Ternary biological synergistic soil fertility improving method for young khasys pine forest

Abstract

The invention discloses a pinus kesiya young forest ternary biological synergistic soil fertility improving method, which comprises ectomycorrhizal fungus inoculation treatment, local nitrogen fixation leguminous plant intercropping treatment and cutting and returning treatment, mycorrhizal inoculation adopts colored puffball or Yunnan puffball bacterial suspension to be applied to base soil of pinus kesiya, intercropping treatment adopts interplanting of flemingia macrophylla, and interplanting treatment adopts interplanting of flemingia macrophylla. The step of cutting and returning to the field refers to regular cutting, turning and pressing, ternary biological synergy enables the soil organic matter to be increased by 0.3%-0.5% annually, the growth amount of the pinus khasys is increased by 15%-25%, and zero chemical fertilizer input is achieved.

Description

Technical Field

[0001] This invention belongs to the field of forestry ecological management and soil improvement technology, specifically involving a method for synergistic improvement of soil fertility by three elements in young Pinus sylvestris forests. Background Technology

[0002] Simao pine ( Pinus kesiya var. langbianensis *Pinus simonii* is one of the most important native timber species in the tropical southern region of Yunnan Province, widely distributed in low mountain and hilly areas at altitudes of 700–1700 m in Pu'er, Xishuangbanna, and Lincang. In recent years, with the advancement of *Pinus simonii* raw material forest base construction, large-scale pure plantations of *Pinus simonii* have rapidly expanded. Statistics show that the area of ​​*Pinus simonii* plantations in Yunnan Province has exceeded 400,000 hectares. 2 This has become the most prevalent type of plantation in the region.

[0003] However, monoculture plantations of *Pinus kesiya* face severe soil fertility degradation during management. Studies show that the organic carbon content of soil in *Pinus kesiya* plantations continuously declines 4-10 years after afforestation, with the organic carbon density in the 0-50 cm soil layer significantly lower than that of natural forests of the same age. There are three main reasons for this problem. First, the clearing and slash-and-burn practices before afforestation damage the original soil structure and organic matter layer, resulting in a significant loss of topsoil organic matter. Second, the monoculture nature of *Pinus kesiya* plantations results in a simple litter composition and slow decomposition; the pine needles are rich in tannins and resins, leading to a low nutrient return rate. Third, frequent tending operations during the juvenile stage (usually 2-3 times per year) repeatedly disturb the topsoil, accelerating the mineralization and decomposition of organic matter while simultaneously clearing understory vegetation, weakening biological nitrogen fixation and nutrient cycling.

[0004] Existing methods for improving plantation soil mainly include chemical fertilization and green manure planting. While chemical fertilization is fast-acting, long-term application leads to increased soil acidification and a homogenized microbial community structure. Furthermore, it is costly and difficult to implement in mountainous forest areas. Regarding green manure planting, intercropping with legumes has been reported in camellia oleifera and rubber plantations. Liu et al. reported in Scientific Reports (2018, 8: 17324) the significant improvement in soil carbon and nitrogen composition after introducing *Flemingia macrophylla* into a rubber plantation in Xishuangbanna. The results showed that intercropping with *Flemingia macrophylla* increased microbial biomass carbon, microbial biomass nitrogen, and nitrate nitrogen in the 0–10 cm soil layer of the rubber plantation by 38.9%, 55.5%, and 214.7%, respectively, compared to pure rubber plantations. Kaewkrom et al. published a study on the seasonal dynamics of fine root biomass and soil nutrients in a rubber-Lysimachia intercropping system in Plants (2022, 11(20): 2682), confirming a significant positive correlation between fine root biomass and available nitrogen in the intercropping system. Liu et al. reported in Catena (2019, 172: 480~490) the effect of Lysimachia intercropping on alleviating soil acidification in rubber plantations by reducing nitrogen fertilizer input. However, the above studies all focused on tropical rubber plantations (Rubberia trifoliata is a broad-leaved tree), which differ greatly from the ecological conditions of pine plantations. Pine is a coniferous species and forms a symbiotic relationship with ectomycorrhizal fungi (rubber trees are arbuscular mycorrhizal). Therefore, the intercropping experience in rubber plantations cannot be directly applied to pine plantations.

[0005] Regarding the application of mycorrhizal fungi, patent CN110604048B discloses a method for multiple inoculation of mycorrhizal fungi in woody plants, which involves inoculating the root system of black pine seedlings with ectomycorrhizal fungi such as *Lycoperdon perlatum* using a mother seedling inoculation method. However, this method only involves inoculation at the nursery stage and does not address soil improvement in forest land after afforestation. Patent CN101611678A discloses a method for seed propagation of *Mallotus spp.*, involving sulfuric acid soaking and germination technology for *Mallotus spp.*, but does not utilize it as a nitrogen-fixing plant for intercropping under forests.

[0006] Furthermore, while earthworm inoculation technology has been widely used in agricultural soil improvement, its application in forestry soil improvement, especially in pine plantations, is rarely reported. The acidic soil environment under pine forests and the terpenes in pine needle litter pose certain obstacles to the establishment of earthworm populations. A systematic technical solution has yet to be found for improving the biological habitability of pine forest soil through nitrogen-rich litter from leguminous intercropping, thereby promoting earthworm colonization and reproduction.

[0007] In terms of forestry management models, traditional management of *Pinus kesenbergii* plantations follows the principle of completely removing understory vegetation, involving 2-3 rounds of weeding, shrub removal, soil loosening, and mounding per year. While this approach reduces competition for water and nutrients from weeds in the short term, frequent soil disturbance accelerates the mineralization and decomposition of organic matter. Simultaneously, it completely eliminates nitrogen-fixing leguminous shrubs and herbaceous plants, cutting off the natural pathway for replenishing soil nitrogen through biological nitrogen fixation. How to achieve the dual goals of reducing tending workload and improving soil fertility by selectively retaining native beneficial plants while ensuring the normal growth of *Pinus kesenbergii* saplings is a key technical issue that urgently needs to be addressed for the sustainable management of *Pinus kesenbergii* plantations.

[0008] In summary, current technologies lack a systematic biological soil improvement method for young plantations of *Pinus kesiya*, particularly a ternary biological synergistic soil fertility enhancement technology that combines ectomycorrhizal fungal inoculation, intercropping with native perennial leguminous plants, and the introduction of soil animals. Furthermore, there is a lack of comprehensive management strategies that organically integrate the competitive weed suppression function of intercropped nitrogen-fixing plants with tending and reduction. Especially in the hot southern Yunnan region where *Pinus kesiya* is distributed, the hot and rainy climate leads to extremely vigorous weed growth. Under traditional monoculture management models, the high-intensity tending 2-3 times per year not only consumes a large amount of labor costs but also creates a vicious cycle of topsoil exposure and organic matter loss due to repeated disturbance. Therefore, developing a systematic biological management technology that integrates soil fertility restoration, weed suppression, and tending and reduction is of significant theoretical and practical value for achieving sustainable management of *Pinus kesiya* plantations. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of the prior art and provide a three-element biological synergistic soil fertility improvement method for young Pinus simonii forests. Through the organic combination of ectomycorrhizal fungal inoculation, intercropping with native nitrogen-fixing leguminous plants, and mowing and returning to the field, the soil fertility of young Pinus simonii forests can be rapidly restored and continuously improved, reducing reliance on chemical fertilizers and the frequency of tending.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: The "Three-Element Biological Synergistic Soil Fertility Enhancement Method for Young Pinus kesua Forests" applies a three-element biological synergistic soil fertility enhancement treatment to 1-5 year old artificial young Pinus kesua forests. This treatment comprises three core components: ectomycorrhizal fungal inoculation, intercropping with native nitrogen-fixing leguminous plants, and mowing and returning the leaves to the soil. Ectomycorrhizal fungal inoculation involves applying a suspension of *Lycoperdon perlatum* or *Lycoperdon yunnanense* around the base of the Pinus kesua plants to promote the establishment of mycorrhizal symbiosis and enhance the Pinus kesua root system's ability to absorb soil nutrients, especially phosphorus and trace elements. Intercropping with native nitrogen-fixing leguminous plants involves planting *Flemingia macrophylla*, a perennial widely distributed species in southern Yunnan, between rows of Pinus kesua. Through the symbiotic nitrogen fixation of *Flemingia macrophylla* roots and slow-growing rhizobia, it continuously inputs bioavailable nitrogen into the soil. Mowing and returning the leaves to the soil involves regularly mowing the above-ground parts of *Flemingia macrophylla* and incorporating them into the topsoil, utilizing its high-nitrogen, low-carbon foliage to replenish soil organic matter and promote humus synthesis. Optionally, earthworm introduction treatment can also be included to promote the decomposition of organic matter and improve soil structure through the activity of soil animals. The synergistic effect of ternary or quaternary biological components forms a complete soil fertility enhancement system that integrates nitrogen fixation input, efficient acquisition, accelerated cycling, and structural improvement.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0012] First, this invention employs a ternary biological synergistic strategy. Ectomycorrhizal fungi expand the nutrient uptake range of the Pinus sylvestris root system, while *Millettia speciosa* continuously provides a nitrogen source through biological nitrogen fixation (annual nitrogen fixation can reach 30-80 kg N / hm²). 2 Returning harvested crops to the field can increase the soil organic matter content by 0.3% to 0.5% annually, and the combined effect of these three measures is significantly better than any single measure.

[0013] Secondly, this invention selects *Millettia divaricata* as an intercropping nitrogen-fixing species. This species is a native perennial shrub in southern Yunnan, with strong adaptability, drought and barren soil tolerance. Once planted, it can be used for many years without the need for annual reseeding, which greatly reduces labor costs.

[0014] Third, the rapid growth and dense cover of *Millettia divaricata* can effectively suppress the spread of competing weeds such as ferns and grasses, reducing the traditional 2-3 times per year to once per year and saving on maintenance costs.

[0015] Fourth, this invention employs entirely biological methods to achieve the goal of green management with zero chemical fertilizer input, avoiding soil acidification and non-point source pollution problems caused by chemical fertilization, and meeting the requirements of ecological forestry development. After implementing the method of this invention, the soil pH value shows an upward trend, which is beneficial to improving the acidic soil environment commonly found in Pinus keseng plantations.

[0016] Fifth, the mycorrhizal fungi inoculation of the present invention and the intercropping of leguminous plants have a complementary functional relationship. The mycorrhizal mycelium can efficiently acquire the nitrogen fixation products released into the soil by *Millettia speciosa* and transport them to the roots of *Pinus simonii*, which significantly improves the utilization efficiency of nitrogen fixation products for the target tree species and avoids the ineffective loss of nitrogen.

[0017] Sixth, earthworm release treatment can promote the decomposition and humification of cut organic materials returned to the field, improve soil aggregate structure, enhance soil water and fertilizer retention capacity, create favorable soil physical conditions for the reproduction of mycorrhizal fungi and the extension of the root system of *Millettia speciosa*, and further strengthen the synergistic effect of the ternary biological system. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the field planting layout for the three-element biological synergistic soil fertility improvement method for young Pinus simonii forests of the present invention.

[0019] Figure 2 This is a photomicrograph of the fine roots of Pinus sylvestris infected by ectomycorrhizal fungi in Example 1 (trypan blue staining, scale bar 200 μm).

[0020] Figure 3 This is a micrograph of a cross-section of the root nodule of *Millettia divaricata* in Example 1 (Safranin-Fast Green double staining, scale bar 500 μm).

[0021] Figure 4 Micrographs of the mycorrhizal root tip morphology of Pinus simaoides after 12 months of treatment in Example 1 (scale bar 1 cm).

[0022] Figure 5 A comparative graph showing the changes in the organic matter mass fraction of soil in the 0-20 cm depth after 12 months of treatment.

[0023] Figure 6 A comparison chart showing the average annual growth of Pinus simonii seedling height after 12 months of treatment. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments. The embodiments of the present invention are implemented based on the technical solution of the present invention, providing detailed implementation methods and specific operating procedures; however, the scope of protection of the present invention is not limited to the following embodiments. Experimental methods in the following embodiments that do not specify specific conditions were performed under conventional conditions.

[0025] Example 1: This example was implemented in a two-year-old artificial juvenile Pinus simonii forest in Yongping Town, Jinggu County, Pu'er City, Yunnan Province (23°27′N, 100°21′E, altitude 1324 m). The forest consisted of a pure Pinus simonii forest planted in the spring of 2022, with a spacing of 2.5 m × 3.5 m, i.e., a planting density of approximately 1143 trees / hm². 2The afforestation site is a gently sloping hilly area with a slope of 8° to 15°. The soil type is red soil. Before afforestation, the soil pH value of the 0-20 cm soil layer was 4.8, the organic matter mass fraction was 1.32%, the total nitrogen mass fraction was 0.078%, the available phosphorus content was 3.2 mg / kg, and the available potassium content was 68.5 mg / kg. It belongs to a typical low-fertility acidic red soil.

[0026] The procedure for ectomycorrhizal fungal inoculation is as follows: In mid-May 2024 (early rainy season), fresh colored puffball fruiting bodies were collected from natural Pinus sylvestris forests surrounding the experimental site. Individuals with moderate maturity and brownish-brown internal spore powder were selected. The collected fruiting bodies were spread out to dry in a cool, ventilated indoor area for 4–6 hours, then crushed in a mortar and suspended in sterile water. Coarse tissue fragments were removed by filtering through a 200-mesh stainless steel sieve. The spore concentration was counted under an optical microscope using a hemocytometer, and adjusted to 5 × 10⁻⁶ spores with sterile water. 6 The concentration of spores per mL should be used within 4 hours. Any suspension exceeding this timeframe should be discarded and re-prepared, as spores have a limited survival time in room temperature water, and prolonged exposure will significantly reduce spore viability. Within a 25 cm radius of the base of each *Pinus kesoniaensis* plant facing upwards, gently scrape away the top 2 cm of pine needles and weed debris with a small hoe to expose the mineral soil surface. Then, measure 80 mL of the inoculum suspension and evenly pour it onto the exposed soil surface, ensuring thorough penetration into the surface pores. Immediately after watering, cover with a 2-3 cm layer of fresh pine needles to maintain soil moisture, stabilize soil temperature, and reduce UV damage to spore viability. All inoculation should be carried out on cloudy days or in the evening to avoid direct sunlight that could inactivate the spores. If there is no rain for 7 days after inoculation, spray water in the inoculated area to maintain a soil surface moisture content above 25%. In this embodiment, a total of 1143 Pinus simonii strains were inoculated, consuming approximately 91.4 L of bacterial suspension. The entire inoculation process was completed by two workers within 3 days.

[0027] The procedure for intercropping native nitrogen-fixing legumes is as follows. Figure 1As shown, *Flemingia macrophylla* seeds were directly sown in the center of the rows of *Pinus simaoensis*, maintaining a safe distance of 1.25–1.75 m between the *Flemingia macrophylla* planting rows and the rows of pine trees on both sides. Direct sowing of *Flemingia macrophylla* seeds was carried out in early June 2024 (after the rainy season stabilized). The seeds were obtained from naturally matured *Flemingia macrophylla* pods in the local natural forest undergrowth. After harvesting, the pods were threshed and winnowed to obtain plump seeds. Due to the dormant nature of *Flemingia macrophylla* seeds, acid etching treatment was required before sowing: the seeds were immersed in 90% concentrated sulfuric acid for 20 minutes, constantly stirring to ensure uniform contact of the acid surface. They were then quickly removed and rinsed 5–6 times with plenty of water until the water was clear and odorless. The treated seeds were then germinated on moist filter paper at 25°C for 48 hours. Sowing was carried out when more than 50% of the seeds showed signs of germination. At the center line between rows of *Pinus simonii* (approximately 1.25–1.75 m from each side of the rows), dig holes at a spacing of 1.0 m × 1.0 m, with a hole diameter of 5 cm and a depth of 2 cm. Sow 4 treated seeds in each hole, cover with 1–2 cm of soil, and gently compact. In this example, approximately 6000 holes of *Flemingia macrophylla* are sown per hectare between rows of *Pinus simonii*, equivalent to a seed usage of approximately 4.5 kg / hm². 2 Seedlings begin to emerge approximately 10-15 days after sowing, with an emergence rate of 75%-85%. When seedlings have 3-4 true leaves (approximately 30 days after sowing), thin them out, leaving 2 strong seedlings per hole, for a final density of approximately 12,000 plants / hm². 2 .

[0028] The procedure for mowing and returning the plants to the field is as follows: In the autumn of the year the large-leaved *Millettia dielsiana* is planted (mid-October), when the plants reach a height of 60-80 cm, the first mowing should be performed. The mowing height should be 20 cm from the ground, retaining sufficient stem base and axillary buds to ensure regeneration. After the cut branches and leaves have been left to dry in place for 2-3 days to allow initial dehydration, they should be evenly spread between rows of *Pinus koraiensis* and lightly tilled to a soil depth of 5-8 cm. From the second year onwards, mowing and returning to the field should be performed twice a year: the first time is from late May to early June (early rainy season), when the large-leaved *Millettia dielsiana* has recovered through winter and spring growth, reaching a height of 80-100 cm, with lush foliage and nitrogen content at its peak for the year, making it the optimal time for mowing and returning to the field; the second time is from late September to early October (late rainy season), when the plants re-enter a vigorous growth period. Each mowing should maintain a height of 15-20 cm from the ground. When harvesting, care should be taken to avoid damaging the root collar and basal dormant buds of *Millettia speciosa* to ensure the regeneration capacity of suckers after harvesting. Calculations show that each harvest of *Millettia speciosa* yields approximately 3000–5000 kg / hm² of fresh biomass. 2 The dry matter content is approximately 800~1300 kg / hm. 2The crude protein content is approximately 15%–18% of the dry matter, and the carbon-to-nitrogen ratio (C / N) is approximately 18–22, classifying it as easily decomposable high-nitrogen organic matter. The total dry matter input from two annual harvests and return to the field is approximately 1600–2600 kg / hm². 2 The equivalent organic carbon input is approximately 720~1170 kg C / hm. 2 The nitrogen input is approximately 38-70 kg N / hm. 2 .

[0029] The operational details of tilling and returning the soil to the field are as follows: After cutting branches and leaves and drying them until the moisture content drops to 40%~50%, first spread them evenly between rows of Pinus sylvestris to form a 3~5 cm thick mulch layer. Then, use a small rotary tiller or manual hoeing to mix the mulch with the top 5~8 cm of soil. Tillage should be carried out 1~2 days before rainfall to utilize rainwater to promote full contact between organic matter and soil and rapid colonization and decomposition by microorganisms. The tillage depth should not exceed 10 cm to avoid damaging the established mycorrhizal network and earthworm habitats.

[0030] This embodiment also implemented a soil animal introduction treatment. Given that the soil organic matter content in the experimental site was only 1.32%, below the threshold of 1.5%, Eisenia fetida was introduced into the area where the first harvest was turned over and incorporated into the soil in mid-July 2024 (peak of the rainy season). One week prior to introduction, fermented and decomposed cow manure (approximately 50% moisture content) was applied to the introduction area as the basic organic material for earthworm colonization at a rate of 800 kg / hm². 2 The application method is to apply it in strips between the rows of *Millettia speciosa*, with a strip width of 30 cm and a depth of 5 cm. Earthworms are introduced at a rate of 30 earthworms per m². 2 The earthworms are densely distributed on the topsoil covered with plowed-in material, extending into the plowed-in strip between rows of large-leaved *Millettia dielsiana*. Distribution should be carried out in the evening or on a cloudy day. Immediately after distribution, cover with a 3-5 cm layer of fallen leaves to maintain suitable temperature and humidity. Initially, maintain soil moisture content in the distribution area between 30% and 50%. In case of prolonged drought, supplemental watering is necessary. After a 3-month acclimatization period, the earthworm population gradually spreads and establishes itself in the surrounding soil. With continued plowing, it can naturally reproduce and maintain its population size.

[0031] Following the implementation of the ternary biological synergistic treatment in this embodiment, the understory management method was also adjusted accordingly. Because the large-leaved *Millettia speciosa* forms 60%–80% ground cover between rows, it effectively suppresses the growth of ferns and grasses. The frequency of tending was reduced from twice a year to once a year (selective tending is carried out once in September or October, removing only vines climbing to the crown of *Pinus yunnanensis* and herbaceous weeds within 0.5 m of the main trunk of *Pinus yunnanensis*), reducing the tending workload by approximately 60%.

[0032] Example 2: This example was implemented in a 3-year-old artificial juvenile Pinus yunnanensis forest in Puwen Experimental Forest Farm, Jinghong City, Yunnan Province (22°25′N, 101°05′E, altitude 960 m). The plantation spacing was 3.0 m × 4.0 m, with a planting density of approximately 833 trees / hm². 2 The soil type of the afforestation site is lateritic red soil. The soil pH value of the 0-20 cm soil layer is 5.1, the organic matter content is 1.68%, and the total nitrogen content is 0.092%.

[0033] In this embodiment, the ectomycorrhizal fungal inoculation treatment used a suspension of *Lycoperdon yunnanensis*. The collected fruiting bodies of *Lycoperdon yunnanensis* were used to prepare the fungal suspension according to the same method as in Example 1, and the spore concentration was adjusted to 1×10⁻⁶. 7 Inoculation rate: 60 mL per pine tree, applied to a 20 cm radius around the base of the plant. Inoculation time: late April 2024.

[0034] In this embodiment, the intercropping treatment with native nitrogen-fixing leguminous plants was carried out using an in-situ retention method. The forest clearing and scavenging before afforestation was not thorough enough; a naturally distributed population of *Millettia speciosa* already existed between rows of *Pinus sylvestris* and at the forest edges, with a density of approximately 2000-4000 plants / hm². 2 During the tending operation in May 2024, only individuals of *Mallotus macrocarpa* within 1.0 m of *Pinus yunnanensis* were removed, while the remaining natural population was preserved. Simultaneously, seeds were re-sown in forest clearings where *Mallotus macrocarpa* was sparsely distributed, using the same method as in Example 1, with a planting density of 1.5 m × 1.5 m. After re-sown, the combined density of the remaining population and the natural population was approximately 6000–8000 plants / hm². 2 .

[0035] In this embodiment, the mowing and returning of the plant to the soil is performed once a year, with uniform mowing in early October, to a height of 15 cm above the ground. After the mowed branches and leaves have been dried for 3 days, they are plowed back into the soil to a depth of 8-10 cm. Due to the wide spacing between rows (4.0 m), the natural population of *Ligustrum lucidum* and the reseeded populations have created good ground cover, with a calculated coverage of 70%-85%. The fresh biomass from each mowing is approximately 4000-6000 kg / hm². 2 The dry matter content is approximately 1000~1500 kg / hm. 2 Because the soil organic matter content (1.68%) in this forest stand was higher than the 1.5% threshold, and the soil microbial activity and decomposition capacity were good, earthworm introduction was not implemented. The in-situ retention method adopted in this embodiment significantly reduced the cost of intercropping and establishment, while preserving mature plants that had established a symbiotic relationship with local rhizobia, whose nitrogen fixation efficiency was higher than that of newly sown plants.

[0036] In the implementation of Example 2, the spatial pattern of the in-situ preserved *Flemingia macrophylla* population was also optimized. Because the natural population distribution is clustered, with some areas being overly dense and others sparse, it was necessary to thin out the overly dense areas during reseeding. Specifically, for *Flemingia macrophylla* populations with a density exceeding 15,000 plants / hm²... 2 In the affected areas, some weaker individuals were selectively removed, reducing the density to 8,000–10,000 plants / hm². 2 For *Millettia divaricata*, the natural density is less than 2000 plants / hm². 2 In areas where plants were missing or completely absent, reseeding was carried out at a spacing of 1.5 m × 1.5 m. After thinning and reseeding, the uniformity of the distribution of *Millettia speciosa* in the whole forest was significantly improved, and the effective coverage area increased from 45% of the inter-row area before treatment to 78% after treatment. The *Millettia speciosa* plants removed by thinning were dug up along with their roots and plowed into the field as organic material to avoid waste.

[0037] Example 3: This example was implemented in a one-year-old artificial sapling forest of Pinus yunnanensis in Linxiang District, Lincang City, Yunnan Province (23°52′N, 100°05′E, altitude 1150m). The plantation spacing was 2.0 m × 3.0 m, with a planting density of approximately 1667 trees / hm². 2 The soil type of the afforestation site is red soil. The soil pH value of the 0-20 cm soil layer is 4.5, the organic matter mass fraction is 0.98%, and the total nitrogen mass fraction is 0.065%, which belongs to extremely barren degraded red soil.

[0038] In this embodiment, the ectomycorrhizal fungal inoculation treatment used colored puffball (Puffball) Pisolithus tinctorius ) and Yunnan hard-skinned puffball ( Scleroderma yunnanense A compound bacterial suspension was prepared from the fruiting bodies of two ectomycorrhizal fungi. The fruiting bodies of these two fungi were separately prepared into suspensions, which were then mixed at a volume ratio of 1:1. The total spore concentration of the mixed suspension was 8 × 10⁶ spores / mL. 100 mL was inoculated per *Pinus kesiao* plant, and the application area was a 30 cm radius around the base of the plant. Inoculation was carried out in early May 2024.

[0039] In this embodiment, the intercropping treatment of native nitrogen-fixing leguminous plants was carried out by direct seeding. Due to the high afforestation density and row spacing of only 3.0 m, *Millettia speciosa* (large-leaved *Millettia speciosa*) was used. Flemingia macrophylla The planting density was appropriately reduced, with a plant spacing of 1.2m × 1.2m and a minimum distance of 1.0m between the plants and the Pinus sylvestris. Approximately 4500 holes were sown per hectare. The sowing method was the same as in Example 1.

[0040] In this embodiment, earthworm release was implemented simultaneously in July 2024. Given that the soil organic matter content was only 0.98%, far below the 1.5% threshold, the earthworm release density was increased to 50 earthworms / m². 2Before application, apply 1000 kg / hm² of fermented and decomposed cow manure. 2 At the same time, release Eisenia fetida (Eisenia fetida). Eisenia fetida ) and William's ringed worm ( Pheretima guillelmi Two species were mixed and released in a 1:1 ratio. *Eisenia fetida* primarily inhabits the surface organic matter, promoting its decomposition and humification; *Pheretima wielderii* can burrow and create holes in deeper soil layers, improving soil porosity and aeration. The two earthworm species play complementary roles in different soil layers. Due to the relatively high afforestation density in this example (1667 trees / hm²),... 2 With relatively small spacing between rows, the competition for light and water between *Millettia speciosa* and *Pinus yunnanensis* requires close monitoring. A heavy pruning (10 cm in height) of *Millettia speciosa* at the end of the first growing season (November) can effectively control its crown expansion rate and avoid excessive shading of young *Pinus yunnanensis*. From the second year onwards, the normal pruning height (15-20 cm) is restored. By this time, the *Pinus yunnanensis* has surpassed the height of *Millettia speciosa*, reducing the risk of shading competition.

[0041] In each example, the weed suppression effect of intercropping with *Millettia divaricata* was quantitatively monitored. In Example 1, after 12 months of treatment, the weed cover between rows decreased from the initial 85%–95% to 15%–25%, and the weed species changed from a competitive population dominated by ferns (*Miscanthus sinensis*, *Miscanthus sinensis*, etc.) and grasses (*Imperata cylindrica*, *Miscanthus sinensis*, etc.) to a low-growing community dominated by shade-tolerant herbs and mosses. The weed biomass increased from 8500 kg / hm² in Comparative Example 1. 2 Reduced to 1200 kg / hm in Example 1 2 The weed control effect was reduced by 85.9%. This weed control effect is mainly attributed to two competitive advantages of *Mallotus macrocarpa*: first, its rapid growth and continuous ground cover formed by multiple tillers inhibit the germination of light-loving weed seeds and the growth of seedlings through a shading effect; second, the flavonoids secreted by its roots (such as isoflavones like malvatinin) have an allelopathic inhibitory effect on some weed seeds. The effective weed control directly supports the feasibility of reducing the number of tending sessions, reducing the annual tending frequency in Example 1 from twice to once.

[0042] Comparative Example 1 This comparative example was set up on the same plot as in Example 1, using two-year-old pure stands of Pinus yunnanensis planted in the same batch. Conventional management was implemented without any ternary biological synergistic treatments. Comprehensive tending was carried out twice a year (in May and September), including weeding, shrub removal, soil loosening, and hilling up the base of the trees; no fertilizer was applied. Conventional tending required the removal of all weeds and shrubs within a 1.0 m radius around the Pinus yunnanensis trees, with weeds between rows removed to a depth of 2-3 cm below ground level, and soil loosened to a depth of 5-8 cm. After each tending, the exposed surface area of ​​the forest land reached over 80%, representing a traditional high-intensity tending management model.

[0043] Comparative Example 2 This comparative example was set up on the same plot as Example 1, where a single ectomycorrhizal fungal inoculation treatment was applied to a 2-year-old monoculture plantation of *Pinus kesiya*. The inoculation method and dosage were the same as in Example 1 (80 mL of *Lycoperdon persicum* suspension per plant), but intercropping with *Pleurotus ostreatus* and mowing were not performed. Routine management was the same as in Comparative Example 1, with comprehensive weeding and soil loosening twice a year. The purpose of this comparative example was to evaluate the soil fertility improvement effect of mycorrhizal inoculation alone and to provide a control against the ternary synergistic treatment. Due to the lack of nitrogen supply from intercropping nitrogen-fixing plants and the continuous input of organic matter, although the mycorrhizal fungi successfully infected the *Pinus kesiya* root system in the early stages of inoculation, their subsequent reproduction and extension of mycelia were constrained by insufficient soil carbon and nitrogen sources. The rate of infection growth and the final level were lower than in the example with intercropping.

[0044] Comparative Example 3 This comparative example was set up on the same plot as in Example 1. Two-year-old pure stands of *Pinus kesiya* were intercropped with *Pyracantha fortuneana* and mowed back into the soil. The intercropping method and mowing frequency were the same as in Example 1, but no ectomycorrhizal fungal inoculation was performed. The *Pinus kesiya* roots relied solely on their own growth and a small number of naturally occurring ectomycorrhizal fungal spores in the soil for natural infection, resulting in a slow infection process. After 12 months, the mycorrhizal infection rate was only 12.5%, far lower than the 65%–78% in artificially inoculated treatments. This indicates that without artificial inoculation intervention, the mycorrhizalization degree of young *Pinus kesiya* trees is extremely low, making it difficult to fully utilize the nutrient resources provided by intercropped nitrogen-fixing plants.

[0045] Comparative Example 4 This comparative example was conducted on the same plot as in Example 1, where chemical fertilization was applied to a 2-year-old monoculture plantation of Pinus yunnanensis. A nitrogen-phosphorus-potassium compound fertilizer (N-P₂O₅-K₂O mass ratio of 15:15:15) was applied twice annually, with the first application of 300 kg / hm² as basal fertilizer in April. 2 The second application of fertilizer was done in August at a rate of 150 kg / hm². 2 Apply the fertilizer in a trench 30 cm uphill from the pine trees. Follow the same routine care and management practices as control group 1.

[0046] Monitoring of the infection process after inoculation with ectomycorrhizal fungi in each embodiment showed that initial signs of mycorrhizal infection could be observed on the fine roots of *Pinus simonii* as early as 30 days post-inoculation, manifested as swelling of the root tips and dichotomous branching. By 60 days post-inoculation, the average mycorrhizal infection rate reached 25%–35%, exhibiting typical ectomycorrhizal morphological characteristics, including the mantle structure and Haty's network (e.g., ...). Figure 2As shown, after trypan blue staining, the blue-stained mycelium can be clearly observed under an optical microscope to form a network structure by invading the intercellular spaces of the root cortex. The infection rate reached 45%–55% on day 90 post-inoculation, and the extracellular mycelium began to spread into the surrounding soil. By day 180 post-inoculation (the 12-month monitoring point in Example 1), the mycorrhizal infection rate remained stable at a relatively high level of 65%–78%. Figure 4 As shown, mature mycorrhizal root tips are club-shaped and swollen, with a dense white mycorrhizal mantle layer on the surface. The mycorrhizal infection rate in the intercropped area of ​​*Millettia speciosa* was consistently about 10-15 percentage points higher than that in the non-intercropped area. This is attributed to the fact that the organic carbon source and nitrogen released from the rhizosphere of *Millettia speciosa* improved the nutrient supply conditions for mycorrhizal fungi.

[0047] The development of root nodules in *Millettia speciosa* is as follows: Root nodule primordia can be observed on the seedling roots as early as 45 days after direct seeding. By day 60, the number of root nodules reaches 5-12 per plant, with a diameter of 2-4 mm. Figure 3 As shown, after double staining with safranin and Fast Green on the cross-section of the root nodules, a clear boundary was observed between the outer cortical region and the internal infected region. The infected cells were filled with rhizobium bacteria, and the cut surface appeared pink (indicating sufficient leghemoglobin content and normal nitrogenase activity). By day 120, the number of root nodules increased to 20-35 per plant, and the nitrogenase activity (determined by acetylene reduction method) reached 15-28 μmol C2H4 / (g fresh root nodules·h). Mature *Millettia dielsiana* plants retained in situ (Example 2) showed more complete root nodule development, with 50-80 root nodules per plant and nitrogenase activity reaching 25-40 μmol C2H4 / (g fresh root nodules·h), significantly higher than that of seedlings sown in the same year.

[0048] In Example 1, population dynamics monitoring after earthworm release showed that the mortality rate was approximately 15%–20% in the initial stage (months 1–2), with surviving individuals mainly concentrated in the top 0–10 cm of soil covered with organic matter. Young earthworms began to appear in the third month after release, indicating that the population had entered the natural reproduction stage. By the sixth month (January 2025, the dry season), due to a decrease in soil moisture content to 15%–20%, earthworm activity significantly decreased, and the population density remained at 15–20 earthworms / m². 2 In the 9th month (after the rainy season in April 2025), with the recovery of soil moisture and the introduction of organic material from the first harvest and return to the field, the earthworm population rapidly recovered and grew to 35-45 individuals / m². 2The density exceeded the initial release density. This indicates that under continuous organic material input, the earthworm population can be maintained and expanded through natural reproduction without the need for repeated release. Earthworm activity also significantly affected soil enzyme activity: in the treatment area of ​​Example 1, the sucrase activity in the 0-10 cm soil increased from the initial 12.5 mg glucose / (g·24 h) to 28.6 mg glucose / (g·24 h), the urease activity increased from 0.38 mg NH3-N / (g·24 h) to 0.85 mg NH3-N / (g·24 h), and the catalase activity increased from 1.25 mL KMnO4 / (g·20 min) to 2.18 mL KMnO4 / (g·20 min), all of which were significantly different from those in Comparative Example 1 (p<0.05).

[0049] No significant negative competitive impact of intercropping *Millettia speciosa* on *Pinus yunnanensis* saplings was observed during the implementation of any of the embodiments. This is mainly due to the following reasons: *Millettia speciosa* is a semi-shade shrub, with a plant height typically maintained at 60-120 cm, far lower than the height of 2-5 year old *Pinus yunnanensis* (120-450 cm), and will not cause top shading for the pine trees; the root system of *Millettia speciosa* is mainly distributed in the 0-30 cm soil layer, while the taproot system of *Pinus yunnanensis* can penetrate to the 40-60 cm soil layer after 2 years, resulting in a misalignment of nutrient competition between the two root systems; the nitrogen input into the soil by *Millettia speciosa* through biological nitrogen fixation is far greater than the amount of nitrogen it absorbs from the soil, making it a net nitrogen contributor rather than a competitor to the surrounding soil. The seedling height and diameter growth of *Pinus yunnanensis* saplings in Examples 1 to 3 were all higher than the corresponding blank control, further confirming from another perspective that the intercropping treatment has a promoting rather than inhibiting effect on the growth of *Pinus yunnanensis*.

[0050] The detection methods are as follows. Soil samples were collected using a five-point sampling method. Five sampling points were randomly set up in each treatment plot. Soil samples were collected from two depths (0–10 cm and 10–20 cm) at each point using a soil auger, and then combined into one sample. Soil organic matter content was determined using the potassium dichromate external heating method (NY / T 1121.6). Total nitrogen content was determined using the Kjeldahl method (NY / T53). Available phosphorus content was determined using the sodium bicarbonate extraction-molybdenum antimony colorimetric method (NY / T 1121.7). Available potassium content was determined using the ammonium acetate extraction-flame photometric method (NY / T 889). Soil pH was determined using the potentiometric method (soil-water ratio 2.5:1). Soil microbial biomass carbon was determined using the chloroform fumigation-potassium sulfate extraction method. Soil sucrase activity was determined using the 3,5-dinitrosalicylic acid colorimetric method, urease activity using the phenol-sodium hypochlorite colorimetric method, and catalase activity using the potassium permanganate titration method. Mycorrhizal infection rate was determined using a modified Phillips-Hayman staining method. Fine root samples of *Pinus kesiao* were incubated in 10% KOH solution at 90°C for 40 min for clearing, then stained with 0.05% trypan blue lactic acid glycerol solution. Infection rate was statistically analyzed using a grid cross-section method under a dissecting microscope, with at least 100 root segment cross-sections observed for each sample. Seedling height of *Pinus kesiao* was measured to an accuracy of 0.1 cm using the pole method, and ground diameter was measured to an accuracy of 0.01 mm using electronic vernier calipers at a height of 5 cm above the ground. Weed cover was investigated using the quadrat method. Five 1 m × 1 m quadrats were randomly set up within each treatment plot, and the percentage of projected weed cover area in each quadrat was visually estimated. Earthworm density was assessed using a hand-collection method. Five 25 cm × 25 cm × 20 cm soil sampling plots were randomly selected in the release area, and earthworm numbers were counted by turning over each layer. Each treatment had three replicates, with each replicate plot area no less than 400 m². 2 A total of 30 fixed observation plants were marked. Data were analyzed using SPSS 26.0 software using one-way ANOVA. Duncan's multiple comparison method was used to compare the means between groups, and the significance level was set at α=0.05.

[0051] In addition, to accurately evaluate the improvement effect of the ternary biological synergistic treatment on soil at different depths, stratified sampling analysis was performed on Example 1 and Comparative Example 1. The results showed that after treatment in Example 1, the organic matter content in the 0–10 cm topsoil increased from an initial 1.56% to 2.15%, with an annual increase of 0.59 percentage points; in the 10–20 cm subsurface layer, it increased from an initial 1.08% to 1.29%, with an annual increase of 0.21 percentage points. The organic matter improvement effect exhibited obvious surface aggregation characteristics, which is consistent with the depth of organic material incorporation (5–10 cm) and the main activity layer of earthworms (0–15 cm). In Comparative Example 1, the organic matter content in both the 0–10 cm and 10–20 cm layers showed a decreasing trend, decreasing to 1.48% and 1.02%, respectively. Regarding total nitrogen content, in Example 1, the total nitrogen mass fraction in the 0–10 cm layer increased from 0.092% to 0.138%, an increase of 50%; in the 10–20 cm layer, it increased from 0.064% to 0.086%, an increase of 34.4%. The available nitrogen content (alkaline-available nitrogen) in the 0–10 cm layer increased from 58.6 mg / kg to 112.3 mg / kg, an increase of 91.6%. This high increase reflects the rapid accumulation and mineralization release of nitrogen-fixing products from *Millettia speciosa* in the topsoil.

[0052] The dynamic changes in soil nutrients varied significantly across different treatments in different seasons. During the rainy season (June to September), increased rainfall and improved soil temperature and humidity enhanced microbial activity and accelerated organic matter decomposition. In the treatment area of ​​Example 1, the available nitrogen content reached its annual peak of 132.5 mg / kg in mid-rainy season (August), an increase of 68.6% compared to the low point in the dry season (78.6 mg / kg in February). Simultaneously, *Millettia dielsiana* exhibited the highest nitrogen fixation rate during its vigorous growth period in the rainy season, and the rapid decomposition of harvested crop residues after returning to the field also concentrated during this period, forming a seasonal coupling between nutrient supply and the growth requirements of *Pinus koraiensis*. Comparative Examples 1 and 4 showed smaller variations in available nitrogen during the rainy season (Comparative Example 1 fluctuated from 42.3 to 58.1 mg / kg throughout the year, and Comparative Example 4 from 55.8 to 82.6 mg / kg), indicating that the ternary biological synergistic treatment not only increased the total soil nutrients but also enhanced the seasonal supply capacity of nutrients, better matching the growth rhythm of *Pinus koraiensis*.

[0053] Twelve months after implementation (May 2025), the soil physicochemical properties and Pinus sylvestris growth indicators for each treatment were tested as follows.

[0054] In Example 1, the organic matter content in the 0-20 cm soil layer increased from 1.32% to 1.72%, an annual increase of 0.40 percentage points. The total nitrogen content increased from 0.078% to 0.112%, an annual increase of 0.034 percentage points. Available phosphorus content increased from 3.2 mg / kg to 5.8 mg / kg. Available potassium content increased from 68.5 mg / kg to 82.3 mg / kg. Soil pH increased from 4.8 to 5.1. Mycorrhizal infection rate reached 72.5%. The average annual height growth of *Pinus kesiaoensis* seedlings was 85.6 cm, an increase of 21.7% compared to Comparative Example 1. The average annual diameter growth was 1.82 cm, an increase of 18.3% compared to Comparative Example 1.

[0055] In Example 2, the organic matter content in the 0-20 cm soil layer increased from 1.68% to 2.01%, an annual increase of 0.33 percentage points. The total nitrogen content increased from 0.092% to 0.121%. The mycorrhizal infection rate reached 65.8%. The average annual growth of *Pinus kesiao* seedlings was 92.3 cm, an increase of 18.5% compared to the corresponding control.

[0056] In Example 3, the organic matter content in the 0-20 cm soil layer increased from 0.98% to 1.48%, an annual increase of 0.50 percentage points. The total nitrogen content increased from 0.065% to 0.102%. The mycorrhizal infection rate reached 78.2%. The average annual growth of *Pinus kesiao* seedlings was 62.8 cm, an increase of 24.3% compared to the corresponding control.

[0057] In Comparative Example 1 (blank control), the organic matter content in the 0–20 cm soil layer decreased from 1.32% to 1.25%, indicating continuous organic matter loss. The total nitrogen content decreased from 0.078% to 0.072%. The average annual height growth of *Pinus kesiao* seedlings was 70.3 cm, and the average annual diameter growth was 1.54 cm.

[0058] In Comparative Example 2 (single mycorrhizal inoculation), the organic matter content in the 0–20 cm soil layer increased from 1.32% to 1.40%, with an annual increase of only 0.08 percentage points. The total nitrogen content increased from 0.078% to 0.085%. The mycorrhizal infection rate was 58.6%. The average annual height growth of *Pinus kesiao* seedlings was 76.2 cm, an increase of 8.4% compared to Comparative Example 1.

[0059] In Comparative Example 3 (single intercropping with soil returning to the field), the organic matter content in the 0–20 cm soil layer increased from 1.32% to 1.58%, with an annual increase of 0.26 percentage points. The total nitrogen content increased from 0.078% to 0.098%. The average annual growth of *Pinus kesiao* seedlings was 78.9 cm, an increase of 12.2% compared to Comparative Example 1.

[0060] In Comparative Example 4 (chemical fertilization), the organic matter content in the 0–20 cm soil layer decreased from 1.32% to 1.28%, showing a decline rather than an increase. The total nitrogen content increased from 0.078% to 0.095%, a smaller increase than in Example 1. The soil pH decreased from 4.8 to 4.5, indicating significant acidification. Available phosphorus content increased from 3.2 mg / kg to 6.5 mg / kg, and available potassium content increased from 68.5 mg / kg to 95.2 mg / kg, showing a significant increase in phosphorus and potassium nutrients, but at the cost of sacrificing soil pH balance. The average annual growth of *Pinus koraiensis* seedlings was 82.1 cm, 16.8% higher than in Comparative Example 1, but slightly lower than the 21.7% in Example 1.

[0061] From the perspective of soil microbial activity, after 12 months of treatment, the microbial biomass carbon content in the 0-10 cm topsoil reached 385.6 mg / kg, which was 107.1% higher than that of Comparative Example 1 (186.2 mg / kg), 68.7% higher than that of Comparative Example 2 (228.5 mg / kg), and 23.4% higher than that of Comparative Example 3 (312.4 mg / kg). This indicates that the ternary biological synergistic treatment not only directly increased the soil organic matter content, but also significantly enhanced the activity and diversity of the microbial community by improving the soil biological habitat.

[0062] From the perspective of soil physical properties, the soil bulk density in the 0-20 cm depth decreased from the initial 1.42 g / cm³ to 1.28 g / cm³ after treatment in Example 1, while the soil porosity increased from 46.4% to 51.7%, and the soil moisture content increased from 18.3% to 23.6%. The soil bulk density in Comparative Example 1 remained essentially unchanged (from 1.42 g / cm³ to 1.40 g / cm³), and the soil bulk density in Comparative Example 4 (chemical fertilizer treatment) even increased slightly (from 1.42 g / cm³ to 1.44 g / cm³), indicating that chemical fertilization failed to improve the soil physical structure. In Example 1, the large pores and aggregates formed by earthworm activity played a crucial role in improving the soil physical properties.

[0063] From an economic benefit analysis perspective, the initial investment cost for the ternary biological synergistic treatment in Example 1 is approximately RMB 2800 / hm². 2 (Including 400 yuan for inoculant preparation, 1200 yuan for seeds and labor for sowing of *Millettia speciosa*, 600 yuan for earthworm purchase and release, and 600 yuan for well-rotted cow manure). From the second year onwards, since *Millettia speciosa* is a perennial plant and does not require reseeding, and earthworms can reproduce naturally to maintain the population, the annual maintenance cost is only about 800 yuan / hm² for the labor cost of mowing and returning the crop to the field. 2 The annual input cost of chemical fertilization in Comparative Example 4 was approximately RMB 1800 / hm². 2 (450 kg / hm² of compound fertilizer) 2×4 yuan / kg), and requires annual investment. Considering that the number of tending times in Example 1 is reduced from twice a year to once, approximately 600 yuan / hm² of tending labor costs can be saved. 2 ·a^-1. Therefore, from the third year onwards, the annual operating cost of Example 1 (800 yuan / hm) 2 Subtract the savings of 600 yuan / hm 2 That is, net cost of 200 yuan / hm 2 The cost was significantly lower than that of chemical fertilization (1800 yuan / hm²). 2 This approach achieves superior ecological benefits and more lasting soil improvement. Based on a rotation period of 25-30 years for a Pinus sylvestris plantation, the cumulative cost of the ternary biological synergistic program is approximately 8,000-10,000 yuan / hm². 2 This is solely based on a chemical fertilization program (45,000~54,000 yuan / hm²). 2 Its economic advantages are extremely significant, accounting for 18% to 22% of the total.

[0064] The comprehensive comparison results show (e.g.) Figure 5 and Figure 6 As shown in the figure, the annual increase in soil organic matter in Example 1 (ternary biological synergistic treatment) (0.40%) was significantly higher than the sum of Comparative Example 2 (0.08%) and Comparative Example 3 (0.26%) (0.34%), exhibiting a significant nonlinear synergistic effect. Based on the synergistic coefficient CI, the CI value was approximately 0.85 (CI less than 1 indicates synergistic enhancement), indicating that the ternary biological synergistic system produced an over-additive effect. Regarding the promotion of Pinus sylvestris growth, the seedling height growth rate of Example 1 (21.7%) was higher than the simple superposition expected value of Comparative Example 2 (8.4%) and Comparative Example 3 (12.2%) (20.6%), but the difference was more significant in the systematic improvement of root health and nutrient utilization efficiency. The mycorrhizal infection rate of Example 1 (72.5%) was significantly higher than that of Comparative Example 2 (58.6%). This is because the intercropping of *Millettia dielsiana* improved soil organic matter content and microbial community structure, creating a more favorable soil microenvironment for the reproduction and infection of mycorrhizal fungi. It is worth noting that Example 1, while achieving better results than fertilizer application (Comparative Example 4), avoided soil acidification (pH increase of 0.3 units vs. decrease of 0.3 units in fertilizer treatment), embodying the concept of green and sustainable management. Example 3, even under extremely barren degraded red soil conditions, still achieved excellent results with an annual increase of 0.50 percentage points in soil organic matter and a high growth rate of 24.3% for Pinus sylvestris seedlings, indicating that the ternary biological synergistic method of this invention has good applicability and stable improvement effects under different soil fertility baseline conditions.

[0065] The reason why the ternary biological synergistic soil fertility enhancement system of the present invention can produce a super-additive effect is that the three biological components—ectomycorrhizal fungi, native nitrogen-fixing legumes, and soil animals—form a multi-level complementary coupling relationship in terms of function.

[0066] At the nitrogen input level, the roots of *Millettia speciosa* form root nodules in a symbiotic relationship with slow-growing rhizobium (Bradyrhizobium spp.), reducing atmospheric N2 to NH4. + It releases available nitrogen into the surrounding soil through root exudates and litter decomposition, with an annual nitrogen fixation capacity of 30-80 kg N / hm². 2 This constitutes the nitrogen input source for the entire system. As a perennial shrub, *Millettia speciosa* has a well-developed and continuously growing root system, and its nitrogen-fixing capacity is far superior to that of annual leguminous green manure crops. Moreover, it can quickly regenerate from the root neck after being cut, ensuring the continuity of nitrogen fixation.

[0067] In terms of nutrient acquisition, ectomycorrhizal fungi infect the fine roots of *Pinus kesiya*, forming Haty's network and extended mycelium. The absorption area of ​​the mycelium can be tens to hundreds of times larger than that of the root system itself, enabling efficient acquisition of nitrogen and phosphorus released into the soil by *Millettia speciosa*. The mycelium can also secrete low-molecular-weight organic acids such as oxalic acid and citric acid, as well as chelating substances such as iron carriers, activating insoluble iron and aluminum phosphates and organic phosphorus in the soil, significantly improving the utilization efficiency of *Pinus kesiya* for intercropping nitrogen fixation products and potential soil nutrients. *Calamus yunnanensis* and *Calamus yunnanensis* are both broad-spectrum ectomycorrhizal fungi that can grow normally in acidic soil environments with a pH of 4.0–6.5, establishing a stable symbiotic relationship with the roots of *Pinus kesiya*.

[0068] At the level of material cycling, earthworm activity in the area where harvested plant material is turned over and returned to the soil promotes the physical breaking down, enzymatic hydrolysis, and microbial degradation of organic matter, accelerating the transformation of organic matter from fresh plant residues to stable humus. The microbial community within the earthworm's gut can secrete cellulase and hemicellulase, efficiently decomposing the cellulose and hemicellulose in the harvested *Millettia speciosa* into small-molecule organic matter that can be further utilized by soil microorganisms. Earthworm castings have a good aggregate structure, are rich in humic acid and various water-soluble nutrients, and are a high-quality natural soil conditioner. The earthworm's burrowing and hole-building activities improve the soil's pore structure and aeration and permeability, creating a favorable physical environment for the extension and growth of mycorrhizal hyphae and the deep extension of *Millettia speciosa* roots.

[0069] At the ecological function level, the three elements constitute a positive feedback loop of nitrogen input—efficient acquisition—accelerated cycle. Intercropping with *Millettia speciosa* also generates additional ecosystem services: its dense aboveground cover effectively intercepts rainfall energy, mitigating slope soil erosion; its well-developed root network stabilizes the topsoil, enhancing slope stability; and its ability to quickly seal the ground between rows inhibits the invasion of ferns and grasses, reducing competitive pressure with young *Pinus koraiensis* trees. This multi-layered functional coupling and ecological feedback mechanism is the fundamental reason for the super-additive effect generated by the ternary biological synergistic system of this invention.

[0070] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A method for synergistic improvement of soil fertility in young Pinus sylvestris forests using a three-element biological approach, characterized in that, Includes the following steps: A three-element biological synergistic soil fertility enhancement treatment was implemented on 1-5 year old artificial young Pinus sylvestris forests. The three-element biological synergistic soil fertility enhancement treatment included ectomycorrhizal fungal inoculation treatment, intercropping treatment with native nitrogen-fixing leguminous plants, and mowing and returning to the field treatment. The ectomycorrhizal fungal inoculation treatment is as follows: An ectomycorrhizal fungal inoculant is applied to the soil surface within a radius of 20-30 cm around the base of the *Pinus kesoniaensis* plant. The ectomycorrhizal fungal inoculant is selected from at least one of *Lycorrhiza glabra* suspension and *Lycorrhiza yunnanensis* suspension. The inoculation amount per *Pinus kesoniaensis* plant is 50-100 mL, and the spore concentration of the fungal suspension is 10. 6 ~10 7 cells / mL; The intercropping treatment of native nitrogen-fixing leguminous plants is as follows: the perennial native nitrogen-fixing plant *Millettia speciosa* is planted between rows of *Pinus simonii* at a spacing of 0.8 m × 0.8 m to 1.5 m × 1.5 m, and the distance between *Millettia speciosa* and the nearest *Pinus simonii* plant is not less than 1.0 m. The cutting and returning to the field treatment is as follows: the large-leaved shrub is cut 1-2 times a year, the cutting height is 15-20 cm from the ground, and the cut branches and leaves are turned back into the field to a soil depth of 5-10 cm.

2. The method according to claim 1, characterized in that, The ectomycorrhizal fungal inoculation treatment is carried out during the early rainy season from April to June each year. The application method is to evenly irrigate the soil surface at the base of the Pinus sylvestris var. spp. and then cover it with a 2-3 cm thick layer of pine needles and fallen leaves.

3. The method according to claim 1, characterized in that, The planting method for the large-leaved shrub is direct seeding. Before sowing, the seeds are soaked in concentrated sulfuric acid (80%~98% by mass) for 15~30 minutes to break up the hardness. 3~5 seeds are sown per hole at a depth of 1~2 cm.

4. The method according to claim 1, characterized in that, The harvesting and returning of crops to the field is carried out during the first harvest in May-June and the second harvest in September-October each year. The harvested crops are left to dry in place for 2-3 days before being turned over and returned to the field.

5. The method according to claim 1, characterized in that, The method also includes soil animal introduction treatment: in plots where the soil organic matter content is less than 1.5%, local earthworms are released near the root zone of *Smilax china* during the rainy season at a density of 20-50 earthworms / m². 2 The local earthworms are selected from at least one of Eisenia fetida and Vertrilia William.

6. The method according to claim 1, characterized in that, The intercropping treatment of native nitrogen-fixing leguminous plants adopts the in-situ retention method, that is, when planting and tending Pinus yunnanensis, the naturally growing population of *Millettia speciosa* in the forest land is retained, and only individuals of *Millettia speciosa* within a distance of less than 1.0 m from the Pinus yunnanensis plants are removed.

7. The method according to claim 1, characterized in that, The planting density of the artificial young forest of Pinus simonii is 2 m × 3 m to 3 m × 4 m, the altitude of the afforestation site is 700~1700 m, and the soil type is red soil or lateritic soil.

8. The method according to claim 1, characterized in that, The preparation method of the ectomycorrhizal fungal inoculant is as follows: the fruiting bodies of colored puffball or Yunnan hard-skinned puffball are crushed and suspended in sterile water, filtered through a 200-mesh sieve, and the spore concentration is adjusted to 106~107 spores / mL. It is prepared and used immediately.

9. The method according to claim 5, characterized in that, Before releasing the earthworms, fermented and decomposed cow manure is applied as a base organic material in the release area at a rate of 500-1000 kg / hm². 2 The moisture content of the fermented and decomposed cow manure is 40% to 60%.

10. The method according to any one of claims 1 to 9, characterized in that, After the implementation of the three-element biological synergistic soil fertility enhancement treatment, the annual increase in soil organic matter content in young Pinus sylvestris forests reached 0.3%~0.5%, the annual increase in total nitrogen content reached 0.02%~0.05%, the average annual growth of Pinus sylvestris seedling height increased by 15%~25% compared with the untreated control, and the average annual growth of diameter at breast height increased by 10%~20% compared with the untreated control.

Images