Use of quercetin in regulating intra- and / or inter-specific root competition in forest trees

Abstract

The application belongs to the technical field of forest competition, and particularly relates to application of quercetin in regulating intra-species and / or inter-species root competition relationship of forest trees. The application shows through experiments that when no phosphorus is supplied, different kinship groups of Cunninghamia lanceolata root systems all secrete quercetin at 30 days, the intra-species kinship group decreases in quercetin secretion amount, while the intra-species non-kinship group increases in quercetin secretion amount; compared with the inter-species non-kinship group which decreases in quercetin secretion amount, the intra-species kinship group and the intra-species non-kinship group both increase in quercetin secretion amount, which shows that Cunninghamia lanceolata may relieve intra-species competition by increasing quercetin secretion amount. The application provides an important theoretical basis for adjustment of neighboring plant competition relationship and related research, and provides an operable technical path for forestry practice, and has scientific innovation and application potential.

Description

Technical Field

[0001] This invention belongs to the field of forest tree competition technology, specifically relating to the application of quercetin in regulating the root competition relationship within and / or between forest tree species. Background Technology

[0002] Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) is a unique and excellent timber tree species for afforestation in the subtropical regions of southern my country. In the southern China fir growing areas, the red soil has severe phosphorus fixation, resulting in low migration rates and easy contact with Al₂O₃. 3+ Fe 3+ or Ca 2 + In addition, competition for limited resources is inevitable within and between plant species, and this survival competition (niche overlap) further exacerbates the scarcity of phosphorus available for absorption. Therefore, the extremely low availability of soil phosphorus resources during the growth and development of Chinese fir (Cunninghamia lanceolata) has become the primary problem limiting the productivity of plantations. Over long-term evolution, Chinese fir has developed a series of ecological adaptation strategies to cope with low-phosphorus stress. Plants exhibit kinship recognition behavior; when encountering closely related individuals, they adopt a more moderate competitive strategy, while facing distantly related or unrelated individuals, they employ a more intense competitive strategy. By adjusting their own growth ecological strategies, plants can promptly alleviate the degree of competition with neighboring plant roots, thereby avoiding unnecessary waste of resources. Currently, the specific pathways and mechanisms of competition between Chinese fir roots and neighboring plants in the ecological adaptation strategies of Chinese fir to low-phosphorus stress have not been thoroughly elucidated. Research on the competition strategies between Chinese fir roots and neighboring plants under low-phosphorus conditions is of great significance for alleviating the practical problem of soil phosphorus deficiency.

[0003] Current research indicates that when Arabidopsis thaliana is immersed in a culture medium containing root exudates from different related individuals, it was found that when exposed to root exudates from closely related individuals, the target individual developed fewer lateral roots, while exposure to root exudates from distantly related individuals resulted in more lateral root formation. This phenomenon disappeared after the addition of root exudate inhibitors. This reflects the important role of root exudates in the competitive strategy of plant roots against neighboring plants, potentially acting as signaling substances to adjust the degree of competition between roots and neighboring plants. However, due to the wide variety of root exudates, our understanding of their functions is still incomplete. Quercetin is an important secondary metabolite in plants, participating in functions such as antioxidation, defense, and signal transduction. Some studies have found that under specific conditions (such as stress environments), plants may secrete small amounts of flavonoids (including quercetin) through their roots to regulate the rhizosphere microbial community or cope with abiotic stress. Under stress conditions, quercetin is not a major component universally or continuously secreted by roots, but its role is very important. Currently, the mechanism of quercetin's role in plant root competition strategies remains unclear. For Chinese fir, when the competitive environmental conditions created by neighboring plants of different phylogenetic relationships differ, whether the secretion of quercetin by the Chinese fir roots and the changes in its content play a crucial role in the strategy by which the Chinese fir roots adjust their level of competition with neighboring plants. Summary of the Invention

[0004] The purpose of this invention is to provide the application of quercetin in regulating the root competition relationship within and / or between forest tree species. Compared with the decrease in quercetin secretion in non-related interspecific groups, the quercetin secretion in both related and non-related intraspecific groups increased, indicating that Chinese fir may alleviate intraspecific competition by increasing quercetin secretion. This invention provides an important theoretical basis for the adjustment of neighboring plant competition relationships and related research.

[0005] This invention provides the application of quercetin in regulating root competition within and / or between forest tree species.

[0006] As a preferred approach, when nutrients are lacking, trees regulate the competitive relationship between the roots of neighboring trees by secreting quercetin.

[0007] As a preferred embodiment, the nutrient element includes phosphorus.

[0008] As a preferred embodiment, the regulation includes increasing quercetin secretion to alleviate intraspecific competition.

[0009] As a preferred option, the trees include Chinese fir and Phoebe zhennan.

[0010] The present invention also provides a method for regulating intraspecific and / or interspecific root competition in forest trees using quercetin, comprising the following steps: treating forest trees in which intraspecific and / or interspecific root competition exists with quercetin.

[0011] As a preferred embodiment, the concentration of quercetin is 0.08–0.48 μg·g. -1 The amount of quercetin used is 0.3 to 0.8 mL.

[0012] As a preferred embodiment, the processing time is 30 to 90 days.

[0013] As a preferred embodiment, the root competition relationship includes root competition relationship caused by nutrient element deficiency, wherein the nutrient element includes phosphorus.

[0014] As a preferred option, the trees include Chinese fir and Phoebe zhennan.

[0015] Beneficial Effects: This invention provides the application of quercetin in regulating intraspecific and / or interspecific root competition in forest trees. Experiments show that, without phosphorus supply, the roots of *Cunninghamia lanceolata* in different phylogenetic groups secreted quercetin after 30 days of treatment. Quercetin secretion decreased in intraspecific phylogenetic groups, while it increased in intraspecific non-phylogenetic groups. Compared to the decrease in quercetin secretion in interspecific non-phylogenetic groups, quercetin secretion increased in both intraspecific phylogenetic and non-phylogenetic groups. This suggests that *Cunninghamia lanceolata* may alleviate intraspecific competition by increasing quercetin secretion. This invention provides an important theoretical basis for adjusting neighboring plant competition and related research.

[0016] This invention demonstrates that by exogenously applying or intervening in quercetin secretion, the competitive relationship between neighboring plant roots can be artificially regulated, optimizing forest spatial layout, reducing resource waste, and improving the productivity of plantations. Simultaneously, this invention provides a new perspective for the study of plant competition mechanisms in forest ecology, and offers a theoretical basis for forestry production (such as mixed forest design and density control), contributing to sustainable forest management. This invention not only deepens the understanding of plant allelopathy and competitive strategies but also provides an operable technical path for forestry practice, possessing both scientific innovation and application potential. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 The amount of root exudates in different phylogenetic relationships of Chinese fir under normal phosphorus supply treatment in Example 1;

[0019] Figure 2 The amount of root exudates of different phylogenetic Chinese fir trees in Example 1 under phosphorus-free treatment;

[0020] Figure 3 The change in the content of Quercetin, a substance secreted by the roots of Chinese fir in Example 1 without phosphorus supply;

[0021] Figure 4 This is a comparison of the aboveground growth of the Chinese fir seedlings in Example 2;

[0022] Figure 5 This is a comparison of the root morphology and structure of the Chinese fir seedlings in Example 2;

[0023] Figure 6 A comparison of the biomass distribution patterns of Chinese fir seedlings in Example 2;

[0024] Figure 7 This is a comparison of phosphorus utilization efficiency in the Chinese fir seedlings of Example 2;

[0025] Figure 8 This is a comparison of the root physiological indicators of the Chinese fir seedlings in Example 2;

[0026] Note: Figures 4-8 In the figure, G, P, and G×P represent the results of two-way ANOVA of the effects of kinship, phosphorus supply level, and their interaction on the relevant indicators of Cunninghamia lanceolata seedlings, respectively; Kin: intraspecific kinship group; Non-kin: intraspecific non-kinship group; Intnon-kin: interspecific non-kinship group; the same uppercase letter indicates that the difference between different kinship groups under the same phosphorus supply level did not reach a significant level (p>0.05); the same lowercase letter indicates that the difference between different phosphorus supply levels under the same kinship group did not reach a significant level (p>0.05). Detailed Implementation

[0027] This invention provides the application of quercetin in regulating intraspecific and / or interspecific root competition in forest trees. As one specific embodiment, when nutrient elements are deficient, forest trees regulate the competitive relationship between neighboring tree roots by secreting quercetin, wherein the nutrient element includes phosphorus. As another specific embodiment, the regulation includes increasing quercetin secretion to alleviate intraspecific competition. The trees mentioned in this invention include Chinese fir and Phoebe zhennan. In a specific embodiment of this invention, the Chinese fir is the Yang 020 clonal line and the Yang 061 clonal line (Yang Zhen, Chen Zhiqiang, Wang Nemin, et al. Effects of phosphorus application method on photosynthetic capacity and phosphorus allocation of Chinese fir seedlings [J]. Journal of Fujian Agriculture and Forestry University: Natural Science Edition, 2019, 48(1):7.DOI:CNKI:SUN:FJND.0.2019-01-006. Lin Decheng, Lu Jia'ao, Li Qi, et al. Effects of underground space crowding degree on endogenous organic acids in the root system of Chinese fir seedlings [J]. Journal of Fujian Agriculture and Forestry University: Natural Science Edition, 2021.DOI:10.13323 / j.cnki.j.fafu(nat.sci.).2021.01.008.), and the Phoebe zhennan is the precious native broad-leaved tree species Phoebe zhennan (Phoebe). (Liao Xiaoli, Xue Kexin, Cai Siying, et al. Effects of exogenous silicon addition on growth and photosynthetic characteristics of Phoebe zhennan seedlings [J]. Journal of Tropical and Subtropical Botany, 2024(6). DOI:10.11926 / jtsb.4910.). One-year-old seedlings of both Cunninghamia lanceolata and Phoebe zhennan were selected for the experiment.

[0028] This invention demonstrates that, under normal phosphorus supply treatment, the roots of *Cunninghamia lanceolata* under different phylogenetic relationships did not secrete quercetin. This phenomenon may be due to the abundant nutrient environment, eliminating the need for *Cunninghamia lanceolata* roots to identify surrounding plant species, thus reducing the need for quercetin secretion. Without phosphorus supply, *Cunninghamia lanceolata* roots from different phylogenetic groups secreted quercetin at 30 days of treatment, but not at 15 and 45 days. This is related to the relatively low intensity of root competition in the early stages of treatment. As treatment time increased, due to environmental nutrient stress, the degree of competition among roots increased, and *Cunninghamia lanceolata* from different phylogenetic groups regulated the degree of competition with neighboring roots by secreting quercetin. The content of quercetin secreted by the roots of *Cunninghamia lanceolata* under phosphorus-free treatment varied under different phylogenetic relationships. The relative quercetin content in the intraspecific related groups was 3.07 times that in the interspecific unrelated groups, while the relative quercetin content in the intraspecific unrelated groups was 1.23 times that in the interspecific unrelated groups. Quercetin secretion decreased in the intraspecific related groups, while it increased in the intraspecific unrelated groups. Compared to the decrease in quercetin secretion in the interspecific unrelated groups, quercetin secretion increased in both the intraspecific related and unrelated groups, suggesting that *Cunninghamia lanceolata* may alleviate intraspecific competition by increasing quercetin secretion.

[0029] This invention investigated the aboveground growth, root morphology, biomass allocation, phosphorus utilization efficiency, and root physiological characteristics of *Cunninghamia lanceolata* seedlings. Significant differences were found in root growth among intraspecific related groups, intraspecific unrelated groups, and interspecific unrelated groups. This indicates that significant root competition exists in *Cunninghamia lanceolata* regardless of intraspecific or interspecific kinship relationships. Furthermore, the presence or absence of quercetin also affected root growth, leading to variations in aboveground seedling growth. Therefore, it can be concluded that quercetin can indeed regulate intraspecific and interspecific root competition in forest trees.

[0030] The present invention also provides a method for regulating intraspecific and / or interspecific root competition in forest trees using quercetin, comprising the following steps: treating forest trees in which intraspecific and / or interspecific root competition exists with quercetin.

[0031] The concentration of quercetin described in this invention is 0.08–0.48 μg·g. -1 The amount of quercetin used is 0.3–0.8 mL. As a specific embodiment, the concentration of quercetin can be 0.08 μg·g⁻¹. -1 0.10 μg·g -1 0.12 μg·g -1 0.14 μg·g -1 0.16 μg·g -1 0.18 μg·g -1 0.20 μg·g -1 0.22 μg·g -1 0.24 μg·g -1 0.26 μg·g -1 0.28 μg·g -1 0.30 μg·g -1 0.32 μg·g -1 0.34 μg·g -1 0.36 μg·g -1 0.38 μg·g -1 0.40 μg·g -1 0.42 μg·g -1 0.44 μg·g -1 0.46 μg·g -1 and 0.48 μg·g -1 Any one of the following. As one specific embodiment, the amount of quercetin used can be any one of 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, and 0.8 mL.

[0032] The processing time described in this invention is 30 to 90 days. As a specific embodiment, the processing time can be any one of 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, and 90 days.

[0033] In one specific implementation, the root competition relationship includes root competition caused by nutrient element deficiency, wherein the nutrient element includes phosphorus. The trees include Chinese fir and Phoebe zhennan. In this specific embodiment of the invention, the varieties of Chinese fir and Phoebe zhennan are as described above and will not be repeated here.

[0034] The embodiments of this invention demonstrate that by exogenously applying or intervening in quercetin secretion, the competitive relationship between neighboring plant roots can be artificially regulated, optimizing forest spatial layout, reducing resource waste, and improving the productivity of plantations. Simultaneously, this invention provides a new perspective for the study of plant competition mechanisms in forest ecology, and offers a theoretical basis for forestry production (such as mixed forest design and density control), contributing to sustainable forest management. This invention not only deepens the understanding of plant allelopathy and competitive strategies but also provides an operable technical path for forestry practice, possessing both scientific innovation and application potential.

[0035] To further illustrate the present invention, the application of quercetin provided by the present invention in regulating the root competition relationship within and / or between forest tree species is described in detail below with reference to embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0036] Unless otherwise specified, the present invention does not have special requirements for the raw materials used in the preparation, and commercially available products well known to those skilled in the art can be used.

[0037] 1. Experimental Location: Greenhouse at Fujian Agriculture and Forestry University Science and Technology Park. This area has a subtropical humid monsoon climate, characterized by short winters and long summers, warm and humid conditions, abundant rainfall, and an average annual temperature of 19℃. The temperature in the potted plant greenhouse was 20–28℃, relative humidity >80%, with 14 h / d of light and a light intensity of approximately 800 μmol·m². -2 ·s -1 .

[0038] 2. Material Preparation

[0039] Potted plant device: An indoor potted plant experiment was conducted using a polyethylene device with a pot opening diameter of 35cm, a pot bottom diameter of 22cm, and a height of 27cm. The device was rinsed with purified water and dried before use.

[0040] Test materials: Yang 020 and Yang 061 Chinese fir clones were selected (Yang Zhen, Chen Zhiqiang, Wang Nemin, et al. Effects of phosphorus application method on photosynthetic capacity and phosphorus allocation of Chinese fir seedlings [J]. Journal of Fujian Agriculture and Forestry University: Natural Science Edition, 2019, 48(1):7.DOI:CNKI:SUN:FJND.0.2019-01-006. Lin Decheng, Lu Jia'ao, Li Qi, et al. Effects of underground space crowding degree on endogenous organic acids in the root system of Chinese fir seedlings [J]. Journal of Fujian Agriculture and Forestry University: Natural Science Edition, 2019, 48(1):7.DOI:CNKI:SUN:FJND.0.2019-01-006. The experimental materials included one-year-old seedlings of the precious native broad-leaved tree species Phoebe bournei (Liao Xiaoli, Xue Kexin, Cai Siying, et al. Effects of exogenous silicon addition on the growth and photosynthetic characteristics of Phoebe bournei seedlings [J]. Journal of Tropical and Subtropical Botany, 2024(6).DOI:10.11926 / jtsb.4910.). The tested clonal line of Cunninghamia lanceolata, Yang 020, has the characteristics of narrow crown and tolerance to poor soil, while the clonal line of Cunninghamia lanceolata, Yang 061, has the characteristics of wide crown, strong resistance and strong ability of roots to seek phosphorus under low phosphorus stress. The selected Phoebe bournei is a common tree species in mixed Cunninghamia lanceolata forests and has the characteristics of fast growth, beautiful tree shape and relatively straight trunk. All the seedlings tested were of uniform growth, healthy and free from pests and diseases. The initial seedling height and ground diameter are shown in Table 1.

[0041] Table 1. Seedling height and diameter at ground level before transplanting of the tested seedlings

[0042] Yang 020 Chinese fir clonal strain 30.16±0.98c 3.86±0.14c Yang 061 Chinese fir clone 34.75±0.54b 4.83±0.08b Nanmu 42.48±1.22a 6.45±0.25a

[0043] Note: The same lowercase letter indicates that the differences among the tested seedlings were not statistically significant (p>0.05).

[0044] Cultivation substrate: The river sand that has been washed, dried and passed through a 2mm mesh screen is mixed with the southern red soil at a volume ratio of 3:1. The chemical nutrient content of the cultivation substrate soil is shown in Table 2.

[0045] Table 2 Chemical element content of potted plant substrate

[0046]

[0047] 3. Data Statistics and Analysis: Two-way ANOVA was performed on the relevant indicators of the tested Cunninghamia lanceolata seedlings. If no significant interaction was found (p>0.05), one-way ANOVA was performed. The Tukey-B and LSD multiple comparison methods were used to test for significance (p=0.05). Simultaneously, the independent samples t-test was used to analyze the differences in relevant indicators under different phosphorus supply levels. Data analysis was performed using SPSS 26.0 software. Graphs were generated using GraphPad Prism 9 software, and results are expressed as mean ± standard error. Heatmaps were generated using TBtools software.

[0048] Example 1

[0049] (I) Test Methods

[0050] 1. Planting and Management

[0051] Three planting patterns were established: an intraspecific related group (clones of *Cunninghamia lanceolata* 'Yang 061' + 'Yang 061'), an intraspecific unrelated group (clones of *Cunninghamia lanceolata* 'Yang 061' + 'Yang 020'), and an interspecific unrelated group (clones of *Cunninghamia lanceolata* 'Yang 061' + *Phoebe zhennan*), to create a competitive environment between neighboring plants with different kinship relationships. KH₂PO₄ was used as the phosphorus source, and each planting pattern was configured with a phosphorus-free setting (P₀, the cultivation substrate contained 0 mg·kg⁻¹). -1 KH2PO4) and normal phosphorus supply (P1, the cultivation substrate contains 12 mg·kg -1 Two phosphorus supply levels were applied (KH2PO4, control group), with each treatment biologically replicated five times. Two seedlings were planted per pot, with a horizontal spacing of 10 cm between the two seedlings, one seedling at each end to create a competitive environment between adjacent plants of different phylogenetic relationships. Treatment P1 was watered with 400 mL of phosphorus solution every 5 days, while treatment P0 was watered with an equal amount of purified water (400 mL) to ensure consistent soil moisture content. The insufficient K2PO4 in treatment P0 was addressed by... + From an equal amount of KCl (12 mg·kg) -1 The modified Hoagland nutrient solution (Table 3) was applied to the roots of the P0 and P1 treatment seedlings every 5 days at a pH of 400 mL. To ensure the seedlings' nutritional needs during potted cultivation, 400 mL of the modified Hoagland nutrient solution (pH 5.5) was applied to the roots of both P0 and P1 treatment seedlings every 5 days. 200 mL of water was also applied to the roots of the seedlings at 18:00 each day. All phosphorus, KCl, and purified water were applied to the roots of each seedling in a clockwise direction. The experiment lasted 45 days.

[0052] Table 3. Improved Hoagland Complete Nutrient Solution Formula

[0053]

[0054] 2. Harvest

[0055] Destructive sampling was employed, with samples taken every 15 days for a total of three harvests (15 days, 30 days, and 45 days after treatment). At harvest, the tested *Cunninghamia lanceolata* clone, Yang 061, was used as the target plant for relevant index measurements. Within the intraspecific phylogenetic group, one *Cunninghamia lanceolata* clone was randomly selected from two planted clones of Yang 061. At harvest, the above-ground height and diameter of the seedlings were measured to calculate the increase in growth. After harvest, the roots were rinsed thoroughly with purified water, and the surface moisture was absorbed using a cotton cloth before subsequent index measurements.

[0056] 3. Determination of root exudates: Three clonal seedlings of *Cunninghamia lanceolata* (Yang 061) from different treatments were selected. Rhizosphere soil (shaking method) and non-rhizosphere soil samples (potting substrate within 2 cm of the center of the cultivation container from the edge of the pot) were collected from each seedling, resulting in a total of 108 soil samples for root exudate determination (Table 4). 0.50 g of each soil sample was weighed into a 1.5 mL centrifuge tube, and 500 μL of extraction buffer (methanol:acetonitrile:acetic acid:water volume ratio 1:1:0.1:2) was added. The mixture was ground at 65 Hz for 90 s, vortexed thoroughly, sonicated at 4℃ for 30 min, then allowed to stand at -40℃ for 1 h, vortexed for 30 s, allowed to stand at 4℃ for 0.5 h, and centrifuged for 15 min (12000 r / min at 4℃). The supernatant was collected. Another 500 μL of extraction buffer was added, and the above steps were repeated to extract the supernatant once more. The extracts obtained from the two experiments were mixed and lyophilized. The lyophilized powder was reconstituted with 300 μL of extraction buffer (methanol:acetonitrile:acetic acid:water volume ratio of 1:1:0.1:2), and 7.5 μL of internal standard (0.14 mg / mL dichlorophenylalanine) was added. 200 μL of the supernatant was transferred for subsequent LC-MS detection and analysis.

[0057] Instrumentation platform: LC-MS (Waters, UPLC; Thermo, Q Exactive); Column: ACQUITY UPLCHSS T3 (2.1 × 100 mm 1.8 μm); Chromatographic separation conditions: Column temperature 40℃; Flow rate 0.3 mL·min -1Mobile phase composition: A: Water (Milli-Q Adwantage A10, Merck KgaA, Germany) + 0.05% formic acid (CNW, Shanghai); B: Acetonitrile (Merck KgaA, Germany); Injection volume: 3 μL; Autosampler temperature: 4℃; Mobile phase gradient elution program is shown in Table 5. Mass spectrometry detection parameters: Heater temperature: 300℃; Sheath gas flow rate: 45 arb; Auxiliary gas flow rate: 15 arb; Tail gas flow rate: 1 arb; Electrospray voltage: 3.0 kV; Capillary temperature: 350℃; S-Lens RF Level: 30%. Scanning modes: Full Scan (m / z 70–1050) and data-dependent secondary mass spectrometry (dd-MS2, TopN = 10); Resolution: 70,000 (primary mass spectrometry) and 17,500 (secondary mass spectrometry). Collision mode: High-energy collisional dissociation (HCD). Compound Discoverer 3.1 software (Thermo Scientific) was used to process the LC-MS data and search the database to obtain qualitative and quantitative results of root exudates. Further data normalization and editing were performed in Excel 2010, and the data was finally organized into a two-dimensional data matrix. Pairwise comparisons between samples are shown in Table 6. Venn diagrams were used to screen for the timing of quercetin appearance, and heatmap analysis was used to reveal changes in quercetin content.

[0058] Table 4 shows the soil sample numbers collected.

[0059]

[0060] Table 5 Mobile Phase Elution Procedure

[0061]

[0062]

[0063] Table 6. Numbering of pairwise comparisons between rhizosphere and non-rhizosphere soils

[0064]

[0065] (II) Results and Analysis

[0066] Root secretions of Chinese fir seedlings

[0067] Figure 1In this study, the root exudates of phosphorus-treated Chinese fir trees differed under competitive conditions among neighboring trees with different phylogenetic relationships. After 15 days of treatment, five identical substances were found in the root exudates of the interspecific related groups, intraspecific unrelated groups, and the interspecific unrelated groups. After 30 days of treatment, no identical substances were found in the interspecific related groups, intraspecific unrelated groups, and the interspecific unrelated groups. After 45 days of treatment, two identical substances were found in the interspecific related groups, intraspecific unrelated groups, and the interspecific unrelated groups.

[0068] Figure 2 In the study, the root exudates of *Cunninghamia lanceolata* under non-phosphorus treatment differed under competition conditions among neighboring plants constructed with different phylogenetic relationships. At 15 days of treatment, two identical substances were found in the interspecific related groups, intraspecific unrelated groups, and interspecific unrelated groups; at 30 days of treatment, one identical substance was found in each of these groups; and at 45 days of treatment, no identical substances were found in any of these groups.

[0069] Table 7 shows that under normal phosphorus supply, the roots of *Cunninghamia lanceolata* under different phylogenetic relationships did not secrete quercetin. This may be because the nutrient environment was abundant, and the *Cunninghamia lanceolata* roots did not need to identify surrounding plant species. Without phosphorus supply, the roots of *Cunninghamia lanceolata* in all phylogenetic groups secreted quercetin at 30 days of treatment, but not at 15 and 45 days. This is related to the relatively low intensity of root competition in the early stages of treatment. As treatment time increased, due to environmental nutrient stress, the degree of competition among roots increased, and *Cunninghamia lanceolata* in all phylogenetic groups regulated the degree of competition with neighboring roots by secreting quercetin.

[0070] Table 7. Common secretions and timing of quercetin appearance in the roots of Chinese fir under different phosphorus supply levels.

[0071]

[0072]

[0073] Figure 3 In the study of *Cunninghamia lanceolata*, the content of quercetin, a root exudate in *Cunninghamia lanceolata* under phosphorus-free treatment, varied under different phylogenetic relationships and competition conditions. Quercetin secretion decreased in intraspecific related groups, while it increased in intraspecific unrelated groups. Compared to the decrease in quercetin secretion in interspecific unrelated groups, quercetin secretion increased in both intraspecific related and unrelated groups, suggesting that *Cunninghamia lanceolata* may alleviate intraspecific competition by increasing quercetin secretion. The relative quercetin content in intraspecific related groups was 3.07 times that in interspecific unrelated groups, and the relative quercetin content in intraspecific unrelated groups was 1.23 times that in interspecific unrelated groups.

[0074] Example 2

[0075] (I) Test Methods

[0076] 1. Planting and management are the same as in Example 1.

[0077] 2. Harvesting: The method is the same as in Example 1. The soil sample numbering is the same as in Example 1. The pairwise comparisons of rhizosphere soil and non-rhizosphere soil are numbered the same as in Example 1.

[0078] 3. Measurement of root morphological growth indicators

[0079] Before potting, the root system was scanned using a digital scanner (STD1600 Epson USA). Morphological scans of the root system were also performed after each harvest. The root system analysis software WinRHizo (Version 4.0 B) was used to quantitatively measure and analyze morphological parameters such as total root length, surface area, volume, and average diameter. The increments of morphological parameters such as root length, root surface area, root volume, and average root diameter were calculated.

[0080] 4. Determination of biomass and phosphorus use efficiency of the tested seedlings

[0081] Fresh weights of the aboveground and underground (root) parts of each tested Cunninghamia lanceolata seedling were weighed separately, and then placed in a 105℃ oven for 30 minutes to kill the enzymes. The temperature was then adjusted to 70℃ and dried to constant weight. Biomass was measured (accurate to 0.001), and the root-to-shoot ratio was calculated. The phosphorus content of the aboveground parts and roots of the tested Cunninghamia lanceolata seedlings was determined using the H₂SO₄-HClO₄ digestion method and inductively coupled plasma atomic emission spectrometry (ICP-AES). Phosphorus utilization efficiency was calculated using the following formula.

[0082] Phosphorus accumulation (mg / stalk) -1 )=Y×PC

[0083] Phosphorus utilization efficiency (g·mg) -1 ) = Y / PA

[0084] Where Y represents the biomass of each organ or the whole plant at harvest (g·plant) -1 PC represents the phosphorus content (mg / g) of each organ or the whole plant at harvest. -1 PA represents the phosphorus accumulation in individual organs or the whole plant at harvest (mg / plant). -1 ).

[0085] 5. Measurement of root physiological activity indicators

[0086] 0.20 g of fresh root sample from each Chinese fir seedling was placed in a 5 mL centrifuge tube and immediately frozen in liquid nitrogen. The sample was then pulverized using a ball mill (45 Hz, 30 s), and mixed with an appropriate amount of phosphate buffer (pH = 7.8) for extraction. After centrifugation for 15 min (4℃, 10000 r / min), the supernatant was extracted. Subsequently, the malondialdehyde (MDA) content was determined using the thiobarbituric acid (TBA) colorimetric method, the soluble protein content was determined using a kit (Suzhou Keming Biotechnology Co., Ltd.), the superoxide dismutase (SOD) content was determined using the nitroblue tetrazolium (NBT) method, and the peroxidase (POD) activity was determined using the guaiacol colorimetric method. Take another 0.10-0.20g of fresh root samples to determine acid phosphatase activity. The specific steps are as follows: wash the roots in distilled water, dry them, and place them in a glass containing 30mL of 200mM sodium acetate buffer (pH 5.0) and 6mM p-nitrophenyl phosphate substrate mixed at a volume ratio of 1:1. Incubate at room temperature for 90min and then terminate the reaction. Determine the root acid phosphatase activity at 405nm absorbance using the p-nitrophenol standard curve.

[0087] (II) Results and Analysis

[0088] 1. Above-ground growth of Chinese fir seedlings

[0089] Figure 4 In this study, the aboveground growth of *Cunninghamia lanceolata* under different phosphorus supply levels and in neighboring plants with different kinship relationships showed differences, with kinship relationships having a significant effect on the aboveground growth of *Cunninghamia lanceolata* (p<0.05). Under normal phosphorus supply treatment, the increase in seedling height in the intraspecific kinship group was significantly greater than that in the intraspecific non-kinship group and the interspecific non-kinship group; the increase in diameter at rootstock in the intraspecific non-kinship group was significantly greater than that in the intraspecific kinship group and the interspecific non-kinship group. Under no phosphorus supply treatment, the increases in seedling height and diameter at rootstock in the intraspecific kinship group and the non-kinship group were both significantly greater than those in the interspecific non-kinship group. The increase in diameter at rootstock in the intraspecific kinship group, the non-kinship group, and the interspecific non-kinship group under the no phosphorus supply treatment were all significantly greater than those under the normal phosphorus supply treatment. For the increases in seedling height and diameter at different treatment stages, under both normal and no phosphorus supply treatments, the increases in the intraspecific kinship group, the non-kinship group, and the interspecific non-kinship group accounted for the largest proportion at 30 days of treatment.

[0090] 2. Root morphology and growth of Chinese fir seedlings

[0091] Figure 5In this study, the root morphology of *Cunninghamia lanceolata* under different phosphorus supply levels and in neighboring species with varying phylogenetic relationships exhibited differences. The effects of phylogenetic relationships, phosphorus supply levels, and their interactions were more pronounced at treatment 45 days. Under normal phosphorus supply, the root length of *Cunninghamia lanceolata* in the intraspecific phylogenetic group was significantly greater than that in the interspecific non-phylogenetic group at treatment 15 days (p<0.05). However, at treatment 30 days, the root length and surface area of ​​both the intraspecific phylogenetic and non-phylogenetic groups were significantly smaller than those in the non-phylogenetic group. At treatment 45 days, the differences in root morphology indicators between the intraspecific phylogenetic and non-phylogenetic groups and between the interspecific non-phylogenetic groups were not statistically significant (p>0.05). Under no phosphorus supply, the differences in root morphology indicators between the intraspecific phylogenetic and non-phylogenetic groups and between the interspecific non-phylogenetic groups were not statistically significant at treatments 15 and 30 days. At treatment 45 days, the root length, surface area, volume, and average diameter of *Cunninghamia lanceolata* in the intraspecific phylogenetic and non-phylogenetic groups were significantly greater than those in the interspecific non-phylogenetic group. Under different phosphorus supply levels, the root length, surface area, volume, and average diameter of the intermolecular relatives of Cunninghamia lanceolata in the non-phosphorus supply treatment were significantly greater than those in the normal phosphorus supply treatment.

[0092] 3. Biomass distribution pattern of Chinese fir seedlings

[0093] Figure 6 In this study, under different phosphorus supply levels, the biomass distribution pattern of *Cunninghamia lanceolata* under competition from neighboring plants with different phylogenetic relationships varied. Phylogenetic relationships significantly affected root biomass at 45 days (p<0.05). Under normal phosphorus supply, the aboveground biomass and total biomass of the intraspecific related groups were significantly greater than those of the intraspecific non-related groups at 45 days. Under no phosphorus supply, there were no significant differences in the biomass of different organs and the total biomass of *Cunninghamia lanceolata* in the intraspecific related and non-related groups, or in the interspecific related groups, throughout the entire treatment period (p>0.05). Under different phosphorus supply levels, the aboveground biomass of the intraspecific related groups under the no-phosphorus supply treatment was significantly greater than that under the normal phosphorus supply treatment.

[0094] 4. Phosphorus utilization efficiency of Chinese fir seedlings

[0095] Figure 7 In this study, phosphorus use efficiency of *Cunninghamia lanceolata* varied under different phosphorus supply levels and in neighboring plants with different kinship relationships. Kinship, phosphorus supply level, and interactions all had significant effects on phosphorus use efficiency in both the aboveground and whole plant parts of *Cunninghamia lanceolata* (p<0.05). Under normal and no phosphorus supply treatments, the phosphorus use efficiency in both the aboveground and whole plant parts of the intraspecific related and non-related groups was significantly higher than that in the interspecific non-related groups at 45 days of treatment. Under different phosphorus supply levels, the whole plant phosphorus use efficiency in the intraspecific non-related group under normal phosphorus supply treatment was significantly higher than that under no phosphorus supply treatment; the whole plant phosphorus use efficiency in the intraspecific related group under no phosphorus supply treatment was significantly higher than that under normal phosphorus supply treatment; and the aboveground and whole plant phosphorus use efficiency in the intraspecific non-related group under normal phosphorus supply treatment was significantly higher than that under no phosphorus supply treatment.

[0096] 5. Physiological characteristics of the root system of Chinese fir seedlings

[0097] Figure 8 In this study, the changes in root physiological indicators of *Cunninghamia lanceolata* under different phosphorus supply levels and in neighboring plants with different kinship relationships were different. Kinship, phosphorus supply level, and interaction had a more significant impact on SOD and POD in *Cunninghamia lanceolata* roots. Under normal phosphorus supply treatment, the acid phosphatase and POD in the roots of the intraspecific related and non-related groups were significantly higher than those in the interspecific non-related groups at 15 days of treatment (p<0.05). The SOD and POD in the roots of the intraspecific related and non-related groups were significantly higher than those in the interspecific non-related groups at 30 days of treatment. The soluble protein content in the roots of the interspecific non-related groups was significantly higher than that in the intraspecific related and non-related groups at 45 days of treatment. Under the phosphorus-free treatment, at 15 days, the root SOD and POD levels in the intraspecific related and non-related groups were significantly higher than those in the interspecific non-related groups. Simultaneously, the root SOD and POD levels in the intraspecific related groups were significantly higher than those in the intraspecific non-related groups. At 30 days, the SOD, POD, and soluble protein content in the roots of *Cunninghamia lanceolata* in the intraspecific related groups were significantly higher than those in the intraspecific non-related groups. At 45 days, the MDA and soluble protein content in the roots of *Cunninghamia lanceolata* in the interspecific non-related groups were significantly higher than those in the intraspecific non-related groups. Under different phosphorus supply levels, at 15 and 30 days, the root SOD and POD levels in the intraspecific related groups under the phosphorus-free treatment were significantly higher than those under the normal phosphorus supply treatment. At 45 days, the root POD and soluble protein content in the intraspecific related groups under the phosphorus-free treatment were significantly higher than those under the normal phosphorus supply treatment. At 45 days, the MDA and soluble protein content in the interspecific non-related groups under the phosphorus-free treatment were significantly higher than those under the normal phosphorus supply treatment.

[0098] By examining indicators such as aboveground growth, root morphology, biomass allocation, phosphorus use efficiency, and root physiological characteristics of *Cunninghamia lanceolata* seedlings, significant differences were found in root growth among intraspecific related groups, intraspecific unrelated groups, and interspecific unrelated groups. This indicates that significant root competition exists in *Cunninghamia lanceolata* regardless of intraspecific or interspecific kinship relationships. Furthermore, the presence or absence of quercetin also resulted in different root growth patterns, leading to variations in aboveground seedling growth. Therefore, it can be concluded that quercetin can indeed regulate intraspecific and interspecific root competition in forest trees.

[0099] Example 3

[0100] (I) Test Materials

[0101] The experimental materials included two clonal lines of Chinese fir, Yang 020 and Yang 061, and one-year-old seedlings of the precious native broad-leaved tree species, Phoebe bournei. The Yang 020 clonal line was characterized by its narrow crown and tolerance to poor soil conditions, while the Yang 061 clonal line was characterized by its wide crown, strong resistance, and strong ability to seek phosphorus in its roots under low phosphorus stress. The selected Phoebe bournei is a common species in mixed Chinese fir forests, characterized by its rapid growth, beautiful tree shape, and relatively straight trunk. All seedlings were required to be uniformly growing, healthy, and free from pests and diseases.

[0102] (II) Experimental Design

[0103] Three planting patterns were established: an intraspecific related group (clones of *Cunninghamia lanceolata* 'Yang 061' + 'Yang 061'), an intraspecific unrelated group (clones of *Cunninghamia lanceolata* 'Yang 061' + 'Yang 020'), and an interspecific unrelated group (clones of *Cunninghamia lanceolata* 'Yang 061' + *Phoebe zhennan*), to create a competitive environment between neighboring plants with different kinship relationships. KH₂PO₄ was used as the phosphorus source, and each planting pattern was configured with a phosphorus-free setting (P 0.0 mg·kg⁻¹). -1 KH2PO4), low phosphorus supply (P1, 0.03 mg·kg -1 KH2PO4), moderate phosphorus supply (P2, 0.5 mg·kg -1 KH2PO4) and normal phosphorus supply (P3, 12 mg·kg) -1 Four phosphorus-supplying treatments (KH2PO4, control group) were used, with quercetin concentrations ranging from 0.08 to 0.48 μg·g⁻¹. -1 Three different dosage treatments of quercetin were designed: 0 mL / plant, 0.3 mL / plant, and 0.8 mL / plant, with each treatment biologically replicated five times. A total of 540 pots were planted. Two seedlings were planted in each pot, with a horizontal spacing of 10 cm between the two seedlings, creating a competitive environment between neighboring plants of different phylogenetic relationships. Every 5 days, each pot was watered with 400 mL of phosphorus solution of different concentrations, with insufficient potassium... + From an equal amount of KCl (12 mg·kg) -1 To ensure the seedlings' nutritional needs during potted cultivation, 400 mL of modified Hoagland nutrient solution (Table 1) with a pH of 5.8 was applied to the roots of the seedlings treated with different phosphorus concentrations every 5 days. 200 mL of purified water was also applied to the roots of the seedlings daily at 18:00 to ensure adequate water supply. All phosphorus solutions, KCl solutions, and purified water were applied to the roots of each seedling in a clockwise direction. Quercetin was applied around the roots of each seedling after watering with the modified Hoagland nutrient solution, once every 5 days.

[0104] The experiment lasted 90 days, with seedlings harvested and relevant indicators measured every 30 days (30 days, 60 days, and 90 days). At harvest, the tested *Cunninghamia lanceolata* clone, Yang 061, was used as the target plant for relevant indicator measurements. Within the intraspecific phylogenetic group, one seedling was randomly selected from two planted *Cunninghamia lanceolata* clones. At harvest, the above-ground height and diameter of the seedlings were measured to calculate the increase in seedling growth. After harvest, the roots were rinsed thoroughly with purified water, and the surface moisture was absorbed using a cotton cloth before subsequent indicator measurements.

[0105] (III) Measurement Method

[0106] 1. Measurement of root growth indicators

[0107] After harvest, the seedling roots were scanned using a Canadian digital scanner (STD1600 EpsonUSA), and the total root length, total surface area, total average diameter, and total volume were quantitatively analyzed using the WinRHizo (version 4.0B) root analysis system software.

[0108] 2. Determination of phosphorus content

[0109] The above-ground and underground parts of the test seedlings were blanched at 108℃ and dried at 80℃ to constant weight. The samples were then pulverized and passed through a 0.2mm sieve. 0.20g of each sample was weighed and digested using the H2SO4-HClO4 digestion method (LY / T1271-1999) in the national standard (GB / T1.1-1993). The phosphorus content (g / kg) of the above-ground and underground parts was determined using ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometer) (manufactured by PerkinElmer Instruments (Shanghai) Co., Ltd., model Optima 8000).

[0110] 3. Physiological characteristic measurement

[0111] 3.1 Assay of Antioxidase Activity

[0112] Superoxide dismutase (SOD) activity was determined using the nitroblue tetrazolium (NBT) photochemical reduction method; peroxidase (POD) activity was determined using the guaiacol colorimetric method; catalase (CAT) activity was determined using the ultraviolet absorption method; and root activity was determined using dehydrogenase (DHA) assay kits (Suzhou Keming Biotechnology Co., Ltd.).

[0113] 3.2 Determination of osmotic regulators and acid phosphatase activity

[0114] The content of malondialdehyde (MDA) was determined by the thiobarbituric acid (TBA) colorimetric method; the content of soluble protein in the root system, proline content, and acid phosphatase (Apase) activity were all determined using a kit.

[0115] (iv) Data Statistical Analysis

[0116] Four-way ANOVA was performed using SPSS 19.0 to investigate the interactions of four factors—treatment time (30d, 60d, 90d), phosphorus supply level (P0, P1, P2, P3), and quercetin treatment (0mL, 0.3mL, 0.8mL)—on various morphological and physiological indicators of *Cunninghamia lanceolata* seedlings. Three-way and two-way ANOVAs were also conducted for any three or two factors. If no significant interaction was found (P>0.05), one-way ANOVA was performed, and Duncan's multiple comparison method or t-test was used to verify significance (P<0.05). All results are expressed as mean ± standard error. All figures and tables in this paper were created using Origin 8.5.

[0117] Therefore, this invention demonstrates through experiments that, without phosphorus supply, the roots of *Cunninghamia lanceolata* in different phylogenetic groups secreted quercetin after 30 days of treatment. Quercetin secretion decreased in intraspecific phylogenetic groups, while it increased in intraspecific non-phylogenetic groups. Compared to the decrease in quercetin secretion in interspecific non-phylogenetic groups, quercetin secretion increased in both intraspecific phylogenetic and non-phylogenetic groups. This suggests that *Cunninghamia lanceolata* may alleviate intraspecific competition by increasing quercetin secretion. This invention provides an important theoretical basis for the adjustment of neighboring plant competition relationships and related research.

[0118] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for regulating root competition within and / or between forest tree species using quercetin, characterized in that, Includes the following steps: Treat trees with quercetin that exhibit intraspecific and / or interspecific root competition. The concentration of quercetin is 0.08~0.48 μg·g. -1 The amount of quercetin used is 0.3~0.8 mL; The root competition relationship includes root competition caused by nutrient element deficiency, wherein the nutrient element includes phosphorus. The trees include Chinese fir and Phoebe zhennan; the Chinese fir is a Chinese fir seedling; the Phoebe zhennan is a Phoebe zhennan seedling.

2. The method according to claim 1, characterized in that, The quercetin treatment time is 30-90 days.

3. The application of the method according to claim 1 or 2 in regulating root competition within and / or between tree species.

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