A method for evaluating resistance of sugarcane germplasm to borer in field based on dynamic sensitive contrast

Abstract

The application discloses a sugarcane germplasm resource borer resistance field evaluation method based on dynamic sensitive contrast, and belongs to the technical field of agricultural pest resistance identification. The application respectively investigates the dead heart seedling number and total seedling number in the first and second generations of borer damage stable period in the sugarcane seedling stage, investigates the borer damage node number and total node number in the mature harvest period, calculates the average borer damage dead heart seedling rate and borer damage node rate in the seedling stage, takes the test material with the highest borer damage rate in the same period investigation as the dynamic sensitive contrast benchmark, calculates the relative borer damage dead heart seedling rate index and relative borer damage node rate index of each test material, and then synthesizes the comprehensive borer damage rate index P, and maps P to a preset nonlinear classification interval to output the borer resistance level. The application effectively eliminates the interference of the borer population base difference between different years and different places on the evaluation result through the dynamic sensitive contrast, realizes the comparability of the evaluation results across years and regions, and provides a reliable basis for sugarcane borer resistance breeding and variety layout.

Description

Technical Field

[0001] This invention belongs to the field of agricultural pest resistance identification technology, specifically involving a field evaluation method for the resistance of sugarcane germplasm resources to stem borer based on dynamic sensitive controls. Background Technology

[0002] sugar cane( Saccharum officinarum Sugarcane borer (L.) is one of my country's most important sugar and economic crops. The sugarcane borer is the most widespread and damaging pest in sugarcane production, causing damage throughout the entire growth cycle: in the seedling stage, it causes dead heart, with a general damage rate of 10%–30%, and in severe cases, it can reach over 50%; in the mid-to-late stages, it causes borer-damaged nodes or dieback, with a node damage rate usually exceeding 20%, leading to yield losses of 5%–20%. Common dominant species in Guangxi sugarcane areas include the sugarcane two-spotted borer (L.). Chilo infuscatellus Snellen ), twig borer ( Chilo sacchariphagus Bojer ) and yellow moth ( Tetramoera schistaceana Snellen Different species of stem borers cause damage at different times and on different parts of the plant. The damage caused by dead heart in the seedling stage and by stem borer infestation in the later stages means that the evaluation of stem borer resistance in sugarcane germplasm must cover the entire growth period.

[0003] For a long time, chemical control has been the main method for controlling sugarcane borers. However, long-term excessive use of pesticides has led to the "3R" problem: resistance, resurgence, and residue, resulting in ecological degradation and a decline in sugarcane quality. Therefore, planting insect-resistant varieties is widely recognized as the safest, most economical, and most effective way to control sugarcane borers. The breeding of insect-resistant varieties relies on a scientific and accurate evaluation of the borer resistance of sugarcane germplasm resources, and the reliability of this evaluation depends first and foremost on the rationality of the comparative benchmark.

[0004] However, sugarcane resistance to stem borers is a quantitative trait controlled by multiple genes, significantly influenced by environmental factors and exhibiting poor stability. Current techniques typically employ natural field surveys, evaluating resistance based on indicators such as seedling dead heart rate, stem borer infestation rate in the mid-to-late stages, stem borer-infested plant rate, and even the number of borer-infested holes. These survey indicators are numerous and inconsistent. The variability in stem borer infestation rates among sugarcane varieties (lines) in the field is often significantly low, affecting the effective evaluation of stem borer resistance. At the operational level, existing studies usually use a fixed susceptible variety (such as Xintai Sugar 22) as a universal control, calculating the resistance index by "infestation rate of tested material / infestation rate of fixed control." However, this method suffers from the following fundamental technical defects in practical applications:

[0005] First, existing methods rely on fixed varieties as sensitive controls, implicitly assuming that the susceptibility of these fixed varieties remains constant across different years and locations, and that they represent the maximum insect pressure in the current season. However, in reality, due to the explosive and fluctuating nature of stem borer populations, the natural occurrence can vary by more than an order of magnitude across different years and locations. The damage rate of the fixed control variety itself fluctuates significantly with the environment (i.e., "benchmark drift"): when the overall stem borer infestation is relatively mild in a given year, the damage rate of the fixed control is lower, leading to an artificially inflated "resistance index" for each tested material, causing many materials to be misclassified as "susceptible"; conversely, when insect pressure is high, the damage rate of the fixed control is higher, compressing the resistance index and causing the material to be misclassified as "resistant." This evaluation distortion caused by unstable benchmarks makes the identification results from different batches and regions completely incomparable, severely restricting the cross-year screening of breeding materials.

[0006] Secondly, the grading standards are inconsistent and lack technical basis. Existing evaluation systems employ diverse grading methods for stem borer resistance (e.g., 4 or 5 levels), and threshold divisions are often based on empirical settings (e.g., simply using a 10% or 15% dead heart rate as the boundary), lacking objective basis based on large-sample statistical analysis. Furthermore, existing grading systems largely fail to consider the differences in damage mechanisms and yield loss weights between dead heart in the seedling stage and stem borer damage in the mid-to-late stages, making it difficult for evaluation results to accurately reflect the overall resistance level of the germplasm.

[0007] Third, the evaluation indicators are too simplistic and cannot reflect resistance throughout the entire growth period. Some methods assess resistance solely based on the seedling dead heart rate, neglecting the hidden borer damage caused by stem borers inside the sugarcane stalks in the mid-to-late stages; while methods that only investigate the rate of borer-damaged nodes overlook the loss of effective stalks due to seedling dead heart. This one-sidedness of a single indicator cannot comprehensively characterize the overall resistance of sugarcane germplasm to stem borers throughout the entire growth cycle.

[0008] Fourth, the lack of systematic and standardized integration in various aspects such as the determination of the investigation time window, the number of samples, the number of repetitions, and the data recording and calculation process means that even the same batch of germplasm materials may yield significantly different conclusions when investigated by different operators with different operating habits.

[0009] In summary, existing technologies cannot effectively eliminate the interference of fluctuations in the base population of stem borers in different years and locations on the evaluation results. At the same time, they lack a systematic evaluation method that covers the entire growth period and has unified grading standards, which seriously restricts the accurate identification of stem borer resistant germplasm and the efficient development of insect-resistant breeding. Summary of the Invention

[0010] In view of the above, this paper proposes a method for investigating and evaluating the natural resistance of sugarcane germplasm resources to stem borer in the field, addressing the technical problems in existing methods for evaluating the resistance of sugarcane germplasm resources to stem borer, such as inconsistent control standards, inconsistent grading standards, incomplete evaluation indicators, and lack of standardized full-process methods.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] A field evaluation method for the resistance of sugarcane germplasm resources to stem borer based on dynamic sensitive controls includes the following steps:

[0013] (1) To obtain a normalized baseline for eliminating the fluctuation of the base population of the borer, the number of dead heart seedlings and the total number of seedlings of each test material were investigated and recorded during the stable period of the first generation of borer damage and the stable period of the second generation of borer damage in the sugarcane seedling stage; during the sugarcane ripening and harvesting period, the number of borer-damaged nodes and the total number of nodes of each test material were investigated and recorded.

[0014] (2) Based on the survey results of step (1), calculate the first-generation stem borer dead heart rate and the second-generation stem borer dead heart rate of each test material, and take the average of the two as the average stem borer dead heart rate A during the seedling stage; and calculate the stem borer node rate D of each test material.

[0015] (3) The test material with the highest stem borer rate in the same period survey was used as the dynamic sensitive control benchmark. The relative stem borer dead heart rate index of each test material was calculated according to the formula PA = A / TsA, and the relative stem borer node rate index of each test material was calculated according to the formula PD = D / TsD. TsA is the average stem borer dead heart rate of the dynamic sensitive control benchmark during the seedling stage, and TsD is the stem borer node rate of the dynamic sensitive control benchmark.

[0016] (4) Synthesize the comprehensive stem borer damage index P = (PA + PD) / 2 for each test material, map the comprehensive stem borer damage index P to a preset nonlinear grading interval, and output the stem borer resistance level of each test material.

[0017] In this invention, further, in step (1), the stable period of damage caused by the first generation of stem borers is in mid-to-late April, the stable period of damage caused by the second generation of stem borers is in mid-to-late June, and the sugarcane ripening and harvesting period is in mid-to-late December.

[0018] In this invention, further, in step (1), the test materials are investigated synchronously in the same field at the same time, and the test material with the highest stem borer damage rate is determined as the dynamic sensitive control benchmark.

[0019] In this invention, further, in step (1), when investigating sugarcane germplasm resources, there are no fewer than 10 test materials, and the number of dead heart seedlings damaged by stem borers in 100-200 sugarcane plants in each material is investigated sequentially; when investigating sugarcane variety regional test materials, each plot is investigated repeatedly 3-4 times; at the sugarcane maturity period or harvest time, the number of stem borer-damaged nodes and the total number of nodes of 15-30 sugarcane plants in each plot are investigated in a random sequence.

[0020] Furthermore, this invention also includes a stability verification step: for test materials whose initial identification results are high-resistance, resistant, or medium-resistance, repeated identification is conducted under the same ecological conditions in the following year. If the two identification results are consistent, the resistance level to stem borers is confirmed; if the two identification results are inconsistent, the lowest resistance level to stem borers in the two years is taken as the final evaluation result.

[0021] In this invention, the stem borer is further specified as the sugarcane two-spotted stem borer (… Chilo infuscatellus Snellen ), twig borer ( Chilo sacchariphagus Bojer ) and / or yellow moth ( Tetramoera schistaceana Snellen ).

[0022] This invention also proposes the application of the above-described methods in the selection of parent lines for sugarcane stem borer resistance breeding or the screening of resistant varieties.

[0023] This invention also proposes a sugarcane resistance to stem borer evaluation system, comprising:

[0024] The data acquisition module is used to input the number of dead heart seedlings, total number of seedlings, number of nodes damaged by stem borers, and total number of nodes from field surveys;

[0025] The index calculation module is used to execute the above-described method and calculate the comprehensive borer damage index P of each test material based on the data input by the data acquisition module.

[0026] The graded output module is used to store preset nonlinear graded intervals, compare the comprehensive stem borer damage index P with the graded intervals, and output the corresponding stem borer resistance level.

[0027] A storage module is used to store the survey data and evaluation results.

[0028] In this invention, the preset nonlinear hierarchical interval in the hierarchical output module is further defined as follows:

[0029] When P ≤ 0.1, output high resistance level; when 0.1 < P ≤ 0.2, output resistance level; when 0.2 < P ≤ 0.4, output medium resistance level; when 0.4 < P ≤ 0.6, output medium sensitivity level; when 0.6 < P ≤ 0.8, output sensitivity level; when P > 0.8, output high sensitivity level.

[0030] The present invention also proposes a computer-readable storage medium having a computer program stored thereon, wherein when the computer program is executed by a processor, the processor performs the steps of the method described above to generate and output the results of the moth resistance level of each test material.

[0031] Compared with the prior art, the present invention has at least the following beneficial effects:

[0032] (1) This invention uses the test material with the highest stem borer damage rate in the same period of investigation as a dynamic sensitive control benchmark, instead of using a fixed variety as a control. This allows the damage rate of the sensitive control to adaptively reflect the maximum damage pressure of the natural stem borer population in the current year and local area. The dynamic benchmark value is used as the denominator for normalization, and the absolute damage rate of each test material is converted into a relative index. This effectively eliminates the interference of differences in stem borer population base between different years and locations on the evaluation results. It overcomes the defect of the fixed control method where data across years and locations are not comparable due to "benchmark drift", and realizes the comparability of evaluation results across years and regions.

[0033] (2) This invention establishes a comprehensive evaluation system covering the seedling dead heart rate and the stem borer rate at maturity. By calculating the average stem borer dead heart rate index and the stem borer rate index at the seedling stage and taking the arithmetic mean of the two as the comprehensive stem borer rate index, it comprehensively reflects the overall resistance level of sugarcane germplasm to stem borers throughout the entire growth cycle, overcoming the one-sidedness of single-index evaluation. At the same time, it proposes a six-level grading standard based on large sample data statistical analysis. Through non-equidistant grading intervals of thresholds 0.1, 0.2, 0.4, 0.6, and 0.8, it realizes the scientific division of stem borer resistance levels, providing a clear quantitative basis for the selection of breeding parents and variety layout.

[0034] (3) The present invention sets up a stability verification mechanism of repeated identification and lowest level confirmation. Materials that show high resistance, resistance or medium resistance in the first identification are repeatedly identified under the same ecological conditions in the following year, and the lowest level in the two years is used as the final evaluation result. This can effectively filter out false resistance materials caused by environmental fluctuations, avoid the omission of excellent resistance materials or the wrong selection of false resistance materials due to environmental fluctuations in a single year, and ensure that the screened resistance germplasm resources have a reliable and stable resistance genetic basis.

[0035] (4) The present invention provides a standardized field survey operation procedure, clarifies key parameters such as survey time window, sample size and number of repetitions, so that the survey results of different operators and different batches are comparable; and the method can realize automated evaluation through computer-readable storage medium and system. After the field survey data is input into the system, index calculation, sensitive control identification, grading judgment and data storage can be completed automatically, which significantly improves the efficiency of large-scale germplasm resource evaluation and avoids errors that may occur in the process of manual calculation and grading. Detailed Implementation

[0036] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0037] 1. Definitions and Terminology

[0038] As described in the background section of this invention, sugarcane borers (including the two-spotted sugarcane borer *Chilo infuscatellus* Snellen, the striped sugarcane borer *Chilo sacchariphagus* Bojer, and the yellow sugarcane borer *Tetramoera schistaceana* Snellen, etc.) can cause damage throughout the entire sugarcane growth cycle. To accurately evaluate the natural resistance of different sugarcane germplasm resources to these borers, this invention provides a field survey and evaluation method based on dynamic sensitive controls.

[0039] To facilitate understanding of this invention, the following definitions are provided for key terms:

[0040] Dead heart rate: The percentage (%) of sugarcane seedlings with dead heart caused by stem borers out of the total number of seedlings surveyed.

[0041] Sugarcane section damaged by stem borer: the percentage (%) of sugarcane sections damaged by stem borer out of the total number of sections surveyed.

[0042] Natural resistance to stem borers: The traits of sugarcane that can reduce or overcome the damage caused by natural populations of stem borers in the field.

[0043] Dynamic sensitive control benchmark: This refers to the material with the highest stem borer infestation rate selected from all tested materials under the same survey time and field conditions. This benchmark is not a pre-set fixed variety, but is dynamically determined based on the actual survey data of the current season and locality, and is used to represent the maximum damage pressure of the natural population of stem borers under the environmental conditions.

[0044] Normalized baseline: This refers to the baseline value used when converting the absolute stem borer infestation rate of each tested material into a relative index with the dynamic sensitive control baseline as the denominator. Normalization eliminates the interference of differences in stem borer population bases across different years and locations on the evaluation results.

[0045] 2. Field survey

[0046] 2.1 Survey period

[0047] The first generation of stem borers reaches a stable period of damage in mid-to-late April, while the second generation reaches a stable period of damage in mid-to-late June. The sugarcane matures and is harvested in mid-to-late December.

[0048] The aforementioned timeframes are the preferred scheme determined based on the occurrence patterns of sugarcane borers in Guangxi. In other sugarcane areas or under different climatic conditions, those skilled in the art can adjust the investigation time within a window after the damage has stabilized and before the symptoms are covered by new tissue, based on the actual occurrence of the borers in the local area. This also falls within the scope of protection of this invention.

[0049] 2.2 Survey on the rate of dead heart seedlings

[0050] The rate of dead heart seedlings was investigated during the sugarcane seedling stage.

[0051] When investigating sugarcane variety regional trial materials, the total number of sugarcane seedlings and the number of dead heart seedlings damaged by stem borers were investigated in each plot (each test material) according to the regional trial design. Each plot was investigated 3 to 4 times (block replication), and the average value of each replication was taken.

[0052] When investigating sugarcane germplasm resources, at least 10 test materials should be used, and the number of dead heart seedlings damaged by stem borers should be investigated sequentially among 100-200 sugarcane plants. During the investigation, all test materials should be investigated simultaneously in the same field at the same time.

[0053] 2.3 Survey on the rate of stem borer damage

[0054] The survey on stem borer damage was conducted during the sugarcane maturity period or at harvest.

[0055] In variety regional trials and germplasm resource trials, the number of nodes damaged by stem borers and the total number of nodes on 15–30 sugarcane plants were randomly and sequentially investigated in each plot.

[0056] 3. Specific Implementation Methods of Data Calculation

[0057] 3.1 Calculation of seedling dieback rate due to stem borer damage

[0058] The rate of dead seedlings due to stem borer damage in each plot was calculated according to formula (1):

[0059] a = b / c × 100% (1)

[0060] In the formula:

[0061] a — Rate of seedlings with dead heart due to stem borer infestation, in percentages (%)

[0062] b — Number of seedlings with dead heart caused by stem borers, in units of plants;

[0063] c — Total number of seedlings surveyed, in units of plants.

[0064] The rate of dead seedlings due to the first generation of stem borer damage is denoted as a1, and the rate of dead seedlings due to the second generation of stem borer damage is denoted as a2.

[0065] The average of the first-generation stem borer dead heart rate and the second-generation stem borer dead heart rate of sugarcane material is taken as the average stem borer dead heart rate during the seedling stage of sugarcane material, and is calculated according to formula (2):

[0066] A = (a1 + a2) / 2 (2)

[0067] In the formula:

[0068] A — Average seedling mortality rate due to stem borer damage during the seedling stage, in percentages (%)

[0069] a1 — First-generation stem borer infestation rate of seedlings with dead heart, in percentage (%)

[0070] a2 — Rate of dead heart seedlings due to second-generation stem borer infestation, in percentage.

[0071] The average seedling mortality rate (A) caused by stem borer during the seedling stage is used as a percentage in subsequent calculations. For example, if a1 = 3.5% and a2 = 4.2%, then A = (3.5 + 4.2) / 2 = 3.85%.

[0072] The significance of taking the average of the dead heart rate over two generations lies in the fact that sugarcane seedlings may be continuously damaged by the first and second generations of stem borers, and the dead heart rate of a single generation may vary greatly due to annual fluctuations in the number of stem borer generations. Taking the average of two generations can more comprehensively reflect the overall degree of stem borer damage during the seedling stage and reduce evaluation bias caused by abnormal occurrences of single-generation stem borers.

[0073] 3.2 Calculation of stem borer damage rate

[0074] The stem borer infestation rate is calculated according to formula (3):

[0075] D = E / F × 100% (3)

[0076] In the formula:

[0077] D — Stem borer infestation rate, in percentages (%)

[0078] E — The number of sugarcane stem nodes damaged by stem borers, measured in nodes;

[0079] F — Total number of survey sections, in sections.

[0080] The investigation and calculation of the stem borer infestation rate should be carried out during the sugarcane ripening and harvesting period, when the stem borer-damaged nodes inside the sugarcane stalk have fully appeared, and the investigation results can reflect the cumulative situation of stem borer-damaged nodes throughout the growing season.

[0081] 4. Determination of dynamic sensitive control benchmarks and index conversion

[0082] The technical principles and specific implementation methods are explained in detail below.

[0083] 4.1 Technical shortcomings of the fixed control method

[0084] As described in the background section, existing technologies typically use a fixed susceptible variety (such as Xintai Sugar 22) as a universal control. This method implicitly assumes that the susceptibility of the fixed control variety remains constant across different years and locations. However, due to the explosive and fluctuating nature of stem borer populations, the natural occurrence rates can vary by more than an order of magnitude across different years and locations. The damage rate of the fixed control variety itself fluctuates significantly with environmental changes (i.e., "baseline drift"), resulting in a lack of comparability of the "damage rate index" calculated using the fixed control as the denominator under different experimental conditions.

[0085] To overcome this technical deficiency, this invention proposes the concept and technical solution of "dynamic sensitive control benchmark".

[0086] 4.2 Determination of Dynamic Sensitive Control Standards

[0087] The material with the highest stem borer infestation rate was selected from all tested materials as the dynamic sensitive control baseline:

[0088] Seedling stage dynamic sensitive control benchmark: Select the test material with the highest average rate of dead heart seedlings due to stem borer damage during the seedling stage (A value), and its A value is denoted as TsA.

[0089] Dynamic sensitive control benchmark at maturity: Select the test material with the highest stem borer infestation rate D value, and its D value is denoted as TsD.

[0090] Using TsA and TsD as the denominators of formulas (4) and (5) respectively, the relative indices of each test material were calculated to eliminate the influence of differences in the population base of stem borers in different years and locations.

[0091] 4.3 Calculation of the seedling dead heart rate index due to stem borer damage

[0092] Using the average stem borer deadhead rate TsA at the seedling stage dynamic sensitive control baseline as the denominator, the relative stem borer deadhead rate index of each tested material was calculated according to formula (4):

[0093] PA = TxA / TsA (4)

[0094] In the formula:

[0095] PA – Rice stem borer infestation rate index;

[0096] TxA — Average seedling mortality rate due to stem borer damage during the seedling stage of the tested materials;

[0097] TsA — the average rate of stem borer damage and dead heart at the seedling stage in a dynamic sensitive control benchmark.

[0098] As shown in formula (4), the PA of the dynamic sensitive control baseline itself is = TsA / TsA = 1.00, which is the maximum value among all tested materials. The PA values ​​of other tested materials are between 0 and 1. The smaller the value, the lower the seedling dead heart rate of the material, that is, the stronger the seedling resistance to stem borer.

[0099] 4.4 Calculation of the stem borer infestation rate index

[0100] Using the stem borer infestation rate TsD at maturity as the denominator, the relative stem borer infestation rate index for each tested material was calculated according to formula (5):

[0101] PD = TxD / TsD (5)

[0102] In the formula:

[0103] PD – Damage Rate Index for Stem Borers;

[0104] TxD — Damage rate of the tested material;

[0105] TsD — the rate of stem borer damage at the dynamic sensitive control baseline.

[0106] Similarly, the dynamic sensitive control baseline itself has a PD of 1.00, which is the highest value among all tested materials. The PD values ​​of other tested materials are between 0 and 1.

[0107] 4.5 Calculation of the Comprehensive Stem Borer Infestation Index

[0108] The sugarcane borer damage rate index is the arithmetic mean of the borer damage dead heart rate index and the borer damage node rate index, calculated according to formula (6):

[0109] P = (PA + PD) / 2 (6)

[0110] In the formula:

[0111] P — Sugarcane borer damage index;

[0112] PA – Rice stem borer infestation rate index;

[0113] PD – Damage Rate Index for Stem Borers.

[0114] The significance of taking the arithmetic mean lies in the fact that seedling-stage dead heart (manifested as PA) and mature-stage borer-damaged nodes (manifested as PD) represent the degree of borer damage at different growth stages of sugarcane. While their impact on sugarcane yield and quality has different weights, in the absence of precise weighting coefficients, the arithmetic mean provides an objective and fair comprehensive evaluation method. By averaging the relative indices of the two growth stages with equal weights, the overall resistance level of sugarcane germplasm to borers throughout the entire growth cycle can be comprehensively reflected, overcoming the one-sidedness of evaluation based on a single indicator.

[0115] 5. Specific implementation methods for graded resistance to stem borers

[0116] 5.1 Technical meaning of nonlinear hierarchical intervals

[0117] This invention maps the comprehensive stem borer damage index P to a preset nonlinear grading interval to determine the stem borer resistance level of each tested material. The "nonlinearity" refers to the fact that the grading thresholds (0.1, 0.2, 0.4, 0.6, 0.8) are not equally spaced (if equally spaced, they would be 0, 0.2, 0.4, 0.6, 0.8, 1.0), but rather a non-equally spaced threshold design is used.

[0118] The technical rationale for this nonlinear design is that, after conducting a systematic statistical analysis of field survey data from 173 sugarcane germplasm materials over two consecutive years, the inventors discovered the following:

[0119] In the low index range (P ≤ 0.4), the resistance differences between different materials are more sensitive, requiring more precise particle size differentiation (divided into three levels: P ≤ 0.1, 0.1 < P ≤ 0.2, and 0.2 < P ≤ 0.4) to accurately identify high-resistance, high-resistance, and medium-resistance materials;

[0120] In the high index range (P > 0.6), the material has shown clear susceptibility to insects, and the granularity of discrimination can be appropriately relaxed (divided into two levels: 0.6 < P ≤ 0.8 and P > 0.8), because in breeding screening, highly susceptible materials usually need to be eliminated or used only as susceptible controls, and the requirements for their granularity of discrimination are relatively low.

[0121] The intermediate sensitivity range (0.4 < P ≤ 0.6) serves as a transition zone between resistance and sensitivity, and is established as a separate level.

[0122] Therefore, the five thresholds of 0.1, 0.2, 0.4, 0.6, and 0.8 are the optimal division points determined based on actual data analysis of the resistance distribution patterns of a large number of germplasm materials. This ensures a more reasonable distribution of varieties in each grade—avoiding the failure situation where "all varieties are concentrated in one or two grades," and also avoiding the vacancy situation where "some grades have almost no varieties." This non-linear grading method is more accurate in distinguishing between moderately resistant and moderately susceptible varieties than equal-interval division, providing a more reliable quantitative basis for the selection of breeding parents.

[0123] 5.2 Grading Standards

[0124] Based on the sugarcane borer damage index P, the natural resistance level of sugarcane germplasm resources in the field was determined according to the grading standards in Table 1.

[0125]

[0126] In the above grading standards, materials of grade 1 (highly resistant) and grade 2 (resistant) have excellent resistance to stem borers and can be used as preferred parents for resistance breeding; materials of grade 3 (moderately resistant) have certain resistance to stem borers and can be used as candidate materials for the promotion and application of resistant varieties in production; materials of grade 4 (moderately susceptible) and grade 5 (susceptible) have relatively weak resistance to stem borers; materials of grade 6 (highly susceptible) are highly sensitive to stem borers and can be used as susceptible controls in breeding.

[0127] 6. Repeated identification and evaluation of stem borer resistance

[0128] Sugarcane resistance to stem borers is significantly affected by environmental factors, and the results of single-year surveys may fluctuate. To overcome this problem, this invention re-evaluates test materials that initially show high resistance (HR), resistance (R), or moderate resistance (MR) under the same ecological conditions the following year—by re-surveying and calculating the comprehensive stem borer damage index P using the same survey method as the first year, and redetermining the resistance level.

[0129] Based on the results of the two assessments, the final evaluation result will be determined according to the following rules:

[0130] (1) If the results of the two assessments are consistent, the resistance level to stem borer is confirmed;

[0131] (2) If the results of the two assessments are inconsistent, the lowest level of resistance to stem borer in the two years shall be taken as the final evaluation result.

[0132] For example, if a material is identified as highly resistant (HR, grade 1) in the first year and moderately resistant (MR, grade 3) in the second year, then the final evaluation result is moderately resistant (MR, grade 3). This rule is based on the priority consideration of resistance stability in breeding practice: only materials that can stably show resistance in different years have reliable breeding value. 7. Specific Implementation Examples

[0134] Example 1:

[0135] This embodiment uses 173 sugarcane germplasm resources as an example to illustrate the complete implementation process of the method of the present invention.

[0136] (1) Test materials

[0137] In February 2016, 173 sugarcane germplasm materials were planted, with each material planted in one row, 7 meters long and 1.2 meters wide, at a planting density of 105,000 buds / hectare, and three replicates (three blocks) were set up. All materials were planted in the same field at the same time to ensure consistent environmental conditions.

[0138] (2) Survey on the rate of dead heart seedlings

[0139] In mid-to-late April 2016 (the stable period of the first generation of stem borer damage), the total number of sugarcane seedlings and the number of dead heart seedlings damaged by stem borer were investigated in each plot (each replicate). The dead heart seedling rate of the first generation of stem borer damage, a1, was calculated according to formula (1).

[0140] In mid-to-late June 2016 (the period when the second generation of stem borers was stable), the same method was used to investigate and calculate the rate of dead seedlings (a2) caused by the second generation of stem borers.

[0141] Calculate the average rate of dead seedlings due to stem borer damage (A) for each material according to formula (2).

[0142] (3) Determination of dynamic sensitive control benchmark

[0143] From 173 materials, the material with the highest average seedling dead heart rate (A) during the seedling stage was selected as the dynamic sensitive control benchmark during the seedling stage, and its A value was denoted as TsA.

[0144] (4) Calculation of the dead heart rate index of stem borer damage

[0145] The relative stem borer damage rate index PA of each test material was calculated according to formula (4).

[0146] (5) Survey and calculation of stem borer damage rate

[0147] In mid-to-late December 2016 (sugarcane ripening and harvesting period), the number of stem borer-damaged nodes and the total number of nodes of 30 sugarcane plants in each plot were investigated sequentially, and the stem borer-damaged node rate D of each material was calculated according to formula (3).

[0148] From 173 materials, the material with the highest stem borer infestation rate D was selected as the dynamic sensitive control benchmark at maturity, and its D value was denoted as TsD. The relative stem borer infestation rate index PD of each tested material was calculated according to formula (5).

[0149] (6) Calculation and classification of comprehensive stem borer damage index

[0150] Calculate the comprehensive stem borer damage index P = (PA + PD) / 2 for each tested material according to formula (6). Determine the stem borer resistance level of each material according to the grading standards in Table 1.

[0151] (7) Results

[0152] The 2016 survey results showed that among 173 sugarcane germplasm materials, the highest seedling dead heart rate was 17.35%, and the lowest was 1.55%. Thirteen varieties (lines) had a dead heart rate exceeding 10% due to stem borer damage, accounting for 7.51% of the total; four varieties (lines) had a dead heart rate below 2% due to stem borer damage, accounting for 2.31% of the total. The highest stem borer damage rate at harvest was 9.94%, and the lowest was 1.36%.

[0153] The evaluation results of representative materials are as follows: CP72-2086 has a comprehensive stem borer damage index P = 0.14, and is rated as level 2 (resistant, R); R22 has a comprehensive stem borer damage index P = 0.22, and is rated as level 3 (medium resistant, MR); GT03-2112 has a comprehensive stem borer damage index P = 0.62, and is rated as level 5 (susceptible, S); GT02-251 has a comprehensive stem borer damage index P = 1.00, and is rated as level 6 (highly susceptible, HS).

[0154] (8) Repeated identification

[0155] In 2017 (the first year of ratoon sugarcane), the 173 materials were re-identified under the same ecological conditions, and the investigation and calculation methods were exactly the same as in 2016.

[0156] The repeated identification results showed that: CP72-2086 had a comprehensive stem borer infestation rate index of P = 0.18 in 2017, and was still rated as Level 2 (resistant, R), consistent with the results in 2016; R22 had a comprehensive stem borer infestation rate index of P = 0.13 in 2017, and was rated as Level 2 (resistant, R), an improvement from Level 3 (moderately resistant, MR) in 2016. According to the rule of "taking the lowest level as the final evaluation result", the final evaluation result of R22 was Level 3 (moderately resistant, MR); Liucheng 03-182 had a comprehensive stem borer infestation rate index of P = 0.73 in 2017, and was rated as Level 5 (susceptible, S); RB83-5486 had a comprehensive stem borer infestation rate index of P = 1.00 in 2017, and was rated as Level 6 (highly susceptible, HS).

[0157] The above results show that the method of the present invention can effectively distinguish the differences in resistance to stem borer among different sugarcane germplasm resources, and through repeated identification and the "lowest level" rule, it can identify materials with unstable resistance expression, providing reliable technical support for breeding.

[0158] Example 2

[0159] This embodiment uses 30 major sugarcane cultivars as materials and conducts simultaneous verification at two different ecological zones, Chongzuo Xinhe and Nanning Dingdang, to demonstrate the cross-point reproducibility and interannual stability of the method of the present invention.

[0160] (1) Experimental materials and design

[0161] Thirty major sugarcane cultivars (including GT11, GT29, GT42, GT44, GT46, GT47, GT49, GT50, GT52, GT55, GT57, GT58, GT59, GT60, GT98-296, Dezhe 09-78, Funong 41, Guiliu 03-1137, Guiliu 05-136, Guiliu 07-150, Haizhe 22, Xintai Sugar 22, Yuetang 00-236, Yuetang 62, Yuetang 93-159, Yunzhe 05-49, Yunzhe 05-51, Yunzhe 08-1609, Zhongzhe 9, and Zhuangtang 6) were selected and planted at the Chongzuo Xinhe experimental site and the Nanning Dingdang experimental site, respectively. Each cultivar was replicated three times.

[0162] A survey of newly planted sugarcane was conducted in 2024, and a repeat identification of the first year's ratoon sugarcane was conducted in 2025. The survey methods, calculation methods, and grading standards for each year were all performed in accordance with steps (1) to (4) of this invention.

[0163] (2) Evaluation results of 30 varieties at the Xinhe test site

[0164] According to the method of this invention, the comprehensive stem borer damage index P and stem borer resistance level of 30 varieties in Chongzuo Xinhe test site in 2024 (newly planted sugarcane) and 2025 (first year of ratoon sugarcane) are shown in Table 2.

[0165]

[0166] Table 2 shows that among the 30 varieties evaluated over two years at the Xinhe test site, 30.0% (9 / 30) maintained the same grade, and 96.7% (29 / 30) experienced a grade change of no more than one grade. Only GT59 showed a grade fluctuation of 2 (from grade 5 to grade 3) over the two years. The significant difference in the evaluation results for GT59 between the two years was mainly attributed to the abnormally high stem borer damage during the new sugarcane planting period in 2024 (P value reached 0.76) and the significant decrease in stem borer damage during the ratooning period in 2025 (P value dropped to 0.36), reflecting the instability of the variety's stem borer resistance between different years. According to the rule of "taking the lowest grade as the final evaluation result" of this invention (see Section 6), the evaluation grade of GT59 was set to 5 (S), effectively avoiding overestimation of resistance due to abnormal data in a single year.

[0167] (3) Evaluation results of 30 varieties at the Dingdang test site

[0168] Following the same method, the comprehensive stem borer damage index P and stem borer resistance level of 30 varieties in 2024 (newly planted sugarcane) and 2025 (first year of ratoon sugarcane) at the Nanning Dingdang test site are shown in Table 3.

[0169]

[0170] As shown in Table 3, among the 30 varieties evaluated over two years at the Dingdang test site, 40% (12 / 30) maintained the same grade, while 76.7% (23 / 30) experienced a grade change of no more than one grade. The variation at the Dingdang test site was slightly higher than that at the Xinhe test site, which is likely related to the more severe stem borer infestation there (the dead heart rate at Dingdang in 2024 ranged from 3.40% to 13.81%, while at Xinhe it ranged from 0.44% to 1.78%), leading to fluctuations in the performance of some varieties under higher stress conditions.

[0171] (4) Cross-point reproducibility verification

[0172] The overall resistance level of the same variety to stem borer was compared at two test sites, Xinhe and Dingdang. The results are shown in Table 4.

[0173]

[0174] Note: "—" indicates that the variety was not fully evaluated at both locations simultaneously.

[0175] Table 4 shows that among the 28 varieties that were evaluated at both test sites:

[0176] Ten varieties had completely identical resistance levels (GT47, GT49, GT52, GT59, Funong 41, Guiliu 05-136, Haizhe 22, Yunzhe 05-49, Yunzhe 05-51, and Zhuangtang 6), accounting for 35.7%; 23 varieties differed by no more than one level, accounting for 82.1%.

[0177] Among them, Zhuangtang 6 showed high susceptibility (level 6, HS) at both test sites, and Yunzhe 05-51 showed susceptibility (level 5, S) at both test sites, indicating that the method of the present invention can stably identify susceptible germplasm resources; GT47, GT52, Funong 41, Guiliu 05-136, Haizhe 22, and Yunzhe 05-49 showed moderate susceptibility (level 4, MS) at both test sites, and the evaluation results were highly consistent.

[0178] Some varieties showed a two-level difference in resistance levels between the two locations (e.g., GT42, GT44, GT50, GT57, GT58, and Yuetang 93-159). The reason for this is that these varieties are highly sensitive to differences in soil type, climate conditions, and the composition of dominant borer populations in Xinhe and Dingdang, leading to variations in their resistance expression levels under different ecological conditions. This invention uses the "lowest level over two years" as the final evaluation result, providing a conservative assessment for these varieties with significant fluctuations across locations, aligning with the priority consideration of resistance stability in breeding practice.

[0179] If a fixed control variety (such as Xintai Sugar 22) is used as the benchmark, the evaluation results of the two sites will be completely incomparable because the damage rate of Xintai Sugar 22 at Dingdang site is significantly higher than that at Xinhe site. However, this invention successfully overcomes this defect by using a dynamic sensitive control benchmark.

[0180] (5) Verification of interannual stability

[0181] The grade changes of the same variety at the Xinhe test site in 2024 and 2025 were statistically analyzed, and the results are shown in Table 5.

[0182]

[0183] Note: GT59 changed from S (Level 5) in 2024 to MR (Level 3) in 2025. According to the rule of "taking the lowest level as the final evaluation result" as specified in Section 6 of this invention, its final evaluation result is Level 5 (S), which reflects the conservative requirement of adversarial stability.

[0184] As shown in Table 5, 30.0% (9 / 30) of the varieties maintained the same level, while 96.7% (29 / 30) changed by no more than one level, indicating that the method of this invention has good stability across different years. GT59, for example, decreased from level 5 (S) to level 3 (MR), a drop of two levels, and its final evaluation result was taken as level 5 (S) according to the rules specified in Section 6 of this invention, reflecting the conservative requirement for resistance stability. The susceptible control material Zhuangtang 6 rose from level 5 (S) to level 6 (HS) over two years, consistently remaining within the susceptible range, indicating that the method of this invention has high stability in identifying extremely susceptible materials.

[0185] Based on the data in Tables 2 to 5: among the 28 varieties evaluated at both experimental sites, 35.7% had completely identical comprehensive grades, and 82.1% differed by no more than one grade, indicating that the present invention effectively eliminated the differences in the base population of sugarcane borers in different ecological zones through a dynamic sensitive control mechanism; 30.0% of the varieties maintained the same grade over two years at the same experimental site, and 96.7% changed by no more than one grade, indicating that the method of the present invention has good interannual stability; Zhuangtang 6 consistently showed a susceptible to highly susceptible (S / HS) grade at both experimental sites and in both years, indicating that the method of the present invention has high reliability in identifying susceptible germplasm resources. These results demonstrate that the method of the present invention can provide reliable technical support for sugarcane borer resistance breeding and variety regional distribution.

[0186] 8. Application of Results

[0187] According to the evaluation results of the method of the present invention, sugarcane germplasm resources with a resistance level of 3 (moderately resistant, MR) or above can be used as resistant varieties or resistant parents in local sugarcane areas. Materials evaluated as 1 (highly resistant, HR) and 2 (resistant, R) can be preserved and utilized as priority parent resources for breeding resistance to sugarcane borers; materials evaluated as 4 (moderately susceptible, MS) or below can be used as susceptible controls in breeding or as appropriate according to other agronomic traits.

[0188] 9. Computer-readable storage media

[0189] In one embodiment of the present invention, all or part of the steps of the above-described investigation and evaluation method can be implemented by a computer program. Specifically, the number of dead heart seedlings, total number of seedlings, number of nodes damaged by stem borers, and total number of nodes obtained from the field survey are input into the computer system, and the computer program automatically performs the following operations:

[0190] (1) Calculate the average rate of dead seedlings and the rate of stem borer damage during the seedling stage for each test material according to formulas (1) to (3);

[0191] (2) The test material with the highest stem borer infestation rate was automatically identified as a dynamic sensitive control benchmark;

[0192] (3) Calculate the comprehensive stem borer damage index P for each tested material according to formulas (4) to (6);

[0193] (4) Automatically output the moth resistance level of each test material according to the grading standards in Table 1;

[0194] (5) Store survey data and evaluation results, and generate evaluation reports.

[0195] The computer program is stored in a computer-readable storage medium and performs the above functions when executed by a processor. The computer-readable storage medium includes, but is not limited to, ROM, RAM, hard disk, optical disk, flash memory, etc.

[0196] Automated processing via computer programs can significantly improve the efficiency of large-scale evaluation of stem borer resistance in germplasm resources, avoid errors that may occur during manual calculation and grading, and facilitate the establishment of a germplasm resource stem borer resistance database, providing data support for long-term breeding work.

[0197] The above embodiments only illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention.

Claims

1. A field evaluation method for the resistance of sugarcane germplasm resources to stem borer based on dynamic sensitive controls, characterized in that, Includes the following steps: (1) To obtain a normalized baseline for eliminating the fluctuation of the base population of the borer, the number of dead heart seedlings and the total number of seedlings of each test material were investigated and recorded during the stable period of the first generation of borer damage and the stable period of the second generation of borer damage in the sugarcane seedling stage; during the sugarcane ripening and harvesting period, the number of borer-damaged nodes and the total number of nodes of each test material were investigated and recorded. (2) Based on the survey results of step (1), calculate the first-generation stem borer dead heart rate and the second-generation stem borer dead heart rate of each test material, and take the average of the two as the average stem borer dead heart rate A during the seedling stage. And calculate the stem borer infestation rate D for each tested material; (3) The test material with the highest stem borer rate in the same period survey was used as the dynamic sensitive control benchmark. The relative stem borer dead heart rate index of each test material was calculated according to the formula PA = A / TsA, and the relative stem borer node rate index of each test material was calculated according to the formula PD = D / TsD. TsA is the average stem borer dead heart rate of the dynamic sensitive control benchmark during the seedling stage, and TsD is the stem borer node rate of the dynamic sensitive control benchmark. (4) Synthesize the comprehensive stem borer damage index P = (PA + PD) / 2 for each test material, map the comprehensive stem borer damage index P to a preset nonlinear grading interval, and output the stem borer resistance level of each test material.

2. The method according to claim 1, characterized in that, In step (1), the first generation of stem borers is infested during the stable period in late April, the second generation of stem borers is infested during the stable period in late June, and the sugarcane is infested during the mature harvest period in late December.

3. The method according to claim 1, characterized in that, In step (1), all the test materials are investigated simultaneously in the same field at the same time, and the test material with the highest stem borer damage rate is determined as the dynamic sensitive control benchmark.

4. The method according to claim 1, characterized in that, In step (1), when investigating sugarcane germplasm resources, there should be no fewer than 10 test materials. For each material, the number of dead heart seedlings damaged by stem borers in 100-200 sugarcane plants should be investigated sequentially. When investigating sugarcane variety regional test materials, each plot should be investigated 3-4 times. At the sugarcane maturity period or harvest time, the number of stem borer-damaged nodes and the total number of nodes of 15-30 sugarcane plants in each plot should be investigated sequentially.

5. The method according to claim 1, characterized in that, It also includes a stability verification step: for test materials whose initial identification results are high resistance, resistant or medium resistance, the identification is repeated under the same ecological conditions in the following year. If the two identification results are consistent, the resistance level is confirmed; if the two identification results are inconsistent, the lowest resistance level in the two years is taken as the final evaluation result.

6. The method according to claim 1, characterized in that, The borer in question is the sugarcane two-spotted borer (Salmonella spp.) Chilo infuscatellus Snellen ), twig borer ( Chilo sacchariphagus Bojer ), Yellow moth ( Tetramoera schistaceana Snellen ).

7. The application of the method according to any one of claims 1 to 6 in the selection of parent lines for sugarcane stem borer resistance breeding or the screening of resistant varieties.

8. A sugarcane resistance to stem borer evaluation system, characterized in that, include: The data acquisition module is used to input the number of dead heart seedlings, total number of seedlings, number of nodes damaged by stem borers, and total number of nodes from field surveys; An index calculation module is used to execute the method described in any one of claims 1 to 6, and to calculate the comprehensive borer damage index P of each test material based on the data input by the data acquisition module; The graded output module is used to store preset nonlinear graded intervals, compare the comprehensive stem borer damage index P with the graded intervals, and output the corresponding stem borer resistance level. A storage module is used to store the survey data and evaluation results.

9. The system according to claim 8, characterized in that, The preset nonlinear hierarchical interval in the hierarchical output module is: When P ≤ 0.1, output a high resistance level; When 0.1 < P ≤ 0.2, output the resistance level; When 0.2 < P ≤ 0.4, output the medium resistance level; When 0.4 < P ≤ 0.6, output a medium sensitivity level; When 0.6 < P ≤ 0.8, the output sensitivity level; When P > 0.8, output a high sensitivity level.

10. A computer-readable storage medium having a computer program stored thereon, characterized in that, When the computer program is executed by the processor, the processor performs the steps of the method as described in any one of claims 1 to 6, generating and outputting the moth resistance level results for each test material.

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