Application of quercetin in regulation of intraspecific and / or interspecific root system competition relationship of forest trees

Abstract

The invention belongs to the technical field of forest competition, and particularly relates to application of quercetin in regulation of intraspecific and / or interspecific root system competition relations of forest trees. Experiments show that when phosphorus is not supplied, all cedarwood root systems in different genetic relationship groups secrete quercetin after being treated for 30 days, the secretion amount of quercetin in an intraspecific genetic group is reduced, and the secretion amount of quercetin in an intraspecific non-genetic group is increased; compared with the reduction of the quercetin secretion amount of the interspecific non-genetic group, the quercetin secretion amounts of both the intraspecific genetic group and the non-genetic group are increased, which indicates that the cunninghamia lanceolata can alleviate intraspecific competition by increasing the quercetin secretion amount. According to the method, an important theoretical basis is provided for adjustment of the competitive relationship of adjacent plants and related research, an operable technical path is also provided for forestry practice, and the method has scientific innovation and application potential.

Description

Technical Field

[0001] The present invention belongs to the technical field of forest tree competition, and particularly relates to the application of quercetin in regulating the intraspecific and / or interspecific root competition relationship of forest trees. Background Art

[0002] Cunninghamia lanceolata (Lamb.) Hook is a high-quality timber afforestation species unique to the subtropical regions of southern my country. In the southern fir growing areas, phosphorus in red soil is severely solidified, has low mobility, and is easily converted to Al. 3+ 、Fe 3+ or Ca 2 + At the same time, competition for limited resources is a common phenomenon that inevitably occurs within and between plant species. This survival competition (niche overlap) makes the phosphorus they can absorb even more scarce. Therefore, the available soil phosphorus resources that can be absorbed and utilized by Chinese fir trees during their growth and development are extremely scarce. Insufficient soil available phosphorus has become the primary problem limiting the productivity of Chinese fir plantations. During its long evolutionary process, Chinese fir has developed a series of ecological adaptation strategies to cope with low-phosphorus stress environments. Plants exhibit kin recognition behavior. When encountering closely related individuals, they adopt a more moderate competition strategy. When facing distantly related individuals or non-related individuals, they adopt an intense competition strategy. By adjusting their own growth ecological strategies, plants can timely alleviate the degree of competition with neighboring roots, thereby avoiding unnecessary waste of resources. At present, the specific pathways and mechanisms of competition between Chinese fir roots and neighboring plants in Chinese fir's ecological adaptation strategies to low-phosphorus stress environments have not been deeply understood. Research on the competitive strategies of Chinese fir roots and neighboring plants in low-phosphorus environments is of great significance to alleviating the practical problem of insufficient soil phosphorus.

[0003] Recent research has shown that when Arabidopsis thaliana plants were immersed in culture medium containing root exudates from individuals with different kinship, they developed fewer lateral roots when exposed to root exudates from closely related individuals, while more lateral roots were formed when exposed to root exudates from distantly related individuals. This phenomenon disappeared after the addition of root exudate inhibitors. This suggests that root exudates play an important role in plant root-neighbor competition strategies, potentially acting as signaling agents to regulate the degree of competition between roots and neighbors. 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, involved in antioxidant, defense, and signal transduction functions. Some studies have found that under specific conditions (such as stressful environments), plants may secrete small amounts of flavonoids (including quercetin) through their roots to regulate rhizosphere microbial communities or cope with adverse stress. Under stressful conditions, quercetin is not a predominant or widespread component of root secretion, but its role is nonetheless significant. Currently, the mechanism of quercetin's role in plant root competition strategies remains unclear. For Chinese fir, whether roots secrete quercetin and whether changes in its content play a crucial role in adjusting the root system's competitive strategy against neighboring plants when competing against them differs from the competition environment created by neighboring plants of different kinship relationships. Summary of the Invention

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

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

[0006] As a preferred option, when nutrients are deficient, trees regulate the competitive relationship with the roots of neighboring plants by secreting quercetin.

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

[0008] As a preferred embodiment, the regulation includes increasing the secretion of quercetin and alleviating intraspecific competition.

[0009] As a preferred solution, the trees include fir and nanmu.

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

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

[0012] As a preferred solution, the treatment time is 30 to 90 days.

[0013] As a preferred solution, the root competition relationship includes a root competition relationship caused by a deficiency of nutrient elements, and the nutrient elements include phosphorus.

[0014] As a preferred solution, the trees include fir and nanmu.

[0015] Beneficial effects: The present invention provides the application of quercetin in regulating intraspecific and / or interspecific root competition among forest trees. Experiments show that when phosphorus is not supplied, the roots of Chinese fir in different relatedness groups all secrete quercetin after 30 days of treatment. The quercetin secretion in the intraspecific relatedness group decreases, while the quercetin secretion in the intraspecific unrelatedness group increases. Compared with the decrease in quercetin secretion in the interspecific unrelatedness group, the quercetin secretion in both the intraspecific relatedness group and the unrelatedness group increases, indicating that Chinese fir may alleviate intraspecific competition by increasing quercetin secretion. The present invention provides an important theoretical basis for the adjustment of competitive relationships between neighboring plants and related research.

[0016] The research of the present invention shows that by exogenously applying or intervening in the secretion of quercetin, the competitive relationship between the roots of neighboring plants can be artificially adjusted, the spatial layout of trees can be optimized, resource waste can be reduced, and the productivity of plantations can be improved. At the same time, the present invention provides a new perspective for the study of plant competition mechanisms in forest ecology, and provides a theoretical basis for forestry production (such as mixed forest design and density control), helping sustainable forest management. The present invention not only deepens the understanding of plant allelopathy and competition strategies, but also provides an operational technical path for forestry practice, with both scientific innovation and application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0018] Figure 1 The amount of root secretion substances of Chinese fir with different genetic relationships under normal phosphorus supply treatment in Example 1;

[0019] Figure 2 is the amount of root secretion substances of different related Chinese fir species in Example 1 under the treatment of no phosphorus supply;

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

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

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

[0023] Figure 6 Comparison of biomass allocation patterns of Chinese fir seedlings in Example 2;

[0024] Figure 7 Comparison of phosphorus utilization efficiency of Chinese fir seedlings in Example 2;

[0025] Figure 8 Comparison of root physiological indicators of Chinese fir seedlings in Example 2;

[0026] Note: Figures 4 to 8 G, P, and G×P represent the results of two-way analysis of variance on the related measured indicators of Chinese fir seedlings based on kinship, phosphorus supply level and their interaction, respectively; Kin: intraspecific kinship group; Non-kin: intraspecific non-kinship group; Intnon-kin: interspecific non-kinship group; the same capital letters indicate that the differences between different kinship groups at the same phosphorus supply level are not significant (p>0.05); the same lowercase letters indicate that the differences between the same kinship groups at different phosphorus supply levels are not significant (p>0.05). DETAILED DESCRIPTION

[0027] The present invention provides the use of quercetin for regulating root competition within and / or between tree species. In one embodiment, when a nutrient deficiency occurs, the tree secretes quercetin to regulate root competition with neighboring trees, where the nutrient includes phosphorus. In one embodiment, the regulation includes increasing quercetin secretion to alleviate intraspecific competition. The forest trees described in the present invention include Chinese fir and nanmu. In a specific embodiment of the present invention, the Chinese fir is the asexual line of Chinese fir No. Yang 020 and the asexual line of Chinese fir No. Yang 061 (Yang Zhen, Chen Zhiqiang, Wang Nemin, et al. Effects of phosphorus application methods on photosynthetic capacity and phosphorus distribution 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 Jiaao, Li Qi, et al. Effects of underground space crowding on endogenous organic acids in the roots 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 nanmu is a 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 nanmu 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 nanmu were selected for the experiment.

[0028] The present invention embodiment shows that the root system of Chinese fir under normal phosphorus supply treatment does not secrete quercetin under the neighboring plant competition conditions of different kinship structures. The reason for this phenomenon may be that due to the abundant nutrient environment, the root system of Chinese fir does not need to identify the surrounding plant species and therefore does not need to secrete quercetin. When phosphorus is not supplied, the root systems of Chinese fir in different kinship groups all secrete quercetin when treated for 30 days, but do not secrete quercetin at 15 days and 45 days, which is related to the fact that the intensity of root competition in the early stage of treatment is not yet strong. As the treatment time increases, due to environmental nutrient stress, the degree of competition between the roots increases, and Chinese fir in different kinship groups all regulate the degree of competition with the neighboring plant roots by secreting quercetin. There are differences in the content changes of quercetin, a substance secreted by the root system of Chinese fir under the neighboring plant competition conditions of different kinship structures when phosphorus is not supplied. The relative quercetin content in the intraspecific kinship group was 3.07 times that of the interspecific unrelated group, and the relative quercetin content in the intraspecific unrelated group was 1.23 times that of the interspecific unrelated group. Quercetin secretion decreased in the intraspecific kinship group, while it increased in the intraspecific unrelated group. Compared with the decrease in quercetin secretion in the interspecific unrelated group, quercetin secretion increased in both the intraspecific kinship and unrelated groups, suggesting that Chinese fir may mitigate intraspecific competition by increasing quercetin secretion.

[0029] The present invention tests indicators such as the aboveground growth of Chinese fir seedlings, root morphology and growth, biomass allocation pattern, phosphorus utilization efficiency, and root physiological characteristics of Chinese fir seedlings. Significant differences in root growth were found among intraspecifically related, intraspecifically unrelated, and interspecifically unrelated groups. This indicates that significant root competition does occur in Chinese fir when different intraspecific and interspecific relationships exist. Furthermore, root growth performance differs in the presence or absence of quercetin, leading to differences in aboveground growth of the seedlings. Therefore, it can be concluded that quercetin can indeed regulate root competition within and between tree species.

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

[0031] The concentration of quercetin in the present invention is 0.08-0.48 μg·g -1 The dosage of quercetin is 0.3-0.8 mL. As a specific embodiment, the concentration of quercetin can be 0.08 μg·g -1 , 0.10μg·g -1 , 0.12μg·g -1 , 0.14μg·g -1 , 0.16μg·g -1 , 0.18μg·g -1 , 0.20μg·g -1 , 0.22μg·g -1 , 0.24μg·g -1 , 0.26μg·g -1 , 0.28μg·g -1 , 0.30μg·g -1 , 0.32μg·g -1 , 0.34μg·g -1 , 0.36μg·g -1 , 0.38μg·g -1 , 0.40μg·g -1 , 0.42μg·g -1 , 0.44μg·g -1 , 0.46μg·g -1 and 0.48 μg·g -1 As a specific embodiment, the amount of quercetin 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 treatment time of the present invention is 30 to 90 days. As a specific implementation manner, the treatment time can be any one of 30 days, 40 days, 50 days, 60 days, 70 days, 80 days and 90 days.

[0033] As a specific embodiment, the root competition relationship includes a root competition relationship caused by a lack of nutrient elements, and the nutrient elements include phosphorus. The trees include Chinese fir and Phoebe nanmu. In the specific embodiment of the present invention, the species of Chinese fir and Phoebe nanmu are the same as described above and will not be repeated here.

[0034] The embodiments of the present invention show that by exogenously applying or intervening in quercetin secretion, the competitive relationship between neighboring roots can be artificially adjusted, the spatial layout of trees can be optimized, resource waste can be reduced, and the productivity of plantations can be improved. At the same time, the present invention provides a new perspective for the study of plant competition mechanisms in forest ecology, and provides a theoretical basis for forestry production (such as mixed forest design and density control), thereby facilitating sustainable forest management. The present invention not only deepens the understanding of plant allelopathy and competition strategies, but also provides an operational technical path for forestry practice, which has both scientific innovation and application potential.

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

[0036] Unless otherwise specified, the present invention has no special requirements for the raw materials, and commercially available products known to those skilled in the art can be used.

[0037] 1. Experimental location: greenhouse in the Science and Technology Park of Fujian Agriculture and Forestry University. The area has a subtropical humid monsoon climate with short winters and long summers, warm and humid weather, abundant rainfall, and an average annual temperature of 19°C. The temperature of the potted greenhouse was 20-28°C, the relative humidity was >80%, the light intensity was 14h / d, and the light intensity was about 800μmol·m -2 ·s -1 .

[0038] 2. Material Preparation

[0039] Potted plant device: A polyethylene device with a pot mouth diameter of 35 cm, a pot bottom diameter of 22 cm, and a height of 27 cm was used for indoor potted plant experiments. The device was rinsed with purified water and dried before use.

[0040] Test materials: Cunninghamia lanceolata clone Yang 020 and Cunninghamia lanceolata clone Yang 061 (Yang Zhen, Chen Zhiqiang, Wang Nemin, et al. Effects of phosphorus application methods on photosynthetic capacity and phosphorus distribution of Cunninghamia lanceolata 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 Jiaao, Li Qi, et al. Effects of underground space crowding on endogenous organic acids in the roots of Cunninghamia lanceolata seedlings [J]. Journal of Fujian Agriculture and Forestry University: Natural Science Edition, 2019, 48(1): 7. One-year-old seedlings of the precious native broad-leaved tree species Phoebe bornei (Liao Xiaoli, Xue Kexin, Cai Siying, et al. Effects of exogenous silicon addition on the growth and photosynthetic characteristics of Phoebe bornei seedlings [J]. Journal of Tropical and Subtropical Botany, 2024(6). DOI:10.11926 / jtsb.4910.) were used as experimental materials. The Yang 020 Chinese fir clone has the characteristics of narrow crown width and resistance to barrenness, while the Yang 061 Chinese fir clone has the characteristics of wide crown width, strong resistance and strong root phosphorus-seeking ability under low-phosphorus stress conditions. The selected Phoebe bornei is a common tree species in mixed Chinese fir forests and has the characteristics of fast growth, beautiful tree shape and relatively straight trunk. All the seedlings tested were uniform in growth, healthy, and free of diseases and insect pests. The initial seedling height and ground diameter are shown in Table 1.

[0041] Table 1 Seedling height and ground diameter of the test seedlings before transplanting

[0042] Test seedlings Seedling height / cm Ground diameter / mm Yang 020 Chinese fir clone 30.16±0.98c 3.86±0.14c Yang 061 Chinese fir clone 34.75±0.54b 4.83±0.08b Phoebe 42.48±1.22a 6.45±0.25a

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

[0044] Cultivation medium: Wash and dry river sand that has been sieved through a 2 mm mesh and mix it with southern red soil in a volume ratio of 3:1. The chemical nutrient element content of the cultivation medium soil is shown in Table 2.

[0045] Table 2 Chemical element content of potting medium

[0046]

[0047] 3. Data Statistics and Analysis: Two-way ANOVA was performed on the relevant parameters of the participating Chinese fir seedlings. If there was no significant interaction (p>0.05), a one-way ANOVA was performed. Tukey's b and LSD multiple comparison methods were used for significant differences (p=0.05). Independent sample T-tests were used to analyze the differences in relevant parameters under different phosphorus supply levels. Data analysis was performed using SPSS 26.0 software. GraphPad Prism 9 software was used for plotting, and results are presented as mean ± standard error. Heat maps were created using TBtools software.

[0048] Example 1

[0049] (1) Test method

[0050] 1. Planting and management

[0051] Three planting patterns were set up: intraspecific related group (Yang 061 Chinese fir clone + Yang 061 Chinese fir clone), intraspecific unrelated group (Yang 061 Chinese fir clone + Yang 020 Chinese fir clone), and interspecific unrelated group (Yang 061 Chinese fir clone + Phoebe nanmu) to create a competitive environment for neighboring plants with different kinship relationships. KH2PO4 was used as the phosphorus source in the experiment. Each planting pattern was set up with no phosphorus supply (P0, the cultivation medium contained 0 mg·kg -1 KH2PO4) and normal phosphorus supply (P1, the cultivation medium contains 12 mg kg -1 Two phosphorus supply levels (KH2PO4, control group) were used for treatment, and each treatment was biologically replicated 5 times. Two seedlings were planted in each pot, with a horizontal spacing of 10 cm between the two seedlings. A seedling was planted at each end to form a competitive environment with neighboring plants of different genetic relationships. Every 5 days in the P1 treatment, 400 mL of phosphorus solution was poured into each pot, and at the same time, an equal amount of pure water (400 mL) was poured into the P0 treatment to ensure consistent soil moisture content. The insufficient K in the P0 treatment + By an equal amount of KCl (12 mg kg -1 ) solution, with 400 mL applied to each pot every 5 days. To ensure the seedlings' growth needs for other nutrients during potting, 400 mL of modified Hoagland nutrient solution (Table 3) (pH 5.5) was applied to the roots of the P0 and P1 seedlings every 5 days. Each day at 6:00 PM, 200 mL of water was applied to the roots of the seedlings. The phosphate solution, KCl solution, and purified water were applied clockwise to the roots of each seedling. The experimental period was 45 days.

[0052] Table 3 Modified Hoagland complete nutrient solution formula

[0053]

[0054] 2. Harvest

[0055] Destructive test samples were harvested every 15 days for a total of three harvests (15, 30, and 45 days after treatment). At harvest, the Yang 061 Chinese fir clone was used as the target plant for relevant index measurements. Within the intraspecific related group, one plant from the two Yang 061 clones was randomly selected. At harvest, the aboveground height and ground diameter of the Chinese fir seedlings were measured to calculate the growth increment. Afterwards, the roots were rinsed with purified water, and the surface moisture was blotted with cotton cloth for subsequent index measurements.

[0056] 3. Root Exudate Assay: Three seedlings of the Yang 061 Chinese fir clone were selected from each treatment. Rhizosphere soil (shaking method) and non-rhizosphere soil samples (potting medium located within 2 cm from the center of the culture container, within the pot edge) were collected from each seedling, yielding a total of 108 soil samples for root exudate assay (Table 4). 0.50 g of each soil sample was weighed into a 1.5 mL centrifuge tube. 500 μL of extraction solution (methanol:acetonitrile:acetic acid:water, volume ratio: 1:1:0.1:2) was added. The sample was ground at 65 Hz for 90 s, vortexed to mix thoroughly, and sonicated at 4°C for 30 min. The sample was then allowed to stand at -40°C for 1 h, vortexed for 30 s, allowed to stand at 4°C for 0.5 h, and centrifuged for 15 min (12,000 rpm at 4°C). The supernatant was collected. Another 500 μL of extraction solution was added, and the above steps were repeated for another extraction. The two extracts were mixed and lyophilized, and the lyophilized powder was reconstituted with 300 μL of extraction solution (methanol:acetonitrile:acetic acid:water volume ratio of 1:1:0.1:2), 7.5 μL of internal standard (0.14 mg / mL dichlorophenylalanine) was added, and 200 μL of the supernatant was transferred for subsequent LC-MS detection and analysis.

[0057] Instrumental analysis platform: LC-MS (Waters, UPLC; Thermo, Q Exactive); chromatographic column: ACQUITY UPLCH SSS T3 (2.1 × 100 mm 1.8 μm); chromatographic separation conditions: column temperature 40°C; 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°C; mobile phase gradient elution program is shown in Table 5. Mass spectrometry detection parameters: heater temperature, 300°C; 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°C; S-Lens RF level, 30%. Scan modes: Full scan (m / z 70–1050) and data-dependent secondary mass spectrometry (dd-MS2, Top N = 10); resolution: 70,000 (MS / MS) and 17,500 (MS / MS). Collision mode: high-energy collisional dissociation (HCD). Compound Discoverer 3.1 software (Thermo Fisher Scientific) was used to process LC-MS data spectra and perform database searches, generating qualitative and quantitative results for root exudates. The extracted data were normalized and post-edited in Excel 2010 before being organized into a two-dimensional data matrix. Pairwise comparisons between samples are shown in Table 6. Venn diagrams were used to identify the occurrence of quercetin, and heatmap analysis revealed changes in quercetin content.

[0058] Table 4 Number of soil samples collected

[0059]

[0060] Table 5 Mobile phase elution program

[0061]

[0062]

[0063] Table 6 Numbering of pairwise comparisons of rhizosphere soil and non-rhizosphere soil

[0064]

[0065] (2) Results and Analysis

[0066] Root exudates of Chinese fir seedlings

[0067] Figure 1In the study, differences in root exudates were observed among Chinese fir trees treated with phosphorus supply under neighboring competition conditions of varying kinship relationships. Five common substances were found in root exudates among the interspecific kinship group, the intraspecific non-kinship group, and the interspecific non-kinship group on day 15 of treatment. No common substances were found among the interspecific kinship group, the intraspecific non-kinship group, and the interspecific non-kinship group on day 30 of treatment. Two common substances were found among the interspecific kinship group, the intraspecific non-kinship group, and the interspecific non-kinship group on day 45 of treatment.

[0068] Figure 2 In the study, the root exudates of Chinese fir trees treated with no phosphorus showed differences under neighboring competition conditions of different kinship relationships. On day 15 of treatment, two identical substances were found among the interspecific kinship group, the intraspecific non-kinship group, and the interspecific non-kinship group. On day 30 of treatment, one identical substance was found among the interspecific kinship group, the intraspecific non-kinship group, and the interspecific non-kinship group. On day 45 of treatment, no identical substances were found among the interspecific kinship group, the intraspecific non-kinship group, and the interspecific non-kinship group.

[0069] As shown in Table 7, roots of Chinese fir trees treated with normal phosphorus supply did not secrete quercetin under conditions of neighboring competition with different kinship relationships. This may be because the nutrient environment is abundant, preventing the roots from needing to identify surrounding plant species. When phosphorus was not supplied, roots of Chinese fir trees in all kinship groups secreted quercetin at 30 days after treatment, but not at 15 or 45 days. This is related to the fact that root competition was not intense during the early stages of treatment. As treatment time increased, due to environmental nutrient stress, root competition increased, and Chinese fir trees in all kinship groups secreted quercetin to regulate the degree of competition with neighboring roots.

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

[0071]

[0072]

[0073] Figure 3 In the study, the content of quercetin, a root exudate of Chinese fir under phosphorus-free conditions, varied under neighboring competition established by different kinship relationships. Quercetin secretion decreased in the intraspecifically related group, while it increased in the intraspecifically unrelated group. Compared with the decrease in quercetin secretion in the interspecifically unrelated group, quercetin secretion increased in both the intraspecifically related and unrelated groups, suggesting that Chinese fir may mitigate intraspecific competition by increasing quercetin secretion. The relative quercetin content in the intraspecifically related group was 3.07 times that of the interspecifically unrelated group, and 1.23 times that of the interspecifically unrelated group.

[0074] Example 2

[0075] (1) Test method

[0076] 1. Planting and management are the same as those 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 numbering of the rhizosphere soil and non-rhizosphere soil for pairwise comparison is the same as in Example 1.

[0078] 3. Determination of root morphological growth indicators

[0079] Roots were scanned using a digital scanner (STD1600 Epson USA) before potting and after each harvest. Root morphology was quantitatively measured and analyzed using WinRHizo (Version 4.0 B), a root analysis system. Root length, surface area, volume, and average diameter were calculated based on the increments in root length, surface area, volume, and average diameter.

[0080] 4. Determination of biomass and phosphorus utilization efficiency of seedlings

[0081] The fresh weight of the aboveground and underground parts (roots) of each participating Chinese fir seedling was weighed, and the seedlings were placed in a 105°C oven for 30 minutes to dry. The seedlings were then dried at 70°C to a constant weight. Biomass was measured (accurate to 0.001), and the root-to-shoot ratio was calculated. Phosphorus content in the aboveground and root parts of the participating Chinese fir seedlings was determined using the H2SO4-HClO4 digestion method and inductively coupled plasma atomic emission spectrometry (ICP-AES). Phosphorus utilization efficiency was calculated according to the following formula.

[0082] Phosphorus accumulation (mg·plant -1 )=Y×PC

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

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

[0085] 5. Determination of root physiological activity indicators

[0086] A fresh root sample (0.20 g) from each participating Chinese fir seedling was placed in a 5 mL centrifuge tube and immediately frozen in liquid nitrogen. The sample was then ball milled (45 Hz, 30 s) and mixed with an appropriate amount of phosphate buffer (pH 7.8). The sample was centrifuged for 15 min (10,000 rpm at 4°C), and the supernatant was extracted. Malondialdehyde (MDA) content was determined using a thiobarbituric acid (TBA) colorimetric assay, soluble protein content was determined using a kit (Suzhou Keming Biotechnology Co., Ltd.), superoxide dismutase (SOD) content was determined using the nitroblue tetrazolium (NBT) assay, and peroxidase (POD) activity was determined using a guaiacol colorimetric assay. Another 0.10-0.20 g fresh root sample was taken to determine the acid phosphatase activity. The specific steps were as follows: the roots were washed in distilled water, dried, and placed in a glass containing 30 mL of a 1:1 mixture of 200 mM sodium acetate buffer (pH 5.0) and 6 mM disodium p-nitrophenyl phosphate substrate. The reaction was terminated after incubation at room temperature for 90 min. The root acid phosphatase activity was determined at an absorbance of 405 nm using a p-nitrophenol standard curve.

[0087] (2) Results and Analysis

[0088] 1. Growth of the aboveground part of Chinese fir seedlings

[0089] Figure 4 In the study, differences in aboveground growth of Chinese fir under different phosphorus supply levels and neighbor competition conditions established by different kinship relationships were observed, with kinship relationships significantly influencing aboveground growth (p < 0.05). Under normal phosphorus supply, the seedling height increment of the intraspecific kin group was significantly greater than that of the intraspecific non-kin and interspecific non-kin groups; the ground diameter increment of the intraspecific non-kin group was significantly greater than that of the intraspecific kin and interspecific non-kin groups. Under no phosphorus supply, the seedling height and ground diameter increments of the intraspecific kin and non-kin groups were significantly greater than those of the interspecific non-kin group. Under no phosphorus supply, the ground diameter increments of Chinese fir in the intraspecific kin, non-kin, and interspecific non-kin groups were significantly greater than those in the normal phosphorus supply treatment. Regarding seedling height and ground diameter increments at different treatment periods, under both normal and no phosphorus supply treatments, the intraspecific kin, non-kin, and interspecific non-kin groups accounted for the largest portion of the increments after 30 days of treatment.

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

[0091] Figure 5In the study, root morphology and structure of Chinese fir differed under different phosphorus supply levels and neighbor competition conditions established by different kinship relationships. The effects of kinship, phosphorus supply level, and their interaction were more pronounced at 45 days. Under normal phosphorus supply, root length of Chinese fir in the intraspecific kinship group was significantly greater than that in the interspecific unkinship group at 15 days (p < 0.05). However, root length and surface area of both the interspecific kinship and unkinship groups were significantly smaller than those of the unkinship group at 30 days. At 45 days, no significant differences in root morphology and structure were found between the intraspecific kinship and unkinship groups, or between the interspecific unkinship groups (p > 0.05). Under no phosphorus supply, no significant differences in root morphology and structure were found between the intraspecific kinship and unkinship groups, or between the interspecific unkinship groups, at 15 and 30 days. However, at 45 days, root length, surface area, volume, and average diameter of Chinese fir in the intraspecific kinship and unkinship groups were significantly greater than those in the interspecific unkinship group. Under different phosphorus supply levels, the root length, surface area, volume and average diameter of the Chinese fir in the intraspecific relatives group under the no phosphorus supply treatment were significantly greater than those under the normal phosphorus supply treatment.

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

[0093] Figure 6 In the study, biomass allocation patterns of Chinese fir differed under different phosphorus supply levels under neighboring competition conditions established by different kinship relationships. Kinship significantly affected root biomass on day 45 of treatment (p < 0.05). Under normal phosphorus supply, aboveground and whole-plant biomass of the intraspecific kin group were significantly greater than those of the intraspecific non-kin group at day 45 of treatment. Under no phosphorus supply, no significant differences in biomass of different organs or whole-plant biomass between the intraspecific kin group, non-kin group, or interspecific kin group were observed throughout the treatment period (p > 0.05). Across different phosphorus supply levels, aboveground biomass of the intraspecific kin group in the no phosphorus supply treatment was significantly greater than that in the normal phosphorus supply treatment.

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

[0095] Figure 7 Phosphorus use efficiency (PUE) of Chinese fir differed under different P supply levels and neighboring competition conditions established by different kinship relationships. Phylogeny, P supply level, and interaction all significantly affected PUE of both aboveground and whole-plant Cunninghamia lanceolata (p < 0.05). Under both normal and no P supply treatments, the PUE of both aboveground and whole-plant Cunninghamia lanceolata in the intraspecific kinship and non-kinship groups was significantly greater than that in the interspecific non-kinship group at 45 days of treatment. Under different P supply levels, the PUE of the whole-plant Cunninghamia lanceolata in the normal P supply treatment was significantly greater than that in the no P supply treatment; the PUE of the whole-plant Cunninghamia lanceolata in the no P supply treatment was significantly greater than that in the normal P supply treatment; and the PUE of both aboveground and whole-plant Cunninghamia lanceolata in the normal P supply treatment was significantly greater than that in the no P supply treatment.

[0096] 5. Physiological characteristics of Chinese fir seedling roots

[0097] Figure 8 In the study, changes in root physiological parameters of Chinese fir under different phosphorus supply levels and neighbor competition conditions of different kinship relationships were observed. Kinship, phosphorus supply level, and their interaction had a more pronounced effect on SOD and POD in Chinese fir roots. Under normal phosphorus supply, root acid phosphatase and POD in the intraspecific kinship and non-kinship groups were significantly higher than those in the interspecific non-kinship group at 15 days (p < 0.05). Root SOD and POD in the intraspecific kinship and non-kinship groups were significantly higher than those in the interspecific non-kinship group at 30 days. Root soluble protein content in the interspecific non-kinship group was significantly higher than that in the intraspecific kinship and non-kinship groups at 45 days. Under the no-P treatment, root SOD and POD in the intraspecific kinship group and the non-kinship group were significantly greater than those in the interspecific non-kinship group at 15 days of treatment. Furthermore, root SOD and POD in the intraspecific kinship group were significantly greater than those in the intraspecific non-kinship group. Root SOD, POD, and soluble protein content in the intraspecific kinship group were significantly greater than those in the intraspecific non-kinship group at 30 days of treatment. Root MDA and soluble protein content in the interspecific non-kinship group were significantly greater than those in the intraspecific non-kinship group at 45 days of treatment. Under different P supply levels, root SOD and POD in the intraspecific kinship group under the no-P treatment were significantly greater than those in the normal-P treatment at 15 and 30 days of treatment. Root POD and soluble protein content in the intraspecific kinship group under the no-P treatment were significantly greater than those in the normal-P treatment at 45 days of treatment. Root MDA and soluble protein content in the interspecific non-kinship group under the no-P treatment were significantly greater than those in the normal-P treatment at 45 days of treatment.

[0098] By testing indicators such as aboveground growth, root morphology, biomass allocation, phosphorus use efficiency, and root physiological characteristics of Chinese fir seedlings, significant differences in root growth were found among intraspecifically related, intraspecifically unrelated, and interspecifically unrelated groups. This indicates that root competition does exist among Chinese fir trees when different intraspecific and interspecific relationships exist. Furthermore, root growth also differs in the presence or absence of quercetin, leading to differences in aboveground growth of the seedlings. Therefore, it can be concluded that quercetin can indeed regulate root competition within and between forest tree species.

[0099] Example 3

[0100] (1) Test materials

[0101] One-year-old seedlings of the valuable native broadleaf tree species, Phoebe bournei, and clones Yang 020 and Yang 061 of Chinese fir were selected as experimental materials. The Yang 020 clone has a narrow crown and is tolerant to barrenness, while the Yang 061 clone has a wide crown, strong resistance, and a robust root foraging capacity under low-phosphorus stress conditions. Phoebe bournei, a common species in mixed Chinese fir forests, is characterized by rapid growth, attractive shape, and a straight trunk. All seedlings were required to be uniform, healthy, and free of pests and diseases.

[0102] (2) Experimental design

[0103] Three planting patterns were set up: intraspecific related group (Yang 061 Chinese fir clone + Yang 061 Chinese fir clone), intraspecific unrelated group (Yang 061 Chinese fir clone + Yang 020 Chinese fir clone), and interspecific unrelated group (Yang 061 Chinese fir clone + Phoebe nanmu) to create a competition environment between neighboring plants with different kinship relationships. KH2PO4 was used as the phosphorus source in the experiment. Each planting pattern was set up with no phosphorus supply (P0, 0 mg·kg -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 KH2PO4, control group) were treated with four phosphorus supply levels, and the concentration of quercetin was 0.08-0.48 μg·g -1 Three different dosages of quercetin were designed, namely: 0 mL / plant, 0.3 mL / plant, and 0.8 mL / plant. Each treatment was repeated 5 times, and 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. A seedling was planted at each end to form a competitive environment with neighboring plants of different genetic relationships. 400 mL of phosphorus solution of different concentrations was poured into each pot every 5 days. The insufficient K + By an equal amount of KCl (12 mg kg -1 ) solution instead. In order to ensure the growth of seedlings during potting for other nutrient elements, 400 mL of the modified Hoagland nutrient solution (Table 1) was applied to the roots of the test seedlings treated with different phosphorus concentrations every 5 days, and the pH of the nutrient solution was 5.8. At 18:00 every afternoon, 200 mL of pure water was poured on the roots of the test seedlings to ensure the water demand for seedling growth. The above-mentioned phosphorus solution, KCl solution and pure water were all applied to the roots of each test seedling in a clockwise direction. Quercetin was applied around the roots of each seedling after pouring the modified Hoagland nutrient solution, and was poured once every 5 days.

[0104] The experimental period lasted 90 days, with seedlings harvested every 30 days (30, 60, and 90 days) and relevant indices measured. At harvest, the participating Chinese fir clone, Yang 061, was used as the target plant for relevant indices. Within the intraspecific related group, one plant from the two planted clones, Yang 061, was randomly selected. At harvest, the aboveground height and ground diameter of the Chinese fir seedlings were measured, and the growth increment was calculated. After harvest, the roots were rinsed with purified water, and the surface moisture was blotted with cotton cloth for subsequent indices measurement.

[0105] (3) Determination method

[0106] 1. Root Growth Index Determination

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

[0108] 2. Phosphorus content determination

[0109] The above-ground and underground parts of the test seedlings were fixed at 108°C, dried at 80°C to constant weight, crushed with a grinder and passed through a 0.2 mm pore size sieve. 0.20 g 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 P content (g / kg) in the above-ground and underground parts was determined using ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometer) (PerkinElmer Instruments (Shanghai) Co., Ltd., model Optima 8000).

[0110] 3. Physiological Characteristics Measurement

[0111] 3.1 Antioxidant enzyme activity assay

[0112] The superoxide dismutase (SOD) activity was determined by the nitroblue tetrazolium (NBT) photoreduction method; the peroxidase (POD) activity was determined by the guaiacol colorimetric method; the catalase (CAT) activity was determined by the UV absorption method; and the root dehydrogenase (DHA) activity was determined by the kit (Suzhou Keming Biotechnology Co., Ltd.).

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

[0114] The malondialdehyde (MDA) content was determined by thiobarbituric acid (TBA) colorimetric method; the root soluble protein content, proline content and acid phosphatase (Apase) activity were determined by kits.

[0115] (IV) Data statistical analysis

[0116] Four-way ANOVAs were performed using SPSS 19.0 to analyze the interaction effects of treatment time (30 days, 60 days, and 90 days), phosphorus supply level (P0, P1, P2, and P3), and quercetin treatment (0 mL, 0.3 mL, and 0.8 mL) on various morphological and physiological parameters of Chinese fir seedlings. Three-way and two-way ANOVAs were also performed between any three or two factors. If there was no significant interaction (P>0.05), one-way ANOVAs were performed, and significant comparisons (P<0.05) were analyzed using Duncan's multiple comparison method or t-test. All data are expressed as mean ± standard error. Figures and tables in this article were created using Origin 8.5.

[0117] Thus, the present invention experimentally demonstrates that, when phosphorus is not supplied, the roots of Chinese fir in different relatedness groups all secrete quercetin after 30 days of treatment. The amount of quercetin secreted by the intraspecific relatedness group decreases, while that of the intraspecific unrelatedness group increases. Compared with the decrease in quercetin secretion in the interspecific unrelatedness group, the amount of quercetin secretion in both the intraspecific relatedness group and the unrelatedness group increases, indicating that Chinese fir may alleviate intraspecific competition by increasing quercetin secretion. The present invention provides an important theoretical basis for the adjustment of competition between adjacent plants and related research.

[0118] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Application of quercetin in regulating root competition within and / or between tree species.

2. The use according to claim 1, characterized in that When nutrients are deficient, trees regulate the competitive relationship with the roots of neighboring trees by secreting quercetin.

3. The use according to claim 2, characterized in that The nutrient elements include phosphorus.

4. The use according to claim 2, characterized in that The regulation includes increasing the secretion of quercetin and alleviating intraspecific competition.

5. The use according to any one of claims 1 to 4, characterized in that: The trees include fir and nanmu.

6. A method for regulating intraspecific and / or interspecific root competition among trees using quercetin, characterized in that: The following steps are involved: Treatment of trees with intraspecific and / or interspecific root competition with quercetin.

7. The method according to claim 6, characterized in that The concentration of quercetin is 0.08-0.48 μg·g -1 The dosage of quercetin is 0.3-0.8 mL.

8. The method according to claim 6, characterized in that The treatment time is 30 to 90 days.

9. The method according to claim 6, characterized in that The root competition relationship includes a root competition relationship caused by a deficiency of nutrient elements, and the nutrient elements include phosphorus.

10. The method according to any one of claims 6 to 9, characterized in that: The trees include fir and nanmu.

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