice was used to maintain their low temperature for testing.

[0020] Endogenous hormones were detected using HPLC.

[0021] As a limitation of the present invention, in step S5, the wheat to be evaluated that has a smaller decrease in gibberellin content and a smaller increase in indoleacetic acid and abscisic acid content compared with sensitive varieties or untreated control groups is evaluated as mesosulfuron-methyl herbicide-resistant wheat.

[0022] As a further limitation of the present invention, in step S5, the wheat to be evaluated that has a smaller increase in creatine phosphate disodium salt content and a smaller decrease in cystine content compared with sensitive varieties or untreated control groups is evaluated as mesosulfuron-methyl herbicide-resistant wheat.

[0023] As a further limitation of the present invention, the phenotypic indicators include wheat plant height and flag leaf morphology;

[0024] The yield and quality indicators are wheat grain yield, thousand-grain weight, number of grains per ear, number of ears, and test weight.

[0025] Furthermore, the method for detecting wheat plant height is to measure the length from the ground to the tip of the main ear;

[0026] Wheat was harvested in time according to the experimental plots at the full maturity stage, and the yield was measured by actual harvesting. Two 500-grain samples were taken, weighed, and the thousand-grain weight was calculated, with the error between the two measurements controlled within 0.4g. The bulk density was measured using a GHCS-1000 bulk density meter, and three grains were randomly selected and the average value was taken.

[0027] The detection indicators for flag leaf morphology include the area, perimeter, length, and width of the flag leaf.

[0028] Furthermore, the height of the plant is not included in the awn height. During the later stage of wheat grain filling, three points are randomly selected from each experimental plot to measure the plant height, and the average value is taken to represent the plant height of the wheat variety to be evaluated.

[0029] Preferably, the method for determining the morphology of wheat flag leaves is as follows: in mid-to-late May, 20 flag leaves are selected from each experimental plot, and the area, perimeter, length and width of the flag leaves are measured using a Yaxin-1241 leaf area meter, and the average value is taken after measurement.

[0030] The flag leaf is a vital organ for wheat's photosynthesis, respiration, and transpiration, and a key factor determining wheat yield. Varieties with poor resistance to mesosulfuron-methyl exhibit leaf deformities after application, thus impacting wheat yield. Therefore, flag leaf area, perimeter, length, and width can serve as effective evaluation indicators for assessing mesosulfuron-methyl tolerance.

[0031] As a further limitation of the present invention, analysis of variance was performed on the test results of physicochemical indicators, phenotypic indicators, and yield and quality indicators to compare the significance level of differences. Specifically, the analysis of variance was performed using DPS statistical analysis software, and a p-value < 0.05 was considered statistically significant.

[0032] As a further limitation of the present invention, wheat is harvested at full maturity, and the actual yield is used to test yield and quality indicators.

[0033] This invention also provides the application of the above evaluation method in the preparation of a kit for detecting tolerability to mesosulfuron-methyl.

[0034] This invention also provides a test kit for evaluating wheat tolerance to mesosulfuron-methyl herbicide, the kit comprising reagents for specifically detecting the levels of endogenous hormones and metabolites in wheat samples:

[0035] The endogenous hormones are gibberellin, indoleacetic acid, and abscisic acid;

[0036] The metabolites are creatine phosphate disodium salt and cystine;

[0037] The kit uses the above evaluation method as the criterion for determining tolerance to mesosulfuron-methyl herbicide.

[0038] By adopting the above technical solution, the technical progress achieved by this invention compared with the prior art is as follows:

[0039] This invention provides an early, systematic, and multi-dimensional evaluation method for wheat resistant to mesosulfuron-methyl herbicide. The determined detection timing is clear, and the operation procedure is standardized. This method establishes for the first time the correlation between changes in the content of three endogenous hormones (gibberellin, indoleacetic acid, and abscisic acid) and two metabolites (creatine phosphate disodium salt and cystine) and the mesosulfuron-methyl resistance of wheat, and uses these as core evaluation indicators. This enables early and objective detection of varietal tolerance, and evaluates and identifies mesosulfuron-methyl resistant wheat varieties Shi 4366 and Shi Mai 30, as well as the relatively mesosulfuron-methyl-sensitive variety Gaoyou 2018.

[0040] This invention establishes a comprehensive evaluation system covering the entire growth cycle by systematically integrating the aforementioned early physiological and biochemical response indicators, mid-term morphological indicators, and final yield and quality indicators. Mid-term phenotypic results showed that mesosulfuron-methyl stress caused significant changes in flag leaf morphology in different varieties, and plant height was inhibited to varying degrees. Yield analysis indicated that the sensitive variety 'Gaoyou 2018' experienced significant yield reduction even at the recommended dosage, while the yield and quality indicators of the tolerant varieties 'Shi 4366' and 'Shimai 30' were less affected.

[0041] The evaluation method of this invention can distinguish the tolerance levels of different wheat varieties to mesosulfuron-methyl. For example, 'Shi 4366' and 'Shi Mai 30' show strong tolerance, 'Shi Luan 02-1' is second, and 'Gao You 2018' is sensitive. This provides a reliable and efficient technical means for the rapid screening of tolerant varieties, the early identification of breeding materials, and the scientific application of pesticides in the field. Attached Figure Description

[0042] Figure 1The image shows the flag leaf of wheat in the water control plot of Example 2 of this invention. From left to right, the wheat varieties are Shi 4366, Shi Luan 02-1, Gao You 2018 and Shi Mai 30.

[0043] Figure 2 0.9 L / hm in Example 2 of this invention 2 Photographs of wheat flag leaves in the mesosulfuron-methyl treated plots. From left to right, the wheat varieties are Shi 4366, Shi Luan 02-1, Gaoyou 2018, and Shimai 30.

[0044] Figure 3 The photos show individual wheat plants in the water control plot of Example 2 of this invention. From left to right, the wheat varieties are Shi 4366, Shi Luan 02-1, Gao You 2018, and Shi Mai 30.

[0045] Figure 4 0.9 L / hm in Example 2 of this invention 2 Photographs of individual wheat plants in a plot treated with mesosulfuron-methyl. From left to right, the wheat varieties are Shi 4366, Shi Luan 02-1, Gao You 2018, and Shi Mai 30. Detailed Implementation

[0046] The present invention will be further described in detail below through specific embodiments. It should be understood that the described embodiments are only for explaining the present invention and do not limit the present invention.

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0048] Mesosulfuron-methyl herbicide was a 30 g / L mesosulfuron-methyl dispersible oil suspension, purchased from Bayer Crop Science China Co., Ltd.; flag leaf morphology was detected using a Yaxin-1241 leaf area meter from Beijing Yaxin; endogenous hormone content was detected using a Thermo Fisher Scientific U3000 HPLC; bulk density was measured using a GHCS-1000 bulk density meter; metabolites were freeze-dried in liquid nitrogen, stored on dry ice, and express-shipped to Wuhan Metawell Biotechnology for metabolite analysis.

[0049] Example 1

[0050] An evaluation method for wheat resistant to mesosulfuron-methyl herbicide includes the following steps performed sequentially:

[0051] S1. Stress treatment: During the greening stage of the wheat varieties 'Shimai 30' and 'Gaoyou 2018', 30 g / L mesosulfuron-methyl dispersible oil suspension was prepared for stress treatment. The dosage was the conventional recommended dosage of 0.45 L / hm². A control plot was set up by spraying an equal amount of water.

[0052] S2. Physicochemical index testing: On the 14th day after the herbicide was sprayed, wheat flag leaves were collected, wrapped in aluminum foil, and quickly frozen in liquid nitrogen and stored in a dry ice environment.

[0053] Samples for metabolomics testing were sent to a professional testing institution for non-targeted metabolomics analysis using liquid chromatography-mass spectrometry to obtain data on the content of creatine phosphate disodium salt and cystine.

[0054] For samples used for endogenous hormone detection, the contents of gibberellin, indoleacetic acid, and abscisic acid were determined by high performance liquid chromatography.

[0055] S3. Phenotypic index detection: On day 21 after herbicide application, morphological indexes were measured. The specific indexes and methods are as follows:

[0056] Plant height measurement: Three points were randomly selected in each experimental plot to measure the vertical height from the ground to the top of the main ear. The measurement range did not include the awn, and the average value was calculated.

[0057] Flag leaf morphology measurement: 20 flag leaves were randomly selected from each plot, and the area, perimeter, length and width of each leaf were measured using a Yaxin-1241 leaf area meter. The average value of each index was calculated.

[0058] S4. Yield and Quality Indicators Testing: After wheat reaches full maturity, it is harvested, threshed, and weighed individually by plot to obtain grain yield. Random samples are taken from each plot, and two 500-grain samples are weighed to calculate the thousand-grain weight. The error between the two weighings should not exceed 0.4g. The grain bulk density is measured three times randomly using a GHCS-1000 bulk density meter, and the average value is taken. The number of grains per ear and the number of ears are observed, counted, and recorded.

[0059] S5. Comprehensive evaluation: All indicator data obtained by the wheat to be evaluated in steps S2, S3, and S4 are compared with the corresponding data of the water control group using DPS statistical analysis software for variance analysis.

[0060] Among them, the results of metabolite detection showed that in the leaves of the sensitive variety 'Gaoyou 2018' treatment group, the content of creatine phosphate disodium salt was significantly increased and the content of cystine was significantly decreased compared with its water control; while in the leaves of the tolerant variety 'Shimai 30' treatment group, the changes in these two metabolites were significantly smaller than those in 'Gaoyou 2018'.

[0061] The results of endogenous hormone detection showed that the gibberellin content in the leaves of the 'Gaoyou 2018' treatment group was significantly lower than that of the control, while the indoleacetic acid and abscisic acid content were significantly increased. In contrast, the changes in the content of these three hormones in the leaves of the 'Shimai 30' treatment group were much smaller than those of 'Gaoyou 2018'.

[0062] The plant height measurement results showed that the average plant height of the 'Gaoyou 2018' treatment group was significantly lower than that of its water control group; while the plant height of the 'Shimai 30' treatment group was not significantly different from that of the control group.

[0063] The results of flag leaf morphology measurements showed that the flag leaf area, perimeter, length and width of the 'Gaoyou 2018' treatment group were significantly increased compared with the control, showing a trend of deformity; while the flag leaf morphology indicators of the 'Shimai 30' treatment group showed no significant changes compared with the control.

[0064] The grain yield of the 'Gaoyou 2018' treatment group was significantly lower than that of its water control, and the thousand-grain weight and test weight were also significantly reduced. The grain yield, thousand-grain weight and test weight of the 'Shimai 30' treatment group were not significantly different from those of its water control, and the yield loss rate was extremely low.

[0065] In summary, under mesosulfuron-methyl stress, 'Gaoyou 2018' initially showed decreased levels of specific endogenous hormones, including gibberellin, and increased levels of indoleacetic acid and abscisic acid, along with increased levels of the metabolite creatine disodium phosphate and decreased cystine levels. In the mid-term, plant height was significantly inhibited, and flag leaf morphology became distorted. Ultimately, yield and quality both significantly decreased. In contrast, 'Shimai 30' under the same stress showed only slight changes in early physiological and biochemical indicators; stable morphological indicators in the mid-term; and ultimately, good yield and quality.

[0066] Therefore, 'Gaoyou 2018' was evaluated as a variety sensitive to mesosulfuron-methyl, while 'Shimai 30' was evaluated as a variety tolerant to mesosulfuron-methyl.

[0067] This embodiment demonstrates, by comparing the wheat varieties 'Shimai 30' and 'Gaoyou 2018', that the evaluation method of the present invention can effectively distinguish the tolerance of wheat varieties to mesosulfuron based on the systematic changes in early physiological and biochemical indicators, mid-term morphological indicators, and final yield and quality indicators.

[0068] Example 2

[0069] This embodiment demonstrates the practical application effect of the evaluation method of the present invention using field trial data of specific varieties. The experiment was conducted at the Zhaoxian Experimental Station of the Shijiazhuang Academy of Agricultural and Forestry Sciences. Photos of wheat flag leaves or individual plants were collected 21 days after the second application of mesosulfuron-methyl, as shown below. Figures 1-4 .

[0070] Tested varieties: Shi 4366, Shi Luan 02-1, Gao You 2018, Shi Mai 30.

[0071] Experimental design: A split-plot design was adopted, with a plot area of ​​12.6 m². 2The plots were 9.0m long and 1.4m wide, with a row spacing of 15cm. The basic seedling density was 220,000 seedlings per mu (approximately 14,000 hectares). Sowing was carried out under sufficient soil moisture. A three-factor split-plot design was used, with the main factor being four wheat varieties. The application time was designated as the split plot, with two application periods: early greening stage (March 9th) and after greening to before jointing (March 25th). The dosage was designated as the sub-split plot, with the conventional recommended dosage of mesosulfuron-methyl being 0.45 L / hm². 2 , 2 times the test dose, 0.9 L / hm 2 Three levels were established, including a control group (Ck group) and a water control group, while other fields were managed according to standard practices.

[0072] The evaluation process is conducted in the following steps:

[0073] S1. Stress treatment: According to the experimental design, each plot was treated with either mesosulfuron-methyl or water spray at different times. The water spray was used as the control group without treatment.

[0074] S2. Physicochemical index testing: On the 14th day after the herbicide was sprayed, wheat flag leaves were collected, wrapped in aluminum foil, and quickly frozen in liquid nitrogen and stored in a dry ice environment.

[0075] Samples for metabolomics testing were sent to a professional testing institution for non-targeted metabolomics analysis using liquid chromatography-mass spectrometry to obtain data on the content of creatine phosphate disodium salt and cystine.

[0076] For samples used for endogenous hormone detection, the contents of gibberellin, indoleacetic acid, and abscisic acid were determined by high performance liquid chromatography.

[0077] The test results showed that 'Gaoyou 2018' was the most sensitive to mesosulfuron-methyl. Compared with the untreated control group (water group), the wheat variety exhibited a greater upregulation of creatine phosphate disodium salt, a greater downregulation of cystine, a greater decrease in gibberellin content, and a greater increase in indoleacetic acid and abscisic acid content. Therefore, it was evaluated as a mesosulfuron-methyl tolerant wheat. See Tables 1-2 for details.

[0078] Table 1. Changes in the content of metabolites of 'Gaoyou 2018' compared to the untreated control group

[0079] Table 2. Changes in endogenous hormone levels in 'Gaoyou 2018' compared to the untreated control group.

[0080] Tables 1 and 2 show that spraying 0.9 L / hm of mesosulfuron-methyl... 2 Subsequently, creatine disodium phosphate was significantly upregulated by 23.25-fold, while cystine was significantly downregulated compared to the control, reaching only 8.7 × 10⁻⁶ of the control. -2The gibberellin content of 'Gaoyou 2018' wheat was significantly reduced by 18.20% compared with the control, while the indoleacetic acid and abscisic acid content were significantly increased by 965.17% and 91.74% respectively. Gibberellin, indoleacetic acid, abscisic acid, creatine phosphate disodium salt, and cystine can be used as biomarkers for detecting the effects of mesosulfuron-methyl on wheat.

[0081] S3. Phenotypic index detection: On day 21 after herbicide application, morphological indexes were measured. The specific indexes and methods are as follows:

[0082] (a) Plant height measurement:

[0083] Three points were randomly selected from each experimental plot to measure the vertical height from the ground to the tip of the main ear. The measurement range did not include the awn, and the average value was calculated. The plant height measurement results are shown in Table 3.

[0084] Table 3. Results and significance analysis of plant height measurement

[0085]

[0086] In Table 3, data in the same column followed by different lowercase letters indicate significant differences between treatments (P<0.05), where *: P<0.05; **: P<0.01, and the same applies to the following tables.

[0087] As shown in Table 3, the timing of application, dosage, and their interaction effects all had extremely significant (P<0.01) effects on wheat plant height, but the degree of inhibition varied among different varieties.

[0088] After application on March 9, the plant height of 'Gaoyou 2018' was significantly reduced compared to the water control at the recommended dose of mesosulfuron-methyl, with an inhibition rate of 5.8%. The plant height of the other three varieties, 'Shi 4366', 'Shiluan 02-1', and 'Shimai 30', did not decrease significantly. The treatment with twice the recommended dose of mesosulfuron-methyl showed the most significant inhibition rate on the plant height of 'Gaoyou 2018', reaching 11.3%, followed by 'Shi 4366', with an inhibition rate of 5.1%. The treatment had no significant effect on the plant height of 'Shiluan 02-1' and 'Shimai 30'.

[0089] After application on March 25, at the recommended dose of mesosulfuron-methyl, the plant height of 'Shi 4366' and 'Shi Mai 30' was not significantly affected. The plant height of the other two varieties, 'Shi Luan 02-1' and 'Gao You 2018', was significantly reduced. 'Gao You 2018' showed the most significant inhibition rate, reaching 11.5%, while the effect on 'Shi Luan 02-1' was relatively small, with an inhibition rate of 5.2%. As the dosage increased, this inhibitory effect gradually strengthened, and the plant height of all four varieties decreased significantly. The treatment with twice the recommended dose of mesosulfuron-methyl showed the most significant inhibition rate on 'Gao You 2018', reaching 37.2%, followed by 'Shi Luan 02-1' at 28.5%, and 'Shi Mai 30' and 'Shi 4366' both at 6.3%.

[0090] The above results indicate that mesosulfuron-methyl has varying effects on plant height among different varieties. Even at the conventional recommended dosage, the plant height inhibition rate of Gaoyou 2018 was over 5.8%. This further demonstrates that Gaoyou 2018 is relatively sensitive to mesosulfuron-methyl, followed by Shilu 02-1; the plant height inhibition rate differed significantly between conventional dosage treatment and delayed application. In contrast, Shimai 30 and Shi 4366 showed stronger tolerance, with no significant difference in plant height between the two application periods under conventional dosage treatment.

[0091] (II) Measurement of flag leaf morphology:

[0092] Twenty flag leaves were randomly selected from each plot, and the area, perimeter, length, and width of each leaf were measured using a Yaxin-1241 leaf area meter. The average values ​​of each indicator were calculated. The results of the flag leaf morphology measurements are shown in Table 4.

[0093] Table 4. Results of flag leaf morphology measurement and significance analysis

[0094]

[0095] As shown in Table 4, the timing of pesticide application, the dosage of pesticide application, and their interaction effects all had extremely significant (P<0.01) effects on wheat leaf area, perimeter, leaf length, and leaf width, and these effects varied among different varieties.

[0096] After application on March 9th, at the recommended dose of mesosulfuron-methyl, the leaf area, perimeter, length, and width of 'Shi 4366' were not significantly affected. However, the leaf area, perimeter, length, and width of 'Shi Luan 02-1' and 'Gao You 2018' were significantly higher than the water control, and the leaf area, perimeter, and length of 'Shi Mai 30' were significantly higher than the water control. Treatment with twice the recommended dose of mesosulfuron-methyl had a significantly greater effect on the leaf area, perimeter, and length of the above four varieties than the water control.

[0097] After application on March 25th, at the recommended dose of mesosulfuron-methyl, the leaf area and length of 'Shi 4366' were significantly affected, while the leaf perimeter and width were not significantly affected. At twice the recommended dose of mesosulfuron-methyl, the effects on leaf area, perimeter, length, and width of 'Shi 4366', 'Shi Luan 02-1', 'Gao You 2018', and 'Shi Mai 30' were significantly different regardless of whether the recommended dose or twice the recommended dose was applied. Among the four varieties, 'Gao You 2018' showed the most significant increase, with leaf area, perimeter, length, and width increasing by 104.3%, 64.1%, 32.6%, and 18.2% respectively compared to the water control; followed by 'Shi Luan 02-1' and 'Shi Mai 30'; the effect on leaf area, perimeter, length, and width of 'Shi 4366' was relatively small, increasing by 8.9%, 8.2%, 9.1%, and 4.2% respectively compared to the water control.

[0098] S4. Yield and Quality Indicators Testing: After wheat reached full maturity, it was harvested, threshed, and weighed individually by plot to obtain grain yield. Random samples were taken from each plot, and two 500-grain samples were weighed to calculate the thousand-grain weight. The error between the two weighings was required to be no more than 0.4g. The grain bulk density was randomly measured three times using a GHCS-1000 bulk density meter, and the average value was taken. The number of grains per ear and the number of ears were observed, statistically analyzed, and recorded. The results are shown in Table 5.

[0099] Table 5. Results and significance analysis of yield and quality indicators

[0100]

[0101] The results showed that variety, application time, application dosage and their interaction effects had extremely significant (P<0.01) or significant (P<0.05) effects on grain yield, thousand-grain weight, number of grains per ear and number of ears.

[0102] After application on March 9th, at the recommended dose of mesosulfuron-methyl, the grain yield and number of spikes of 'Shiluan 02-1' were significantly lower than the control in terms of water. The other three varieties, 'Shi 4366', 'Gaoyou 2018', and 'Shimai 30', showed no significant difference compared to the control. Treatment with twice the recommended dose of mesosulfuron-methyl had no significant effect on the grain yield of 'Shimai 30', while the grain yields of the other three varieties, 'Shi 4366', 'Shiluan 02-1', and 'Gaoyou 2018', were all significantly lower than the control in terms of water. Specifically, the number of spikes in 'Shi 4366' and 'Shiluan 02-1' was significantly reduced, while the thousand-grain weight and number of grains per spike in 'Gaoyou 2018' were significantly reduced, and the number of spikes increased.

[0103] After application of the herbicide on March 25, at the recommended dosage of mesosulfuron-methyl, the grain yield of 'Shimai 30' was not significantly different from the water control. However, the grain yields of the other three varieties, 'Shi 4366', 'Shiluan 02-1', and 'Gaoyou 2018', were all significantly lower than the water control. Among them, the number of ears of 'Shi 4366' and 'Shiluan 02-1' was significantly reduced, while the number of grains per ear of 'Gaoyou 2018' was significantly reduced, but the number of ears per ear increased.

[0104] After treatment with twice the dose of mesosulfuron-methyl, the grain yield of all four varieties decreased to varying degrees. 'Gaoyou 2018' showed the most significant decrease, with a reduction of 25.43%, followed by 'Shiluan 02-1' at 11.73%. 'Shi 4366' and 'Shimai 30' experienced relatively smaller reductions, at 4.19% and 3.29%, respectively. 'Gaoyou 2018' and 'Shiluan 02-1' showed significant decreases in thousand-grain weight and grain number per ear, but an increase in the number of ears. 'Shi 4366' showed no significant difference in thousand-grain weight and grain number per ear, but a significant decrease in the number of ears. 'Shimai 30' showed no significant difference in thousand-grain weight, but a decrease in the number of grains per ear and an increase in the number of ears.

[0105] On March 9 and March 25, the treatment with mesosulfuron significantly reduced the grain test weight of 'Shi 4366'. For 'Shi Mai 30', only the grain test weight under the conventional dose treatment on March 9 was not significantly different from the water control, while the other treatments significantly reduced the grain test weight. On March 25, the treatment with twice the dose of mesosulfuron significantly reduced the grain test weight of 'Gaoyou 2018' and 'Shi Luan 02-1', while the other treatments did not show significant differences.

[0106] S5. Comprehensive Evaluation: The responses of the four varieties to mesosulfuron-methyl varied with the delay in application and the increase in dosage. 'Gaoyou 2018', under stress, initially exhibited drastic changes in specific endogenous hormones and metabolites; in the middle stage, it showed significant inhibition of plant height and severe distortion of flag leaf morphology; ultimately, it resulted in a significant decrease in yield and quality. In contrast, 'Shi 4366', under the same stress, showed relatively mild changes in early physiological and biochemical indicators; morphological indicators were less affected in the middle stage; and the final decrease in yield and quality was limited.

[0107] Therefore, 'Gaoyou 2018' was evaluated as a variety sensitive to mesosulfuron-methyl, 'Shiluan 02-1' as a moderately sensitive variety, and 'Shi 4366' and 'Shimai 30' as varieties tolerant to mesosulfuron-methyl.

[0108] In other embodiments, the dosage of the mesosulfuron-methyl herbicide can be adjusted according to the evaluation purpose. For example, in some embodiments, the conventional recommended field dosage can be used, while in others, higher test dosages, such as 0.6 L, 0.75 L, or 0.9 L per hectare, can be used to assess the variety's response to high-intensity stress. Application should be performed during the wheat's greening stage, as stress treatment at this time effectively elicits differences in tolerance among varieties.

[0109] The detection time point is selective, ranging from day 10 to day 15 after application. For example, leaf samples can be taken on days 10, 12, 14, or 15. Practice has shown that sampling at any of these time points, and detecting the levels of three endogenous hormones (gibberellin, indoleacetic acid, and abscisic acid) and two metabolites (creatine phosphate disodium salt and cystine), can effectively capture specific early changes caused by mesosulfuron-methyl stress. The detection of endogenous hormones is not limited to a single method; high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS) can be used. The detection of metabolites is typically performed using non-targeted or targeted LC-MS techniques.

[0110] Phenotypic marker testing can be performed within a time window of 18 to 24 days after application, such as on day 18, day 21, or day 24.

[0111] The comprehensive evaluation involves comparing various indicator data of the variety under evaluation with a reference system. This reference system can be a self-control without pesticide application or a variety with a known tolerance level. For example, the known sensitive variety 'Gaoyou 2018' and the known tolerant varieties 'Shi 4366' and 'Shimai 30' can be used as references. During the evaluation, tolerant varieties typically exhibit the following characteristics compared to sensitive references: a smaller decrease in gibberellin content in early-stage endogenous hormones (e.g., less than 15%), and a smaller increase in indoleacetic acid and abscisic acid content; a smaller increase in creatine disodium phosphate and a smaller decrease in cystine in early-stage metabolites; lower plant height inhibition rate and flag leaf distortion in mid-stages; and less loss in final yield, thousand-grain weight, and test weight. Through this multi-dimensional, multi-stage systematic comparison, an objective judgment can be made regarding the tolerance of the variety under evaluation.

[0112] The software that can be used for analysis of variance in comparative analysis includes, but is not limited to, DPS, SPSS or SAS. The significance level is usually set to 0.05, but sometimes a more stringent 0.01 level can be used.

[0113] In other embodiments, a test kit for evaluating wheat tolerance to mesosulfuron-methyl herbicide is provided. This kit may contain reagents for the specific quantitative detection of the five target analytes, such as corresponding standards, antibodies, probes, or dedicated chromatography-mass spectrometry (GC-MS) components. The instruction manual for the kit will specify the tolerance assessment criteria based on the "content change comparison" principle established by this invention.

[0114] It should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for evaluating wheat resistant to mesosulfuron-methyl herbicide, characterized in that, This includes the following steps performed sequentially: S1. Stress treatment: Apply mesosulfuron-methyl to the wheat to be evaluated during the greening stage; S2. Physicochemical index detection: On days 10-15 after application of mesosulfuron-methyl, the content of endogenous hormones and metabolites were detected. S3. Phenotypic index detection: Phenotypic index detection was performed on days 18-24 after application of mesosulfuron-methyl; S4. Yield and quality index testing: Harvest the wheat to be evaluated and conduct yield and quality index testing; S5. Comprehensive evaluation: Take the test results of the wheat to be evaluated and compare them with the control group of wheat that has not been treated or wheat known to be resistant to mesosulfuron-methyl herbicide to evaluate the tolerance of the wheat to be evaluated to mesosulfuron-methyl herbicide; The endogenous hormones include gibberellin, indoleacetic acid, and abscisic acid; The metabolites include creatine phosphate disodium salt and cystine.

2. The evaluation method for wheat resistant to mesosulfuron-methyl herbicide according to claim 1, characterized in that, In step S5, wheat varieties that show a smaller decrease in gibberellin content and a smaller increase in indoleacetic acid and abscisic acid content compared to sensitive varieties or the untreated control group are evaluated as mesosulfuron-methyl herbicides-tolerant wheat.

3. The evaluation method for wheat resistant to mesosulfuron-methyl herbicide according to claim 2, characterized in that, In step S5, wheat varieties that show a smaller increase in creatine phosphate disodium salt content and a smaller decrease in cystine content compared to sensitive varieties or the untreated control group are evaluated as mesosulfuron-methyl herbicide-resistant wheat.

4. The evaluation method for wheat resistant to mesosulfuron-methyl herbicide according to claim 3, characterized in that, The phenotypic indicators include wheat plant height and flag leaf morphology; The yield and quality indicators are wheat grain yield, thousand-grain weight, number of grains per ear, number of ears, and test weight.

5. The evaluation method for wheat resistant to mesosulfuron-methyl herbicide according to claim 4, characterized in that, The method for detecting wheat plant height is to measure the length from the ground to the top of the main ear of wheat. The detection indicators for flag leaf morphology include the area, perimeter, length, and width of the flag leaf.

6. A method for evaluating wheat resistant to mesosulfuron-methyl herbicide according to any one of claims 1 to 5, characterized in that, Analysis of variance was performed on the test results of physicochemical indicators, phenotypic indicators, and yield and quality indicators to compare the significance level of differences.

7. The evaluation method for wheat resistant to mesosulfuron-methyl herbicide according to claim 6, characterized in that, Wheat is harvested at full maturity, and the actual yield is used to test yield and quality indicators.

8. The application of the evaluation method for wheat resistant to mesosulfuron-methyl herbicide as described in claim 7 in the preparation of a kit for detecting mesosulfuron-methyl tolerance.

9. A test kit for evaluating wheat tolerance to mesosulfuron-methyl herbicide, characterized in that, The kit contains reagents for the specific detection of endogenous hormones and metabolites in wheat samples: The endogenous hormones are gibberellin, indoleacetic acid, and abscisic acid; The metabolites are creatine phosphate disodium salt and cystine; The kit uses the evaluation method of claim 2 or 3 as the criterion for determining tolerance to mesosulfuron-methyl herbicide.

Images