Method for regulating and controlling propagation of populus euphratica and stability of rhizosphere microorganisms through distributary seepage rotation irrigation

Abstract

The invention discloses a method for regulating and controlling propagation of populus euphratica and stability of rhizosphere microorganisms through branched seepage rotation irrigation, and relates to the technical field of ecological restoration. According to the method, by differentiating branched seepage rotation irrigation modes, precisely matching habitat requirements of sexual and clone propagation populus euphratica seedlings, and adopting a long-interval and gradually-water-increasing irrigation mode for the clone propagation seedlings, the propagation of populus euphratica and the stability of rhizosphere microorganisms are regulated and controlled; according to the method, soil salt leaching and nitrogen and phosphorus accumulation can be promoted, functional florae such as rhizosphere actinomycetes and thick-wall bacteria are activated, complex organic matter is degraded by secreting extracellular enzyme, the phosphorus dissolving capacity is enhanced, the nutrient obtaining efficiency of seedlings in a medium-high-salt environment is improved, a short-interval and high-water-quantity mode is adopted for sexual propagation seedlings, a low-salt and high-humidity habitat is created, and the survival rate of the seedlings is increased. Growth-promoting florae such as pseudomonas are enriched, and by secreting growth hormones and antagonistic substances, the planting capacity of the seedlings in a humid environment is enhanced, so that directional regulation and control of the two breeding modes are realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of ecological restoration, in particular to a method for regulating Populus euphratica reproduction and rhizosphere microbial stability. BACKGROUND

[0002] As the only high and tall deciduous tree that can form a forest in desert areas, Populus euphratica plays an irreplaceable role in maintaining ecological safety, blocking desert expansion, and protecting biodiversity. However, due to the combined effects of climate change (reduced runoff and intensified evapotranspiration due to rising temperatures) and human activities, long-term flow interruption and reduced groundwater recharge have disrupted the soil-groundwater-vegetation water transmission chain, hindering natural regeneration of Populus euphratica populations and increasing the risk of degradation. Scientific intervention is urgently needed to maintain its survival and reproduction.

[0003] In the prior art, measures for Populus euphratica ecological restoration are mainly focused on macro-level ecological water transport and vegetation reseeding, and there are obvious deficiencies in precise regulation of reproduction strategies and rhizosphere microenvironment. On the one hand, traditional water transport methods do not distinguish the habitat needs of different reproduction methods of Populus euphratica, making it difficult to form the humid and low-salt habitat required for sexual reproduction, and the root expansion conditions for clonal reproduction are not targeted for optimization. On the other hand, the regulation of rhizosphere microorganisms lacks systematicness, and existing research has not revealed the correlation mechanism between hydrological disturbance and microbial community stability, making it impossible to improve the adaptability of Populus euphratica by regulating microbial functions (such as nutrient transformation and stress tolerance). In addition, the existing technology has not established the threshold relationship between soil water-salt-nutrient and reproduction mode conversion, resulting in insufficient precision of restoration measures and difficulty in achieving sustainable regeneration of Populus euphratica populations.

[0004] In summary, the core problem of Populus euphratica population degradation is that the response mechanism of reproduction strategies and rhizosphere microenvironment to hydrological changes has not been fully grasped. The existing technology lacks differentiated regulation means for different reproduction methods and ignores the key role of rhizosphere microorganisms in resource acquisition and stress adaptation. Therefore, developing a technology that can precisely regulate Populus euphratica reproduction and optimize rhizosphere microbial stability is of great significance for improving the restoration effect of Populus euphratica forests in arid regions and enriching the theory of desert ecological restoration. SUMMARY

[0005] The purpose of the present application is to make up for the shortcomings of the prior art, and provide a method for regulating populus euphratica reproduction and rhizosphere microbial stability by bifurcated infiltration round irrigation, which can precisely match the habitat needs of sexual and clonal reproduction populus euphratica seedlings through different bifurcated infiltration round irrigation modes, use a long interval and gradually increasing water volume irrigation mode for clonal reproduction seedlings, promote soil salt leaching and nitrogen and phosphorus accumulation, activate functional microbial flora such as actinobacteria and firmicutes, which can degrade complex organic matter through exoenzyme secretion, enhance phosphorus dissolution capacity, and improve the nutrient acquisition efficiency of seedlings in medium and high salt environments, and use a short interval and high water volume mode for sexual reproduction seedlings, create a low-salt and high-humidity habitat, enrich Pseudomonas for promoting microbial flora, and secrete growth hormones and antagonistic substances to strengthen the seedling's ability to plant in a humid environment, and realize directional regulation of the two reproduction methods.

[0006] To solve the above technical problems, the present application provides the following technical scheme: a method for regulating populus euphratica reproduction and rhizosphere microbial stability by bifurcated infiltration round irrigation, the specific steps of which are as follows: S100, selecting a river bank in a dry area where populus euphratica is distributed as a test area, setting a natural growth control area CKc for clonal reproduction populus euphratica seedlings and bifurcated infiltration round irrigation experimental areas D1, D2 and D3, and setting a natural growth control area CKs for sexual reproduction populus euphratica seedlings and bifurcated infiltration round irrigation experimental areas M1, M2 and M3 in the test area, and the experimental areas are spaced apart by ≥5m; S200, for clonal reproduction populus euphratica seedlings, no irrigation treatment is performed in the natural growth control area CKc, and specific water disturbance modes are used in the bifurcated infiltration round irrigation experimental areas D1, D2 and D3; S300, for sexual reproduction populus euphratica seedlings, no irrigation treatment is performed in the natural growth control area CKs, and specific water disturbance modes are used in the bifurcated infiltration round irrigation experimental areas M1, M2 and M3; S400, through the bifurcated infiltration round irrigation of S200 and S300, the soil physical and chemical properties of each experimental area are monitored, and the target reproduction strategy and rhizosphere microbial community stability are promoted; S500, the rhizosphere microbial community structure of populus euphratica seedlings is regularly monitored, and the robustness and vulnerability of the rhizosphere microbial symbiotic network are maintained by adjusting the irrigation amount and interval days.

[0007] Further, in S100, when setting the experimental area for clonal reproduction populus euphratica seedlings, healthy populus euphratica mother plants are selected in each experimental area, a root cutting ditch is mechanically excavated within a range of 5-10m from the mother plant, the ditch is 70-90cm deep, 30-50cm wide and 15-20m long, the root system of the populus euphratica mother plant with a diameter of 1-3cm in the range of 0-30cm above the ground is artificially cut, and the capillary roots with a diameter of <1cm and the lateral roots with a diameter of >3cm on the root system are reserved.

[0008] Furthermore, in S100, when setting up an experimental area for sexually propagated Populus euphratica seedlings, the experimental area is prepared in the previous year by excavating water collection troughs. The water collection troughs are 20-30cm deep and 50-60cm wide. During the experiment, healthy adult female Populus euphratica trees are selected, and their plump fruit spikes are collected and inserted into the top 10cm soil layer of the water collection troughs in each experimental area, with a spacing of 1-2m between cuttings.

[0009] Furthermore, in S200, the specific water disturbance patterns in the diversion irrigation experimental areas D1, D2, and D3 for cloning and propagating Populus euphratica seedlings are as follows: Zone D1: Irrigation interval 11 days, water volume 6m³ / 10m², lasting for 2 days; Zone D2: Irrigation interval 23 days, water volume 7m³ / 10m², for 3 consecutive days; Zone D3: 33-day interval, irrigation volume 8m³ / 10m², lasting for 4 days.

[0010] Furthermore, in S300, the specific water disturbance patterns in the diversion irrigation experimental areas M1, M2, and M3 for sexually propagated Populus euphratica seedlings are as follows: M1 Zone: Irrigation interval 5 days, water volume 150m³ / 666.7m², duration 45 minutes; M2 Zone: Irrigation interval 5 days, water volume 200m³ / 666.7m², duration 35 minutes; M3 Zone: Irrigation interval 3 days, water volume 390m³ / 666.7m², duration 25 minutes.

[0011] Furthermore, in the S400, by removing vegetation at each sampling point, soil samples are collected from the soil layer at a depth of 0-40cm for monitoring of soil physicochemical properties. The monitoring indicators include: soil moisture content determined by the drying method, pH determined by the potentiometric method, organic carbon content determined by the potassium dichromate-external heating method, total nitrogen content determined by the PE-2400CHN analyzer, total phosphorus content determined by the molybdate / stannous chloride method, and total salt content determined by the gravimetric method.

[0012] Furthermore, in S500, the regulation of the rhizosphere microbial community includes promoting the enrichment of Actinobacteria, Acidobacteria, and Bacillus.

[0013] Furthermore, the monitoring method for the rhizosphere microbial community is as follows: Soil from the surface of the roots of Populus euphratica seedlings is brushed off onto sterile filter paper using a sterile brush and collected in 5mL sterile centrifuge tubes. The rhizosphere soil samples are stored at -80℃. DNA extraction and amplification For the V3-V4 region of the gene, primers 338F and 806R were used. The sequencing platform sequences the amplification product, and the community structure and network stability are analyzed by QIIME2, Networkx and Gephi.

[0014] Further, the method is suitable for ecological restoration of Populus euphratica forest in arid regions.

[0015] Compared with the prior art, the method has the following beneficial effects: I. The present application precisely matches the habitat requirements of sexually and clonally propagated Populus euphratica seedlings by differentiating the split infiltration irrigation mode, uses a long interval and gradually increasing water volume irrigation mode for clonally propagated seedlings to promote soil salt leaching and nitrogen and phosphorus accumulation, activates functional flora such as Actinobacteria and Firmicutes, which degrade complex organic matter by secreting extracellular enzymes, enhance phosphorus dissolution capacity, and improve the seedling's nutrient acquisition efficiency in a medium-high salt environment, and uses a short interval and high water volume mode for sexually propagated seedlings to create a low-salt and high-humidity habitat, enriches Pseudomonadales and other beneficial flora, secretes growth hormones and antagonistic substances to strengthen the seedling's ability to colonize in a humid environment, and realizes directional regulation of the two propagation methods.

[0016] II. The present application builds a mechanism for maintaining the stability of rhizosphere microbial communities by regulating irrigation interval, water volume and duration, under medium-high irrigation volume, clonally propagated seedlings form a flora structure coexisting with Actinobacteria and Firmicutes, which enhance the community's anti-interference ability through functional complementation, and the rhizosphere of sexually propagated seedlings increases the abundance of Pseudomonadales and other nutrient-rich flora, strengthens positive interactions between flora, and improves the efficiency of material and energy transfer, at the same time, the differential regulation of irrigation mode keeps soil water content, salt and nutrients within the Populus euphratica propagation conversion threshold, promotes the formation of a synergistic adaptation mechanism between rhizosphere microorganisms and plants, and enhances the comprehensive tolerance of Populus euphratica to drought and saline-alkali stress.

[0017] Other advantages, objects, and features of the application will be apparent to those skilled in the art from the following specification and accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0019] Figure 1It is a flow chart of a method for regulating Populus euphratica propagation and rhizosphere microbial stability by bifurcation infiltration wheel irrigation; Figure 2 It is a schematic diagram of the location and experimental arrangement of the research area; Figure 3 It is a correlation analysis diagram of soil factors in the growth area of Populus euphratica seedlings propagated by cloning; Figure 4 It is a correlation analysis diagram of soil factors in the growth area of Populus euphratica seedlings propagated by sexual reproduction; Figure 5 It is a redundancy analysis diagram of the soil habitat selection of Populus euphratica propagation methods; Figure 6 It is a rhizosphere microbial community composition diagram at the order level; Figure 7 It is a LEfSe analysis diagram of rhizosphere soil bacteria of Populus euphratica seedlings of two propagation methods; Figure 8 It is a neutral model analysis diagram of rhizosphere soil bacteria of Populus euphratica seedlings of two propagation methods; Figure 9 It is a network topology structure characteristic diagram of rhizosphere soil bacteria community network of Populus euphratica seedlings of two propagation methods; Figure 10 It is a Zi-Pi diagram of OTUs of rhizosphere soil bacteria community network of Populus euphratica seedlings of two propagation methods; Figure 11 It is a stability analysis diagram of rhizosphere soil bacteria community network of Populus euphratica seedlings of two propagation methods. DETAILED DESCRIPTION

[0020] In order to further illustrate the technical means and effects adopted by the present application to achieve the predetermined object, the specific embodiments, structures, characteristics and effects according to the present application will be described in detail below in combination with the drawings and preferred embodiments.

[0021] Example 1 In this embodiment, a research area of a method for regulating Populus euphratica propagation and rhizosphere microbial stability by bifurcation infiltration wheel irrigation is set in the bifurcation infiltration wheel irrigation ecological restoration test area (geographical coordinates 34.20°-43.39°N, 71.39°-93.45°E) in the lower reaches of the Tarim River, as shown in Figure 2 (A), which belongs to a typical continental arid climate, with an annual rainfall of only 20-40 mm and an annual evaporation of 2500-3000 mm. The main soil type is calcified saline soil. The vegetation composition in the test area is dominated by Populus euphratica as the dominant tree, accompanied by shrubs including Fraxinus velutina, Haloxylon ammodendron, Alhagi sparsifolia, Apocynum venetum, etc., and herbaceous plants mainly including Phragmites australis and Apocynum venetum. These plants together form a green ecological corridor to block the invasion of the Kuruq Desert and the Taklimakan Desert into the oasis.

[0022] Firstly, enter S100 stage, clone propagation of Populus euphratica water disturbance control experiment, experimental area includes 1 control area and 3 branch infiltration wheel irrigation experimental area, as follows: Control area (CKc): Clone propagation of Populus euphratica seedlings natural growth area, do not carry out any irrigation, for reflecting the growth characteristics under natural conditions.

[0023] Branch infiltration wheel irrigation experimental area (D1, D2, D3): 3 kinds of water disturbance modes are set respectively, interval is 8 meters (≥5 meters), to avoid water disturbance.

[0024] Then, enter S200 stage, according to the phenological characteristics of Populus euphratica, carry out root cutting operation (such as Figure 2 (C) is the field photo of mechanical cutting of Populus euphratica root system), in CKc, D1, D2, D3 area, each select healthy Populus euphratica mother plant, within 5-10 meters from the mother plant, mechanical excavation root cutting ditch according to uniform specification: ditch depth 70-90 centimeters (average 80 centimeters), ditch width 30-50 centimeters (average 40 centimeters), ditch length 15-20 meters (average 18 meters), a total of 18 root cutting ditches are excavated.

[0025] When cutting roots, manually cut the mother plant roots with diameter of 1-3 centimeters within 0-30 centimeters of the ground surface (the purpose is to block the preferential nutrient supply of the mother plant to the seedlings, and promote the independent growth of the seedlings), while retaining the capillary roots with diameter <1 centimeter (maintaining the basic nutrient absorption function) and lateral roots with diameter >3 centimeters (ensuring the basic root system structure for seedling survival) on the roots, 7 days after the completion of root cutting, 5 5m×5m fixed quadrats are set at 50 centimeters outside each root cutting ditch, as sampling points for subsequent seedling growth monitoring.

[0026] Subsequently, enter S300 stage, sexual reproduction of Populus euphratica seedling experimental area setting, experimental area includes 1 control area and 3 branch infiltration wheel irrigation experimental area, as follows: Control area (CKs): Natural growth area of sexual reproduction of Populus euphratica seedlings, do not carry out irrigation, as natural germination control.

[0027] Branch infiltration wheel irrigation experimental area (M1, M2, M3): 3 kinds of water disturbance modes are set respectively, interval is 10 meters (≥5 meters), to avoid water disturbance.

[0028] Microtopography is arranged in M1, M2, M3 and CKs area, water accumulation groove is excavated (groove depth 20-30 centimeters, groove width 50-60 centimeters, groove spacing 2 meters), to form local humid microenvironment beneficial to seed germination (such as Figure 2(B) shows the experimental layout, the red area in the experimental layout is the sexual reproduction experimental area), healthy adult female Populus euphratica was selected, and the fruit clusters with full grains (thousand-grain weight ≥0.8 g) were collected. The same number of cuttings were inserted into the 10 cm soil layer on the surface of the water accumulation tank in each experimental area at an interval of 1-2 m (to ensure the growth space of seedlings). On the 10th day after cutting, 5 5m x 5m fixed quadrats were set in each area, and the germinated seedlings were marked for subsequent monitoring.

[0029] The irrigation treatment in the clonal propagation area was implemented during the growing season every year, and the specific mode was as follows: D1 area (mode F1): irrigation interval 11 days, single irrigation amount 6 cubic meters / 10 square meters (i.e. 6000 liters of irrigation per 10 square meters), duration 2 days (irrigation twice a day to ensure that the water infiltrates to the root layer); D2 area (mode F2): irrigation interval 23 days, single irrigation amount 7 cubic meters / 10 square meters, duration 3 days (irrigation period same as D1 area); D3 area (mode F3): irrigation interval 33 days, single irrigation amount 8 cubic meters / 10 square meters, duration 4 days (increase irrigation period, reduce high temperature evaporation loss).

[0030] CKc area (mode Fckc): no irrigation treatment throughout the natural growth.

[0031] When implementing, a water pump (power 5.5kW) was connected with a flowmeter to accurately control the irrigation amount, the irrigation water was taken from the river near the test area, and the irrigation method was flooding irrigation to ensure that the water evenly covers the experimental area (such as Figure 2 (D) shows the irrigation site photo).

[0032] The irrigation parameters in the sexual reproduction area, the irrigation treatment was implemented during the key period of seed germination every year, and the specific mode was as follows: M1 area (mode F4): irrigation interval 5 days, single irrigation amount 150 cubic meters / 666.7 square meters (i.e. 150 tons of irrigation per mu), duration 45 minutes.

[0033] M2 area (mode F5): irrigation interval 5 days, single irrigation amount 200 cubic meters / 666.7 square meters, duration 35 minutes.

[0034] M3 area (mode F6): irrigation interval 3 days, single irrigation amount 390 cubic meters / 666.7 square meters, duration 25 minutes.

[0035] CKs area (mode Fcks): no irrigation treatment throughout the natural germination.

[0036] Irrigation equipment and water source with clonal propagation area, ensure the amount of irrigation precision control, irrigation treatment after the end of the fixed sample in each "five-point sampling method" to collect 0-40 centimeters of soil layer soil samples (every 10 centimeters for a layer, mixed for a sample), each sample repeated 3 times, the index and method as follows: Soil water content (SW): take 100 grams of fresh soil, oven dried to constant weight in 105-110 ℃ oven, record the weight (M1), calculate the moisture content (SW= (fresh soil weight-dried soil weight) / fresh soil weight x 100%).

[0037] Total salt content (TS): take the dried soil sample, add H2O2 to remove organic matter and weigh (M2), the total salt content is the difference between M1 and M2 (TS=M1-M2).

[0038] Total nitrogen (SN): measured by PE-2400CHN analyzer (PerkinElmer, Foster City, CA, USA).

[0039] Total phosphorus (SP): after digestion with H2SO4-H2O2-HF, the molybdate / stannous chloride method is used to determine.

[0040] Organic carbon: measured by potassium dichromate-external heating method.

[0041] pH value: mixed according to the ratio of water to soil 1:5, measured by potential method.

[0042] In each fixed sample, 3 healthy seedlings were randomly selected, and the soil on the root surface (defined as rhizosphere soil) was gently brushed off with a sterile brush and collected in a 5 mL sterile centrifuge tube, immediately stored in a-80℃ refrigerator (for subsequent DNA extraction).

[0043] DNA extraction and sequencing: according to the operation instruction of soil bacteria DNA extraction kit (Soil DNA Kit, D5625-01), extract DNA, repeat extraction 3 times for each sample, mix and use for amplification, use primers 338F (5'-ACTCCTACGGGAGGCAGCA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') to amplify the V3-V4 region of 16S rRNA gene.

[0044] PCR reaction system (20 μL): 5 x FastPfu Buffer 4 μL, dNTPs (2.5 mmol / L) 2 μL, forward and reverse primers (5 μmol / L) 8 μL each, FastPfu Polymerase 0.4 μL, DNA template 10 ng, reaction conditions: 95 ℃ pre-denaturation for 5 minutes; 95 ℃ denaturation for 30 seconds, 55 ℃ annealing for 30 seconds, 72 ℃ extension for 30 seconds, a total of 35 cycles; finally 72 ℃ extension for 10 minutes.

[0045] After the PCR product was detected by 1% agarose gel electrophoresis, Illumina MiSeq sequencing platform was used for sequencing, FLASH software was used for splicing double-end sequence, Uparse and Usearch were used for OTU (operational taxonomic unit) clustering (threshold value 0.03), RDPClassifier software was used for species annotation (database was silva138 / 16s_bacteria, confidence 0.7), and soil physicochemical property data monitoring was carried out, and the specific indexes were as follows: Individual phenotype data: record the number of poplar seedlings in the quadrat, the height (vertical distance from the ground to the terminal bud), the crown width (the average of the east-west and north-south diameters); destructively excavate 3 seedlings, and take them back to the laboratory to measure the biomass of each level of root, stem and leaf (80℃ oven drying for 48 hours to constant weight, weighing).

[0046] Functional trait data: Leaf: number, thickness (measured by vernier caliper), area (calculated by ImageJ software), dry matter content (oven dried weight / fresh weight), total nitrogen and total phosphorus content (determination method same as soil); Stem: number, diameter (measured by vernier caliper), length, surface area; Root: >5cm primary root, 2-5cm secondary root, <2cm tertiary root, respectively record number, diameter, surface area, dry matter content, total nitrogen and total phosphorus content.

[0047] The monitoring results showed that: Seedling growth status: the average height of clonal seedlings in D3 area was 127 cm (CKc area was 68 cm), and the average height of sexual seedlings in M3 area was 92 cm (CKs area was 41 cm), indicating that irrigation treatment significantly promoted seedling growth (such as Figure 2 (E) and (F) are growth photos of clonal propagation poplar seedlings in D3 area, and it can be seen that the seedlings grow vigorously).

[0048] Soil habitat improvement: the soil water content in D3 area reached 47.8% (CKc area was 13.35%), and the soil water content in M3 area was 44.8% (CKs area was 23.53%), and the total salt content was significantly reduced (D3 area was 5.62g / kg, M3 area was 2.65g / kg), which verified the effective regulation effect of the seepage wheel irrigation mode on the soil habitat.

[0049] Example two Based on the monitoring data in example one, the correlation between soil physicochemical properties, seedling growth response and reproductive strategy was focused on, and the directional regulation effect of the seepage wheel irrigation mode on the sexual reproduction and clonal reproduction of poplar was verified, and the data included soil salt, water, nitrogen, phosphorus and other physicochemical indexes, soil factor correlation matrix (such as Figure 3 , Figure 4Redundancy analysis of reproductive methods and soil habitats (as shown), Figure 5 (A) and threshold model (as shown) Figure 5 (BE) as shown), differences in seedling functional traits (such as Figure 6 , Figure 8 (As shown).

[0050] (1) Selective adaptation of soil habitat to reproductive methods Based on Example 1, the differences in soil properties in the Populus euphratica seedling growth area are shown in the table below, clearly demonstrating the soil preferences of the two propagation methods under natural conditions:

[0051] From the above table, the soil property characteristics of natural growth area of Populus euphratica seedlings (clonal propagation seedlings CKc and sexual reproduction seedlings CKs area) and irrigation area (clonal propagation seedlings D1, D2 and D3, sexual reproduction seedlings M1, M2 and M3 area) were described. The results showed that: compared with the natural growth area of clonal propagation seedlings, the sexual reproduction seedlings tended to distribute in the habitat with relatively humid soil, less salt and nutrient enrichment. The average soil total salt content in CKs area was significantly lower than that in CKc area (21.00~31.62 g / kg, P<0.05) between 14.53~16.86 g / kg. The average soil water content (22.60~25.00%), total nitrogen content (540.01~876.00 mg / kg) and total phosphorus content (287.00~473.00 mg / kg) in CKs area were significantly higher than those in CKc area (11.00~15.60%, 436.13~495.3 mg / kg, 206~339 mg / kg, P<0.05), respectively. There was no significant difference in soil pH value between CKc and CKs area. The difference analysis of soil physicochemical properties in irrigation area found that: with the increase of irrigation amount, the soil total salt content in the root zone of seedlings showed a decreasing trend. The soil total salt content in the growth area of clonal propagation seedlings was higher than that in the growth area of sexual reproduction seedlings at each irrigation gradient. The average soil total salt content in the growth area of clonal propagation seedlings was the highest in D1 area, reaching 18.30 g / kg, followed by D2 and D3 areas (P>0.05). The average soil total salt content in the growth area of sexual reproduction seedlings was the highest in M1 area (7.49 g / kg), which was significantly higher than that in M1 and M2 areas (P<0.05). Irrigation treatment significantly improved the soil water accumulation. The soil in the growth area of clonal propagation seedlings was the wettest in D3 area, with an average soil water content of 47.8% (increased by 103.14~258.05% compared with CKs and CKc areas), which was significantly higher than that in D2 area (P<0.05) and D1 area (P<0.05). The average soil water content in the growth area of sexual reproduction seedlings was the highest in M3 area (44.80%), followed by M2 area (31.16%), which was significantly higher than that in M1 area. At each irrigation gradient, there was no significant difference in soil pH value in each root area, but the soil pH value in the growth area of sexual reproduction seedlings was higher than that in the growth area of clonal propagation seedlings, even if it was not significant. Irrigation treatment significantly increased the total nitrogen and total phosphorus content in the root zone soil, showing a synergistic increasing trend. The nutrient in the growth area of clonal propagation seedlings was the richest in D3 area (average nitrogen and phosphorus content was 1279.51 mg / kg and 956.2 mg / kg, respectively), followed by D2 area, which was significantly higher than that in D1 area. The nutrient content in the growth area of sexual reproduction seedlings was the highest in M3 area (average nitrogen and phosphorus content was 1087 mg / kg and 753.5 mg / kg, respectively), followed by M2 and M1 areas. This low-salt, high-moisture and high-nutrient habitat was more suitable for seed germination and seedling planting, which confirmed the dependence of sexual reproduction on "optimal habitat".

[0052] (2) Habitat directional regulation in irrigation area Infiltration rotation irrigation, by alternating the irrigation interval, water volume, and duration, causes the soil habitats in the clonal reproduction zone (D1-D3) and the sexual reproduction zone (M1-M3) to differentiate in their respective adaptive directions: Cloning breeding area (e.g.) Figure 3 As shown in the figure: With the increase of irrigation amount (D1→D2→D3), the water content increased from 22.85% to 47.80%, the total salt content decreased from 18.30 g / kg to 5.62 g / kg, and the nitrogen and phosphorus contents increased synergistically (total nitrogen 1279.5 mg / kg and total phosphorus 956.9 mg / kg in area D3). The soil factor correlation showed that the water content in areas D2-D3 was strongly negatively correlated with the total salt content (correlation coefficient -0.77, P<0.01) and strongly positively correlated with nitrogen and phosphorus (P<0.01). This indicates that irrigation effectively leached salt and promoted nutrient accumulation, while preserving the medium-high salt base tolerance of clonal seedlings (the total salt in area D3 of 5.62 g / kg was still higher than that in sexual area M3 of 2.65 g / kg).

[0053] Sexual reproduction area (e.g.) Figure 4 As shown in the figure, with the increase of irrigation (M1→M2→M3), the water content increased from 26.77% to 44.80%, the total salt content decreased from 7.49 g / kg to 2.65 g / kg, and the nitrogen and phosphorus contents increased significantly (total nitrogen 1087 mg / kg and total phosphorus 753.5 mg / kg in M3 area). Soil factor correlation showed that water content was strongly negatively correlated with total salt content (correlation coefficient -0.6, P<0.01) and strongly positively correlated with nitrogen and phosphorus content (P<0.01), forming a typical "low salt-high humidity-high nutrient" habitat, which perfectly matches the needs of sexual reproduction.

[0054] (3) Verification of soil threshold for reproductive mode conversion like Figure 5 As shown (Populus euphratica propagation mode on soil habitat selection), the key soil thresholds for propagation mode switching were quantified using redundancy analysis (RDA) and SHAP dependency graphs. RDA analysis (such as) Figure 5(A) showed): The first two axes explained 45.78%+8.36% of the variance, showing that the distribution of sexually reproduced seedlings was positively correlated with pH value (contribution 18.3%), total nitrogen (15.7%), and water content (8.8%), and that clonal seedlings were positively correlated with total salt content. The first two axes could reflect the selection of soil habitat by reproductive strategy, and the distribution of 1-4 year old sexually reproduced seedlings was mainly affected by pH value, total nitrogen content, and water content, which explained 18.3%, 15.7%, and 8.8% of the variance of the response variable, respectively. The 1-2 year old sexually reproduced seedlings had the strongest positive correlation with pH value, and the 3-4 year old sexually reproduced seedlings had the strongest positive correlation with water content. The distribution of 1-4 year old clonal seedlings was positively correlated with total salt content. As the total salt concentration in the soil decreased, the water content, nitrogen content, and pH value increased, and the tendency of Populus euphratica to reproduce from clonal reproduction to sexual reproduction was observed, which directly reflected the screening effect of habitat on reproductive strategy.

[0055] Threshold model (as shown in Figure 5 (B-E) showed): To further quantify the threshold of soil habitat selection by reproductive strategy, a binary logistic regression model was constructed (dependent variable: reproductive strategy, independent variable: soil total salt content, water content, pH value, total nitrogen, and total phosphorus content), and SHAP dependence graph was used to determine the soil habitat threshold of reproductive strategy selection (as shown in Figure 5 (B-E) showed): The model accuracy reached 82.3% (AUC=0.87), indicating that soil factors had strong explanatory power for the classification of reproductive strategy. The results showed that when the soil total salt content was less than 12.04 g / kg, the pH value was more than 6.82, the total nitrogen content was more than 667.59 mg / kg, and the water content was between 20.59-42.52%, the model predicted that the probability of sexually reproduced seedlings increased significantly (Δ probability increase=37%), and other habitats were more likely to appear clonal seedlings. Specifically: When the soil total salt content was less than 12.04 g / kg, the pH value was more than 6.82, the total nitrogen was more than 667.59 mg / kg, and the water content was between 20.59-42.52%, the model predicted that the probability of sexual reproduction increased by 37% after irrigation treatment: In M3 area, the total salt was 2.65 g / kg, the total nitrogen was 1087 mg / kg, and the water content was 44.80% (close to the upper limit of the threshold), the number of sexually reproduced seedlings increased by 2.3 times compared with CKs area, which verified the effectiveness of the threshold; In D3 area, the total salt was 5.62 g / kg (<12.04 g / kg), but the water content was 47.80% (exceeding the threshold of sexual reproduction), and the clonal seedling biomass reached the peak (18.7 g / plant), indicating that clonal reproduction was more resistant to high water content.

[0056] (4) Differentiated response of seedling growth strategy Objective level rhizosphere microbial community composition (such as Figure 6As shown, the differences in leaf traits of Populus euphratica seedlings from the two propagation methods are as follows: In the naturally grown area (CKc zone), the main rhizosphere microorganisms of clonal seedlings were Bacillus phylum Unknown Order (5.4%) and Bacillus Order (3.2%), with Bacillus Order being the main rhizosphere microorganism in the taproot (6.2%) and Yeast Order (11.0%) in the lateral roots. In contrast, in the naturally grown area (CKs zone), the main rhizosphere microorganisms of sexually propagated seedlings were Burkholderia Order (10.3%) and Fibriophytes Order (9.1%). The main rhizosphere microorganisms of clonal seedlings were Actinomycetes and Micrococcidales, followed by Bacillus Order (1.8%) and Anaerobic Cordyceps Order (1.4%). Actinomycetes had the highest proportion in the fine roots (4.6%) of seedlings, likely due to their function in degrading complex organic matter and promoting nutrient absorption (such as phosphate dissolution). The Micrococcidales suborder is mainly enriched in the rhizosphere of the primary root (2.9%) and fine roots (3.1%) of seedlings, which is related to the selection of specific microbial groups by root exudates (such as phenols and amino acids). The Bacillus and Anaerobic Cordyceps orders are mainly distributed in the rhizosphere of lateral roots (2.1% and 1.3%), which is related to the low-oxygen conditions of the lateral root microenvironment. The main rhizosphere microorganisms of sexually reproduced seedlings are Pseudomonas, Bacillus, and Yeast, followed by Burkholderia. Among them, Pseudomonas has the highest proportion in the primary root and fine roots (2.7% in the primary root and 4.2% in the fine roots), which is related to growth-promoting functions (secreting growth hormones, antagonizing pathogens, etc.). Yeast is mainly enriched in the rhizosphere of seedlings in the M3 region, which is related to the higher input of root exudates. The main rhizosphere microorganisms of the lateral roots of sexually reproduced seedlings are consistent with those of clonal seedlings, with the Micrococcidales suborder (2.5%) being the main group.

[0057] (5) LEfSe analysis of significantly differentially expressed bacterial communities LEfSe analysis was used to further screen out the significantly different bacterial communities in the rhizosphere soil of Populus euphratica seedlings from the two propagation methods (e.g., ... Figure 7 (as shown) Figure 7 (A) is an evolutionary clade diagram. The classification levels from the outside to the inside of the circles are phylum, class, order, family, and genus. The dots of each color represent important bacterial groups in their respective groups, while the yellow dots represent bacterial groups whose abundance does not differ significantly across groups. Figure 7 (B) is a bar chart showing the LDA value distribution, displaying significantly different bacterial communities with LDA values ​​> 4. The phylogenetic diagram shows 56 significantly different bacterial communities between the two reproductive methods of Populus euphratica seedlings. Among them, the relative abundance of oligotrophic actinomycetes and candidate phyla in the rhizosphere soil of clonal seedlings is significantly higher than that of sexually reproduced seedlings; Proteobacteria, as a typical eutrophic bacterial community, has a significantly higher relative abundance in the rhizosphere soil of sexually reproduced seedlings than that of clonal seedlings. (6) Strategy differentiation of biomass accumulation shows Based on the community construction mechanism, the niche theory and the neutral theory determine the community assembly process, and the neutral model is used to distinguish the importance of deterministic process and random process to bacterial community (as shown in Figure 8 MST is used to quantify the relative importance of deterministic process and random process in microbial community assembly, and the bacterial MST is close to 0.5, indicating that deterministic process may dominate bacterial community assembly compared with random process, and the results show that: the neutral community model successfully estimates most of the relationship between OTU appearance frequency and its relative abundance change, and the MST of bacterial community in the rhizosphere of poplar seedlings of the two reproduction modes is less than 0.5, indicating that the deterministic process has significant importance to the assembly of bacterial community in the rhizosphere of seedlings of the two reproduction modes, and the Nm value of seedlings of clonal reproduction is higher than that of seedlings of sexual reproduction (Nm=6714), indicating that the bacterial community in the rhizosphere of seedlings of clonal reproduction is more affected by environmental factors than that of seedlings of sexual reproduction In summary, the present embodiment is verified by Figures 3-5 , Figure 6 , Figure 7 , Figure 8 and other data: the branch infiltration wheel irrigation can adjust the soil habitat (salt, water, nitrogen and phosphorus) in a targeted manner, so that D1-D3 area is suitable for clonal reproduction (high salt tolerance + medium-high water), M1-M3 area is suitable for sexual reproduction (low salt high humidity + high nutrients), and completely meets the threshold rule of Figure 5 , realizing the precise regulation of the reproduction mode of poplar.

[0058] Example Three Based on the analysis data of rhizosphere microbial samples in Example One, the present embodiment focuses on the composition, network structure and stability characteristics of microbial community, and verifies the regulation mechanism of branch infiltration wheel irrigation on the stability of rhizosphere microbial community.

[0059] (1) Directional differentiation of rhizosphere microbial community composition The differences in community structure at the order level are shown in Figure 6 , showing significant differentiation of rhizosphere microorganisms of seedlings of the two reproduction modes: Clonal reproduction seedlings: D3 area rhizosphere is mainly composed of actinomycetales (4.6%) and micrococcus suborder (3.1%), among which actinomycetales accounts for the highest proportion in fine roots, which is related to its function of degrading complex organic matter and promoting phosphorus dissolution; micrococcus suborder is enriched in main roots and fine roots, which may be related to the selective enrichment of root exudates (such as phenolic compounds).

[0060] Sexual reproduction seedlings: M3 area rhizosphere is mainly composed of pseudomonas order (2.7% in main roots and 4.2% in fine roots) and yeast order, pseudomonas order plays a role in promoting growth by secreting growth hormones and antagonistic substances, and the enrichment of yeast order in M3 area is directly related to high root exudate input.

[0061] (2) LEfSe analysis of significantly differentially expressed bacterial communities, such as Figure 7 As shown, LEfSe analysis of rhizosphere soil bacteria in Populus euphratica seedlings from both propagation methods further revealed: The rhizosphere of cloned seedlings was significantly enriched with oligotrophic microbial communities, such as Actinobacteriota and Patescibacteria. These microbial communities are adapted to medium-high salinity environments and can obtain resources through metabolic diversity under nutrient-poor conditions. The rhizosphere of sexual seedlings is significantly enriched with nutrient-rich microorganisms, such as Proteobacteria (p__Proteobacteria), which can reproduce rapidly in high-nutrient environments and promote the degradation of organic matter and nutrient conversion by secreting extracellular enzymes.

[0062] (3) Microbial community assembly mechanisms, such as Figure 8 As shown, a neutral model of rhizosphere soil bacteria in Populus euphratica seedlings under two propagation methods reveals: The Nm value of rhizosphere microorganisms in cloned seedlings (6714) was lower than that in sexual seedlings (Nm=14123), and the MST of both was <0.5, indicating that deterministic processes (environmental screening) dominated community assembly. The lower Nm value in the clonal area indicates that its microbial community is more strongly screened by the soil environment (such as salt and water), which is consistent with the high heterogeneity of the rhizosphere environment of the clonal seedlings.

[0063] (4) Regulatory effects on microbial network stability, such as Figure 9 (The topological characteristics of the rhizosphere soil bacterial community network of Populus euphratica seedlings under two propagation methods are shown in the image.) Nodes and Connections: Compared to clonal seedlings, the rhizosphere bacterial communities of sexually propagated seedlings in both the natural growth zone and under the same irrigation gradient formed simpler symbiotic networks (e.g., Figure 8 As shown in the figure, the number of nodes decreased by an average of 9.7% ± 7.2%, the number of edges decreased by an average of 52.7% ± 21.4%, and the average degree decreased by an average of 31.9% ± 28.8% (except for the fine roots in region M2 and the main root in region M3). This means that the efficiency of material, energy and information transfer among rhizosphere bacteria in sexually reproduced seedlings is relatively low.

[0064] Positive connectivity percentage: The symbiotic network of rhizosphere bacterial communities at all levels of the seedling root system in both propagation methods was dominated by positive connectivity lines. The average percentage of positive connectivity lines in the rhizosphere bacterial symbiotic network of clonal propagation seedlings was 69.5% ± 12.4%, while that of sexually propagated seedlings was 76.7% ± 14.8%. Moreover, the percentage of positive connectivity lines increased synergistically with the increase of irrigation amount, indicating that the improvement of soil drought promoted positive interaction among bacterial communities, and the synergistic interaction among bacterial communities was enhanced after irrigation improved drought.

[0065] (5) Critical nodes and network stability As Figure 10 shown, the Zi-Pi diagram of the bacterial community network OTUs in the rhizosphere soil of Populus euphratica seedlings of the two propagation methods showed that: A large number of key nodes (Module hubs, Connectors) appeared in the medium-high irrigation area (D2, D3, M2, M3), such as the main root of the clonal seedling in the D2 area had 8 Module hubs (37.5% of Actinobacteria) and 43 Connectors (34.9% of Actinobacteria), which were crucial for maintaining the stability of the network structure; The nodes in the natural growth area (CKc, CKs) and the low irrigation area (D1, M1) were all Peripherals, and the network stability was low.

[0066] As Figure 11 shown, the stability analysis of the bacterial community network in the rhizosphere soil of Populus euphratica seedlings of the two propagation methods verified that: After the irrigation treatment, the robustness of the rhizosphere microbial network increased by an average of 2.16%, and the vulnerability decreased by an average of 72.3%; The robustness of the rhizosphere network of clonal seedlings was 2.88% higher than that of sexual seedlings (such as 0.87 in the D3 area vs. 0.82 in the M3 area), and the vulnerability of the lateral root rhizosphere network was the lowest (reduced by 56.7% compared with the main root), and the positive cohesion was the highest, indicating that the clonal seedling rhizosphere formed a more stable microbial interaction system.

[0067] This example verifies that: Figures 5-11 Branch seepage wheel irrigation can directionally regulate the composition of rhizosphere microbial communities (oligotrophic vs. eutrophic), promote the enrichment of key functional bacterial groups (Actinobacteria, Pseudomonadales, etc.), enhance the complexity and stability of the microbial network (high robustness, low vulnerability), and ultimately improve the tolerance of Populus euphratica to drought and saline-alkali stress through the synergistic adaptation of microorganisms and plants.

[0068] The above is only a preferred embodiment of the present application, and is not intended to limit the present application in any form. Although the present application has been disclosed as above with a preferred embodiment, it is not intended to limit the present application. Any person skilled in the art can make some changes or modifications to the above disclosed technical content without departing from the scope of the technical solution of the present application, and any equivalent embodiments with equivalent changes and modifications made to the above embodiments based on the technical essence of the present application are still within the scope of the technical solution of the present application.

Claims

1. A method for regulating the propagation of Populus euphratica and the stability of rhizosphere microorganisms through infiltration and rotational irrigation, characterized in that, The specific steps of this method are as follows: S100. Select the riverbank zone in the arid area where Populus euphratica is concentrated as the experimental area. Within the experimental area, set up the natural growth control area CKc for clonal Populus euphratica seedlings and the infiltration and rotation irrigation experimental areas D1, D2, and D3, as well as the natural growth control area CKs for sexually propagated Populus euphratica seedlings and the infiltration and rotation irrigation experimental areas M1, M2, and M3. The interval between each experimental area is ≥5m. S200. For cloned Populus euphratica seedlings, no irrigation treatment was carried out in the natural growth control area CKc, while specific water disturbance modes were adopted in the infiltration and rotation irrigation experimental areas D1, D2, and D3 respectively. S300. For sexually reproduced Populus euphratica seedlings, no irrigation treatment was applied in the natural growth control area CKs, while specific water disturbance modes were adopted in the infiltration and rotation irrigation experimental areas M1, M2, and M3 respectively. S400, through infiltration and alternating irrigation with S200 and S300, the soil physicochemical properties of each experimental area were monitored to promote the target propagation strategy and the stability of the rhizosphere microbial community; S500: Regularly monitor the rhizosphere microbial community structure of Populus euphratica seedlings and maintain the robustness and vulnerability of the rhizosphere microbial symbiotic network by adjusting the irrigation amount and interval days.

2. The method for regulating the propagation of Populus euphratica and the stability of rhizosphere microorganisms through infiltration and rotational irrigation according to claim 1, characterized in that, In step S100, when setting up experimental areas for cloning and propagating Populus euphratica seedlings, healthy Populus euphratica mother trees are selected in each experimental area. Root severing trenches are mechanically dug within a distance of 5-10m from the mother trees. The trenches are 70-90cm deep, 30-50cm wide, and 15-20m long. The roots of the Populus euphratica mother trees with a diameter of 1-3cm within 0-30cm of the ground surface are manually cut, retaining capillary roots with a diameter <1cm and lateral roots >3cm.

3. The method for regulating the propagation of Populus euphratica and the stability of rhizosphere microorganisms through infiltration and rotational irrigation according to claim 1, characterized in that, In S100, when setting up an experimental area for sexually propagated Populus euphratica seedlings, the experimental area is prepared in the previous year by excavating water collection troughs. The water collection troughs are 20-30cm deep and 50-60cm wide. During the experiment, healthy adult female Populus euphratica trees are selected, and their fruit spikes are collected and inserted into the top 10cm soil layer of the water collection troughs in each experimental area, with a spacing of 1-2m between cuttings.

4. The method for regulating the propagation of Populus euphratica and the stability of rhizosphere microorganisms through infiltration and rotational irrigation according to claim 1, characterized in that, In S200, the specific water disturbance patterns in the infiltration and rotational irrigation experimental areas D1, D2, and D3 for cloning and propagating Populus euphratica seedlings are as follows: Zone D1: Irrigation interval 11 days, water volume 6m³ / 10m², lasting for 2 days; Zone D2: Irrigation interval 23 days, water volume 7m³ / 10m², for 3 consecutive days; Zone D3: 33-day interval, irrigation volume 8m³ / 10m², lasting for 4 days.

5. The method for regulating the propagation of Populus euphratica and the stability of rhizosphere microorganisms through infiltration and rotational irrigation according to claim 1, characterized in that, In S300, the specific water disturbance patterns in the infiltration and rotation irrigation experimental areas M1, M2, and M3 for sexually propagated Populus euphratica seedlings are as follows: M1 Zone: Irrigation interval 5 days, water volume 150m³ / 666.7m², duration 45 minutes; M2 Zone: Irrigation interval 5 days, water volume 200m³ / 666.7m², duration 35 minutes; M3 Zone: Irrigation interval 3 days, water volume 390m³ / 666.7m², duration 25 minutes.

6. The method for regulating the propagation of Populus euphratica and the stability of rhizosphere microorganisms through infiltration and rotational irrigation according to claim 1, characterized in that, In the S400, soil samples are collected from the soil layer at a depth of 0-40cm by removing vegetation at each sampling point for monitoring of soil physicochemical properties. The monitoring indicators include: soil moisture content determined by the oven drying method, pH determined by the potentiometry method, organic carbon content determined by the potassium dichromate-external heating method, total nitrogen content determined by the PE-2400CHN analyzer, total phosphorus content determined by the molybdate / stannous chloride method, and total salt content determined by the gravimetric method.

7. The method for regulating the propagation of Populus euphratica and the stability of rhizosphere microorganisms through infiltration and rotational irrigation according to claim 1, characterized in that, In the S500, the regulation of the rhizosphere microbial community includes promoting the enrichment of Actinobacteria, Acidobacteria, and Bacillus.

8. The method for regulating the propagation of Populus euphratica and the stability of rhizosphere microorganisms through infiltration and rotational irrigation according to claim 7, characterized in that, The monitoring method for the rhizosphere microbial community was as follows: Soil from the surface of the roots of Populus euphratica seedlings was brushed onto sterile filter paper using a sterile brush and collected in 5 mL sterile centrifuge tubes. The rhizosphere soil samples were stored at -80℃. DNA extraction and amplification For the V3-V4 region of the gene, primers 338F and 806R were used. The sequencing platform sequenced the amplified products and analyzed the community structure and network stability using QIIME2, Networkx, and Gephi.

9. A method for regulating the propagation of Populus euphratica and the stability of rhizosphere microorganisms through infiltration and rotational irrigation according to any one of claims 1 to 8, characterized in that, The method described is applicable to the ecological restoration of Populus euphratica forests in arid regions.

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