Method for evaluating defoliating insect resistance of insect-resistant transgenic poplar in field environment and application of defoliating insect resistance of insect-resistant transgenic poplar in field environment

Abstract

The invention relates to a defoliating pest resistance evaluation method for insect-resistant transgenic poplar in a field environment and application, and belongs to the technical field of transgenic poplar breeding and environmental safety evaluation. According to the method, insect-resistant transgenic poplar and non-transgenic poplar are used as materials, a three-level classification standard based on damage area proportion is established through an orientation equilibrium sampling method by utilizing environmental monitoring and leaf damage detection, and fine evaluation of the defoliating insect resistance of the insect-resistant transgenic poplar is further realized. Field defoliating pest resistance evaluation is an important part of insect-resistant transgenic organism safety evaluation, and it is proved that insect-resistant transgenic poplars have good defoliating pest resistance. Through standardized sampling, high-precision detection and a scientific grading system, the problems of insufficient sample representativeness, inaccurate damage quantification and unscientific grading standard of an existing method for evaluating the field defoliating pest resistance of the transgenic insect-resistant poplar are solved, and a reliable basis is provided for biological safety evaluation and insect-resistant variety breeding of the transgenic poplar.

Description

Technical Field

[0001] The present invention relates to a method for evaluating the performance of insect-resistant transgenic poplars against leaf-eating pests in the field environment and its application, belonging to the technical fields of transgenic poplar breeding and environmental safety evaluation. Background Art

[0002] Populus L. is an important fast-growing timber and ecological protection tree species globally and plays an irreplaceable role in carbon sink forestry and ecological restoration. However, its large-scale planting is often severely threatened by leaf-eating pests such as Hyphantria cunea and Lymantria dispar. Although traditional chemical control can short-term control the pest population density, there are ecological risks such as excessive pesticide residues, accidental killing of non-target organisms, and the evolution of pest resistance. In this context, cultivating insect-resistant transgenic poplars through genetic engineering means has become a key technical path to achieve green pest control.

[0003] However, during the large-scale popularization and planting of insect-resistant transgenic poplars, their biosafety issues need to be focused on. Conducting insect resistance evaluation on target pests is an important part of the biosafety evaluation of insect-resistant transgenic poplars. Currently, the main method for evaluating the performance of poplars against leaf-eating pests is a single indoor insect feeding experiment. Although it can initially screen resistant materials, there are certain limitations in actual production, ignoring the dynamic changes of the pest community and the synergistic effects of other environmental factors under natural conditions. At present, no specific and feasible standardized evaluation system for the performance of insect-resistant transgenic poplars against leaf-eating pests in the field environment has been established.

[0004] Field Insect Resistance Performance refers to the comprehensive ability of plants to resist pest attacks through their own genetic characteristics under natural field environmental conditions. Different from short-term stress tests under laboratory conditions, Field Insect Resistance Performance is closer to the actual planting scenario and can truly reflect the insect resistance persistence and broad-spectrum nature of transgenic plants in complex ecosystems, which is a key indicator for evaluating their commercial application value.

[0005] Therefore, establishing a simple, easy-to-implement, scientific and effective method for evaluating the performance of poplars against leaf-eating pests in the field, with accurate damage quantification and scientific grading criteria, is of great significance for promoting the variety approval, ecological risk control and market promotion of insect-resistant transgenic poplars. Summary of the Invention

[0006] One of the objectives of the present invention is to overcome the shortcomings of insufficient sample representativeness, inaccurate damage quantification, and unscientific grading criteria in the method for evaluating the performance of insect-resistant transgenic poplars against leaf-eating pests in the field, and to provide a systematic and standardized comprehensive evaluation method for the field insect resistance performance of transgenic poplars.

[0007] The above object of the present invention is achieved by the following technical solutions:

[0008] 1. A method for evaluating the performance of insect-resistant transgenic poplars against leaf-eating pests in the field environment, comprising the following steps:

[0009] (1): Sample collection

[0010] During the tree growth season and during the outbreak of Hyphantria cunea, using insect-resistant transgenic poplars and non-transgenic poplars as test materials, and adopting the azimuth balanced sampling method, in the upper-middle part of the canopy of the east, west, south, and north four main azimuths of each test tree;

[0011] (2): Leaf damage detection

[0012] Digitally process the collected leaves, and accurately measure the original area S0 of a single leaf and the insect-damaged area S1 of a single leaf;

[0013] (3): Leaf damage classification and calculation

[0014] Establish a three-level classification standard according to the damage degree:

[0015] Grade I (mild damage): 0% < S1 / S0 × 100% ≤ 30%;

[0016] Grade II (moderate damage): 30% < S1 / S0 × 100% ≤ 70%;

[0017] Grade III (severe damage): 70% < S1 / S0 × 100% ≤ 100%;

[0018] Calculate the loss rate (Loss Rate, L) of each grade of leaves:

[0019] L = [(N Ⅰ × 15% + N Ⅱ × 50% + N Ⅲ × 85%) / N 总 × 100%;

[0020] In the formula, N Ⅰ、 N Ⅱ、 N Ⅲ are the numbers of leaves of each grade respectively, and N 总 is the total number of leaves;

[0021] 15%, 50%, and 85% are the median loss values of the corresponding grades;

[0022] (4): Evaluation of the performance of insect-resistant transgenic poplars against leaf-eating pests

[0023] One-way ANOVA was used to compare the significant differences in the leaf loss rates between insect-resistant transgenic poplars and non-transgenic poplars (p < 0.05). At the same time, the percentage increase in the performance of insect-resistant transgenic poplars against leaf-eating pests (Percentage, P) was calculated as follows:

[0024] P = [(L 对照 - L 转基因 ) / L 对照 × 100%.

[0025] Preferably, in step (1), in the azimuthal balanced sampling method, the current-year branches intercepted in each azimuth are located in the upper-middle part of the canopy and have no mechanical damage. Current-year branches with a random intercepted length of 80 cm are selected, and 30 intact leaves are selected in each azimuth. The total sample size per plant is 120 leaves.

[0026] Preferably, the specific process of step (2) is as follows:

[0027] First, lay the leaves flat on the scanning table to ensure no overlap or wrinkles. Identify and calculate the original area S0 of a single leaf and the pest damage area S1 of a single leaf, while excluding non-pest damages such as natural breaks in the leaf veins and fungal lesions.

[0028] Preferably, in step (1), the insect-resistant transgenic poplars are Populus × euramericana cv. 'Zhonglin 46' transformed with BtCry1Ac, Populus × euramericana cv. 'Zhonglin 46' transformed with BtCry1Ac, and Populus × euramericana cv. 'Zhonglin 46' transformed with BtCry1Ac.

[0029] Preferably, in step (2), a leaf area scanner is used to digitally process the collected leaves to detect the leaf damage area, and the measurement accuracy error of the leaf area scanner is ≤ 1%.

[0030] Another object of the present invention is to provide the application of the method for evaluating the performance of insect-resistant transgenic poplars against leaf-eating pests in a field environment in the evaluation of the performance of insect-resistant transgenic poplars against leaf-eating pests in the field.

[0031] The above object of the present invention is achieved by the following technical solutions:

[0032] The application of the method for evaluating the performance of insect-resistant transgenic poplars against leaf-eating pests in a field environment in the evaluation of the performance of insect-resistant transgenic poplars against leaf-eating pests in the field.

[0033] Through the above experiments and result analysis, the present invention has improved the evaluation criteria and system for the performance of insect-resistant transgenic poplars against leaf-eating pests in a field environment.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] 1. By the azimuthal balanced sampling method, the uniformity and representativeness of the sample spatial distribution are ensured;

[0036] 2. Use precise instrument measurement to replace manual visual inspection, significantly improving the objectivity of data.

[0037] 3. Establish a four-level resistance evaluation standard to achieve refined classification of the performance of leaf-eating pest resistance.

[0038] 4. The equipment required for the present invention is simple and easy to operate, with obvious technical advantages.

[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this does not mean limiting the protection scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a schematic diagram of the field insect resistance situation of transgenic BtCry1Ac insect-resistant poplar Jingxing No. 1 and non-transgenic European black poplar in Example 1 of the present invention.

[0041] Figure 2 It is a diagram showing the damage grading in Example 1 of the present invention.

[0042] Figure 3 It is a comparison chart of the leaf damage rates of transgenic BtCry1Ac insect-resistant poplar and non-transgenic poplar in Example 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0043] The present invention will be further described below with reference to specific embodiments. The following embodiments are not described in detail. It should be understood that the following text is only used to describe one or several specific embodiments of the present application, and does not strictly limit the protection scope of the specific request of the present application. Without conflict, the embodiments in the present application and the features in the embodiments can be combined with each other.

[0044] Example 1

[0045] A method for evaluating the performance of transgenic BtCry1Ac insect-resistant poplar against leaf-eating pests in a field environment, the steps are as follows:

[0046] 1 Experimental materials and methods

[0047] 1.1 Experimental materials

[0048] Select the 2-year-old BtCry1Ac transgenic poplars 'Jingxing1' (Populus×euramericana cl.‘Jingxing1’), 'Jingxing2' (Populus×euramericana cl.‘Jingxing2’), and 'Huabei 1' (Populus×euramericanacl.‘Huabei 1’) (Forestry Technology Approval No.

[2023] 22) for environmental release and productive trials. The control is non-transgenic Populus nigra L. (CK). The experiment adopts a randomized block design, with 3 biological replicates for each strain. The row and plant spacing is 3m×4m. The experimental site is located at the Luanan Experimental Forest Farm in Hebei Province (118°43′E, 39°21′N), with sandy loam soil, an average annual temperature of 11.2°C, and an annual precipitation of 620mm.

[0049] 1.2 Determination of the time for environmental monitoring and investigation

[0050] During the growing season, regularly monitor and record the population dynamics of Hyphantria cunea every 15 days to ensure that the insect density remains above 50 individuals per plant during the experiment. The investigation time is selected in September 2024, during the outbreak period of Hyphantria cunea, when the impact of the pest on poplars is most significant, facilitating the observation and collection of data.

[0051] 1.3 Collection of leaf samples from BtCry1Ac transgenic poplars and non-transgenic poplars

[0052] Adopt the azimuth balanced sampling method. Randomly intercept current-year branches with a length of 80 cm in the upper-middle part of the canopy in the four main azimuths (east, west, south, and north) of each experimental tree. Select 30 intact leaves (without mechanical damage or diseases) from each azimuth. The total sample size per plant is 120 leaves.

[0053] 1.4 Detection of leaf damage in BtCry1Ac transgenic poplars and non-transgenic poplars

[0054] Use the HOLDER YMJ-S leaf area scanner (accuracy ±0.001 cm 2 ) to perform the following operations:

[0055] a. Lay the leaf flat on the scanning table to ensure no overlapping or wrinkling.

[0056] b. Identify and calculate:

[0057] Per Leaf Original Surface Area (S0).

[0058] Per Leaf Pest-Induced Damaged Surface Area, S1

[0059] c. Exclude non-pest damages such as natural vein breaks and fungal lesions (automatically filtered by image morphology algorithms);

[0060] 1.5 Leaf Damage Grading and Calculation

[0061] Establish a three-level classification standard according to the damage degree:

[0062]

[0063]

[0064] Calculate the leaf loss rate of each grade:

[0065] L = [(N Ⅰ × 15% + N Ⅱ × 50% + N Ⅲ × 85%) / N 总 × 100%;

[0066] In the formula, N Ⅰ , N Ⅱ , N Ⅲ are the numbers of leaves of each grade respectively, and N 总 is the total number of leaves;

[0067] 15%, 50%, and 85% are the median loss values for the corresponding grades;

[0068] 1.6 Calculation of the Performance Improvement Rate of Insect-Resistant Transgenic Poplars against Leaf-Eating Pests

[0069] Use one-way ANOVA to compare the significant differences in leaf loss rates between insect-resistant transgenic poplars and non-transgenic poplars (p < 0.05), and at the same time calculate the percentage of the performance improvement of insect-resistant transgenic poplars against leaf-eating pests:

[0070] P = [(L 对照 - L 转基因 ) / L 对照 × 100%;

[0071] 2 Results and Analysis

[0072] 2.1 Evaluation of Leaf Damage Rates of BtCry1Ac Insect-Resistant Transgenic Poplars and Non-Transgenic Poplars

[0073] Field observation of insect resistance showed that under the same pest environment, the leaf integrity of the BtCry1Ac insect-resistant transgenic poplar Jingxing No. 1 was significantly better than that of the control strain (see Figure 1) The damage rates of the leaves of transgenic BtCry1Ac insect-resistant poplars (transgenic BtCry1Ac insect-resistant poplar Jingxing 1, transgenic BtCry1Ac insect-resistant poplar Jingxing 2, transgenic BtCry1Ac insect-resistant poplar Huabei 1) and non-transgenic poplars (CK) were measured. It was found that the damage degrees of the transgenic lines at the II and III damage levels were significantly lower than those of the control lines (see Table 1), indicating that the leaves of transgenic BtCry1Ac insect-resistant poplars have better resistance to Hyphantria cunea.

[0074] Table 1 Proportion of damaged leaf area of transgenic BtCry1Ac insect-resistant poplars and non-transgenic poplars

[0075]

[0076] Note: Different letters ab in the same row indicate significant differences between the transgenic lines and the non-transgenic control lines (one-way ANOVA, p < 0.05).

[0077] 2.2 Evaluation of the performance of transgenic BtCry1Ac insect-resistant poplars and non-transgenic poplars against leaf-eating pests

[0078] The performance of the transgenic lines against leaf-eating pests was evaluated based on the total loss rate (L). The results showed that the total loss rates of transgenic lines Jingxing 1, Jingxing 2, and Huabei 1 were 1.96% ± 0.74%, 2.04% ± 0.55%, and 1.63% ± 0.50% respectively, while the total loss rate of the control line was 15.36% ± 4.77% (see Figure 3 ). Through one-way ANOVA, it was found that the total leaf loss rates of the transgenic lines (Jingxing 1, Jingxing 2, Huabei 1) were significantly lower than those of the control line (p < 0.05); at the same time, compared with the control line, the improvement rates of the performance of Jingxing 1, Jingxing 2, and Huabei 1 against leaf-eating pests were 87.25%, 86.71%, and 89.42% respectively. This result fully indicates that in the evaluation of the performance against leaf-eating pests in the field, the insect-resistant effect of the transgenic lines is significant and they have good performance against leaf-eating pests.

[0079] From the above process, it can be concluded that transgenic BtCry1Ac insect-resistant poplars have high performance against leaf-eating pests; the method for evaluating the performance of the insect-resistant transgenic poplars of the present invention against leaf-eating pests in the field environment can be widely applied to the evaluation process of the insect-resistant ability of insect-resistant poplars.

[0080] Taking insect-resistant transgenic poplars and non-transgenic poplars as materials, this invention uses environmental monitoring and leaf damage detection, and further refines and improves the grading criteria, establishing a systematic and standardized comprehensive evaluation method for the performance of transgenic poplars against defoliating pests in the field. The evaluation of the performance against defoliating pests in the field is an important part of the biosafety evaluation of insect-resistant transgenic organisms. This invention proves that insect-resistant transgenic poplars have good performance against defoliating pests, solves the problems of insufficient sample representativeness, inaccurate damage quantification, and unscientific grading criteria in the current evaluation method for the performance of insect-resistant transgenic poplars against defoliating pests in the field, and plays a guarantee role in the biosafety evaluation of transgenic poplars.

[0081] Although the above has elaborated on the purpose concept and embodiments of the present invention in detail, those of ordinary skill in the art can recognize that various improvements and transformations can still be made to the present invention without departing from the scope defined by the claims, and such improvements and transformations should still fall within the protection scope of the present invention.

Claims

1. A method for evaluating the performance of insect-resistant transgenic poplars against leaf-eating pests in the field environment, comprising the following steps: (1): Sample collection Using insect-resistant transgenic poplars and non-transgenic poplars as experimental materials, by means of azimuth-balanced sampling method, in the upper-middle part of the canopy of the four main azimuths of east, west, south, and north of each experimental tree; (2): Leaf damage detection Digitally process the collected leaves, and accurately measure the original area S0 of a single leaf and the insect-damaged area S1 of a single leaf; (3): Leaf damage grading and calculation Establish a three-level classification standard according to the damage degree: Level I (mild damage): 0% < S1 / S0 × 100% ≤ 30%; Level II (moderate damage): 30% < S1 / S0 × 100% ≤ 70%; Level III (severe damage): 70% < S1 / S0 × 100% ≤ 100%; Calculate the leaf loss rate (Loss Rate, L) of each level: L = [(N Ⅰ × 15% + N Ⅱ × 50% + N Ⅲ × 85%) / N 总 × 100%; Where N Ⅰ、 N Ⅱ、 N Ⅲ are the number of blades of each grade respectively, and N 总 is the total number of blades; 15%, 50%, and 85% are the median loss values for the corresponding levels; (4): Evaluation of the performance of insect-resistant transgenic poplars against leaf-eating pests Use one-way analysis of variance to compare the significant differences in the leaf loss rates between insect-resistant transgenic poplars and non-transgenic poplars (p < 0.05), and at the same time calculate the percentage increase in the performance of insect-resistant transgenic poplars against leaf-eating pests (Percentage, P): P = [(L 对照 - L 转基因 ) / L 对照 × 100%.

2. The method for evaluating the performance of the insect-resistant transgenic poplar according to claim 1 against defoliating pests in a field environment, characterized in that, In step (1), in the azimuth-balanced sampling method, the current-year branches intercepted in each azimuth are located in the upper-middle part of the canopy and have no mechanical damage. Randomly intercept current-year branches with a length of 80 cm, select 30 complete leaves in each azimuth, and the total sample size per plant is 120 leaves.

3. The method for evaluating the performance of the transgenic insect-resistant poplar according to claim 2 in the field environment against defoliating pests, characterized in that, The specific process of step (2) is as follows: First, lay the leaves flat on the scanning table to ensure no overlap or fold, identify and calculate the original area S0 of a single leaf and the insect-damaged area S1 of a single leaf, and at the same time exclude non-insect damage such as natural breaks in leaf veins and fungal lesions.

4. The method for evaluating the performance of the insect-resistant transgenic poplar according to claim 3 against leaf-eating pests in the field environment, characterized in that, In step (1), the insect-resistant transgenic poplars are Populus × euramericana cv. 'Jingxing 1' transformed with BtCry1Ac, Populus × euramericana cv. 'Jingxing 2' transformed with BtCry1Ac, and Populus × euramericana cv. 'Huabei 1' transformed with BtCry1Ac.

5. The method for evaluating the performance of the insect-resistant transgenic poplar according to claim 4 in the field environment, characterized in that In step (2), use a leaf area scanner to digitally process the collected leaves and detect the leaf damage area. The measurement accuracy error of the leaf area scanner is ≤ 1%.

6. The application of the evaluation method according to any one of claims 1-5 in the evaluation of the field performance of insect-resistant transgenic poplars against leaf-eating pests.

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