Composite culture medium for vaccinium uliginosum community recovery and application thereof

Abstract

The invention discloses a composite culture medium for vaccinium uliginosum community recovery and application thereof, and belongs to the technical field of vaccinium uliginosum community recovery. The method aims at solving the problems that existing vaccinium uliginosum population degeneration ecological restoration lacks technical mature culture media and vaccinium uliginosum planting in similar habitat land parcels is difficult. The invention provides a compound culture medium formula for vaccinium uliginosum community recovery. The compound culture medium formula comprises the following steps: diluting 1Kg / m < 3 > of agriculture and forestry water-retaining agent gel after 150 times of water absorption and an Ericoid mixed bacterium solution of which the conidium concentration reaches 108 spores / mL by 1000 times according to the dosage of 0.2 L / m < 3 >, and spraying and inoculating to a compound culture medium, wherein the volume ratio of edible fungus waste containing hardwood components to fallen leaf litters is 3: 2; 50% phoxim missible oil or 30% trichlorfon missible oil is fully mixed according to 2.5 L / m < 3 >, and water is supplemented to make the water content of the composite culture medium reach 70%. The frozen soil regeneration and the sphagnum layer recovery are promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of restoration of blueberry colonies, and in particular relates to a composite cultivation substrate for restoration of blueberry colonies and application thereof. Background Art

[0002] Vaccinium vulgaris (V.uliginosum L.), also known as wild blueberry, has significant medicinal value and commercial development potential because its fruit is rich in a variety of biologically active substances, which is also the main reason driving people to rush to develop its wild population. However, since Vaccinium vulgaris grows in the high-level peat moss swamps in the forest, it has the ecological characteristics of being humid, acidic (pH3.5-5.5), and high in organic matter (not less than 10%). At the same time, due to the slow decomposition rate of organic matter in the acidic and humid soil environment, digestion is inhibited, and plant growth is often limited by nitrogen supply. In order to improve nitrogen absorption, Vaccinium vulgaris can form symbiotic mycorrhizal ERM with ericoid fungi, which is extremely important for the growth and development of Vaccinium vulgaris. However, due to the narrow and harsh growth conditions of Vaccinium vulgaris, it is easily affected by changes in the external environment. Therefore, in recent years, with the frequent occurrence of climate warming and drought events, the degradation of Vaccinium vulgaris communities has been very serious. Therefore, utilizing the symbiotic ericoid mycorrhizal fungi associated with Vaccinium vitis-idaea, creating suitable living conditions for the mycorrhizal fungi, developing and applying the matrix are of great significance for achieving the restoration of Vaccinium vitis-idaea communities in degraded areas and similar ecological planting.

[0003] Peat is the most widely used and most effective blueberry cultivation medium in the world. However, the peat mining process will cause serious damage to the ecological environment. In addition, peat has a long accumulation period and is non-renewable in the short term. Uncontrolled mining will lead to the depletion of peat resources. At the same time, due to the large distribution area of ​​​​Vaccinium vitis viniferum (3 million acres), the volume of peat moss used in the resource recovery process is about 400 million m 3 The existing resource storage and mining volume is far from enough for production needs. Therefore, it is particularly important to seek a substitute for peat moss and apply it to the restoration of Vaccinium vitis vinifera communities.

[0004] At the same time, with the rapid development of the edible fungus industry, the waste of edible fungus bags has become a new source of environmental pollution due to its low utilization value, high pH and EC values, and the production of a large number of spores (more than 2 million tons per year in the two provinces). Therefore, how to use the waste of edible fungus bags with low energy consumption and greenness has become an urgent problem that governments at all levels in the main edible fungus producing areas need to solve. Summary of the invention

[0005] The purpose of the present invention is to address the deficiency of lack of stable, mature and easy-to-promote cultivation substrates and application methods in the process of restoring the blueberry community. The present invention provides a composite cultivation substrate for restoring the blueberry community and an application method thereof, which solves the problem of lack of technically mature cultivation substrates and difficulty in planting blueberry in similar habitat plots in the existing ecological restoration of degraded blueberry populations, and also solves the problem of low energy consumption and green utilization of edible fungus bag waste.

[0006] The invention provides a composite cultivation matrix formula for restoring the blueberry community, which is composed of the following components: a volume ratio of edible fungus waste containing hardwood components to fallen leaves and litters of 3:2, and 1 kg / m3 of agroforestry water retaining agent gel after absorbing 150 times of water. 3 The Ericoid mixed bacterial solution with a conidia concentration of 108 spores / mL was diluted at least 1000 times at 0.2L / mL. 3 Spray inoculate into the composite cultivation matrix, 2000 times of phoxim 50% emulsifiable concentrate or 300 times of trichlorfon 30% emulsifiable concentrate at 2.5L / m 3 Mix thoroughly and add water to make the moisture content of the composite cultivation medium reach 70%.

[0007] It is further defined that the content of the hardwood component is 30% crude fiber, 10% crude protein and 7% crude fat.

[0008] It is further defined that the fallen leaves are the fallen leaves of pine needles on the ground of larch or mixed coniferous and broad-leaved forests.

[0009] It is further defined that the gel component of the agricultural and forestry water-retaining agent is a high molecular weight water-absorbing resin.

[0010] It is further defined that the composition of the Ericoid mixed bacterial solution is a volume ratio of Phialocephala fortinii: Meliniomyces variabilis: Oidiodendron maius of 1:1:1.

[0011] It is further defined that the dosage of the Ericoid mixed bacterial solution inoculated in the formula is 0.2L / m 3 .

[0012] The present invention provides a method for restoring a Vaccinium vitis-idaea colony, and the specific steps of the method are as follows:

[0013] Step 1: In the degraded area of ​​​​the blueberry community, before applying the substrate, use a brush cutter to cut off the competitive shrubs in the blueberry community, and spread the shrubs on the blueberry plants after cutting. Before freezing and snowing in November, spread the composite cultivation substrate formula described in any one of claims 1 to 6 on the blueberry community, with a minimum thickness of 20 cm, and apply it continuously for 3 years;

[0014] Step 2: In a forest clearing with a snow thickness of more than 40 cm in winter, after clearing the forest and before the snow falls in winter, spread the composite cultivation matrix formula of any one of claims 1 to 6 on the plot to be planted with a thickness of 20 cm, use a micro rotary tiller to rotary till the land to make a 1m wide × 0.2m high bed, cover and fix it with a 3-needle shade net after the snow melts in the spring of the second year, and plant the vaccinium nutritious pot seedlings in the middle of the planting bed at a spacing of 50 cm from late May to early mid-June. When planting, cut the shade net crosswise with a wallpaper knife, dig holes with a small shovel, the depth of which is equivalent to the height of the nutritious pot, and then cover and tamp it down, and cover the cut shade net tightly with surrounding soil or matrix.

[0015] It is further defined that the competing shrubs in step 1 are Betula serrata, Salix vaccinium and Rhododendron.

[0016] The present invention provides an application of the above formula in the restoration breeding of Vaccinium vitis viniferum population.

[0017] Further defined, the use also includes the generation of peat moss and the promotion of use in permafrost.

[0018] Beneficial effects: The present invention changes the previous crown cutting, light transmission and rejuvenation mode from the perspective of plants in the process of cultivating blueberry. The present invention starts from the perspective of understanding the mechanism of blueberry community degradation caused by climate warming, permafrost melting, accelerated organic matter mineralization, and excessive development of competing plants. For the first time, the composite cultivation matrix developed is combined with targeted cultivation methods such as removing competing plants to create a cultivation environment with high organic matter, low temperature, high humidity and acidity suitable for the growth of blueberry communities. At the same time, the environment will also inhibit the growth of other plants, fundamentally solving the problem of difficulty in restoring blueberry populations and similar cultivation difficulties.

[0019] The invention not only fundamentally solves the problem of difficulty in ecological restoration of degraded Vaccinium vitis-idaea population, but also promotes the regeneration of frozen soil and the recovery of peat moss layer, thus forming a virtuous cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1The results of the application of different cultivation substrates on plant biomass (a), plant nitrogen content (b) and mycorrhizal infection rate (c) are shown in the figure; Degraded-compound substrate: application of compound substrate in the degraded area, Degraded-Funguswaste: application of single edible fungus waste in the degraded area, Similar-compound substrate: application of compound substrate in similar habitats, Similar-Fungus waste: application of single edible fungus waste in similar habitats; asterisks indicate significant differences between treatments at the same location (*P<0.05, **P<0.01, ***P<0.001); HJY: indicates the experimental location, Hanjiayuan Forestry Bureau Family Forest Farm in Daxing'anling area, XQ: indicates the experimental location, Quanlin Forest Farm in Xinqing Forestry Bureau in Xiaoxing'anling area;

[0021] Figure 2 The results of the application of different cultivation substrates on the abundance of three symbiotic ericoid mycorrhizal fungi, Phialocephala fortinii (a), Meliniomyces variabilis (b) and Oidiodendron maius (c); different letters indicate significant differences among treatments (n = 3, P < 0.05);

[0022] Figure 3 This is the result diagram of the effect of applying different cultivation substrates on the activity of rhizosphere substrate-related enzymes of Vaccinium vitis-idaea; different letters indicate significant differences among treatments (n=3, P<0.05);

[0023] Figure 4 This is the result diagram of the effect of applying different cultivation substrates on the biomass of peat moss;

[0024] Figure 5 The results of the application of different cultivation substrates on the temperature at a depth of -20 cm from the ground surface at two test sites: Hanjiayuan Forestry Bureau (a) and Xinqing Forestry Bureau (b). DETAILED DESCRIPTION

[0025] The gel component of agricultural and forestry water-retaining agent is polymer water-absorbent resin (a water-swellable polymer Super Absorbent Polymer, SAP) containing strong hydrophilic groups such as carboxyl and hydroxyl groups.

[0026] Example 1. Selection and formulation of composite cultivation substrate

[0027] 1. A composite cultivation matrix for restoring the blueberry community is composed of edible fungus waste materials containing hardwood components (30% crude fiber, 10% crude protein and 7% crude fat) of black fungus, oyster mushroom and pine ganoderma after cultivation, ground pine needle litter of larch forest or larch / coniferous and broad-leaved mixed forest in a volume ratio of 3:2, and 1 kg / m3 of agricultural and forestry water retaining agent gel after 150 times water absorption. 3 , the conidia concentration reached 10 8 The Ericoid mixed bacterial solution containing 1000 spores / mL was diluted 1000 times (the lowest dilution factor) at 0.2L / mL. 3 Spray inoculate into the composite cultivation matrix, 2000 times of phoxim 50% emulsifiable concentrate or 300 times of trichlorfon 30% emulsifiable concentrate 2.5L / m 3 Mix thoroughly and add water to make the moisture content of the composite cultivation medium reach 70%.

[0028] 2. Preparation of Symbiotic Ericoid Mycorrhizal Fungal Suspension

[0029] After removing the soil and attachments from the surface of the fresh roots of Vaccinium vitis-idaea, rinse them with distilled water for 3 times, 5% NaOCl for 5 min, 75% ethanol for 5 min, and sterile water for 3 times. After removing the residual water on the sample surface with sterilized filter paper, cut the roots into small sections of about 0.5-1.0 cm, disinfect the surface with 0.1% mercuric chloride, and then inoculate on peptone medium (maltose 20 g / L, peptone 1 g / L, agar 15 g / L, chloramphenicol 200 mg / L) for cultivation. During this period, single colonies of different forms were picked, and after multiple plate purifications, they were inoculated on slant medium, one was stored at 4°C for standby use, and one was sequenced for molecular identification. After sequence splicing, similarity comparison analysis was performed at NCBI to find standard strains with high similarity to the target gene sequence. It is generally believed that those with a similarity of more than 97% are of the same species, and those with a similarity of more than 95% are of the same genus. Finally, the aligned sequence files were used to construct a phylogenetic tree using MEGA6.06 software (using the Neighbour-joining method to build the tree, and the bootstrap value was set to 1000 repetitions).

[0030] After molecular level identification, about 7 days before the preparation of the composite matrix, the three identified Ericoid fungal strains, Oidiodendron maius, Meliniomyces variabilis and Phialocephala fortinii (references: Duan, Y.; Fu, X.; Zhou, X.; Gao, D.; Zhang, L.; Wu, F. Removal of Dominant Species Impairs Nitrogen Utilization in Co-Existing Ledum palustre and Vaccinium uliginosum Communities Subjected to Five-Year Continuous Interruptions. Agronomy 2022, 12, 932. https: / / doi.org / 10.3390 / agronomy12040932) were inoculated into liquid peptone medium (maltose 20 g / L, peptone 1 g / L, chloramphenicol 200 mg / L) and cultured with shaking at 20°C. The conidia concentration reached 10 8 After the conidia suspensions of the three Ericoid fungal strains were concentrated to spores / mL, equal volumes of the conidia suspensions were thoroughly mixed and set aside.

[0031] Example 2. A method for restoring a Vaccinium vitis-idaea colony

[0032] 1. Processing method of composite cultivation matrix formula

[0033] In the Changbai Mountain area of ​​Jilin and the Greater and Lesser Xing'an Mountains of Heilongjiang Province, the edible fungus waste after cultivation in mid-to-late October was crushed with a 1mm aperture grinder, and then fully mixed with larch needles and 150 times water-absorbed agricultural and forestry water-retaining agent gel according to the above ratio in (1). At the same time, during the mixing process, Ericoid mixed bacteria solution diluted 1000 times and 2000 times of 50% phoxim emulsifiable concentrate or 300 times of 30% trichlorfon emulsifiable concentrate (for the prevention and control of grub pests) were sprayed to make the water content of the mixed matrix reach 70%. After fully mixing, the matrix was piled and stewed for about 12-24 hours to allow the larch needles to be fully soaked.

[0034] 2. Application method of composite cultivation medium

[0035] ① In the degradation area of ​​​​the Vaccinium vitis-infected community, before applying the substrate, first use a brush cutter to cut off the competitive shrubs such as birch, bilberry willow, and azalea in the Vaccinium vitis-infected community. After the shrubs are cut, they are spread on the Vaccinium vitis-infected plants, which can increase organic matter and overwhelm the Vaccinium vitis-infected plants, and prevent the winter snow from being too thick and freezing the top flower buds. Before the snow falls in November, the prepared composite cultivation substrate is manually spread on the Vaccinium vitis-infected community with a thickness of 20 cm. In the spring of the second year, due to factors such as winter snow pressure and wind, the thickness of the substrate layer will drop to 10 cm; with the summer rain soaking and microbial decomposition, the thickness of the substrate layer will drop to 5 cm in autumn. After three years of continuous application, the thickness of the organic matter layer will reach 10-15 cm, which will not only increase the surface organic matter and isolate solar radiation, but also raise the height of winter snowfall, which will promote the safe wintering of Vaccinium vitis-infected.

[0036] ②In the wet open space in the forest with a snow thickness of more than 40cm in winter or in a place similar to the habitat of blueberry (with sufficient water source), before the autumn freeze, after clearing the forest, spread the above-mentioned composite cultivation matrix flat on the plot to be planted with blueberry to a thickness of 20cm, and use a micro-rotary tiller to make a 1m wide × 0.2m high bed. After the snow melts in the spring of the second year, cover and fix it with a 3-needle shade net, which can not only keep moisture but also promote the formation of moss. From late May to early mid-June, plant the blueberry seedlings in the nutrient pots in the middle of the planting bed with a spacing of 50cm. When planting, cut the shade net with a wallpaper knife cross-shaped, dig a hole with a small shovel, the depth is equivalent to the height of the nutrient pot, and then cover and tamp it. At the same time, cover the cut shade net tightly with surrounding soil or matrix.

[0037] Example 3.

[0038] The test sites of this embodiment are the family forest farm of Hanjiayuan Forestry Bureau in Daxinganling and Quanlin Forest Farm of Xinqing Forestry Bureau in Lesser Khingan Mountains, where the Vaccinium vitis-idaea plant community has been severely degraded. Among them, the family forest farm of Hanjiayuan Forestry Bureau in Daxinganling is located at the southeast foot of Daxinganling (52°04′48″N; 125°22′30″E, 376M above sea level), in the northwest of the Hanjiayuan Forestry Farm operation area, bordering the Shibazhan Forestry Bureau in the north and the Xinlin Forestry Bureau in the west. The terrain in the farm is relatively flat, with an average altitude of about 350 meters. It has a cold temperate continental climate and a frost-free period of about 100 days throughout the year. The forest farm operation area covers an area of ​​33,000 hectares, of which the wild blueberry cultivation base covers an area of ​​100 hectares. The main vegetation at the sampling points includes Vaccinium vitis-idaea, Ledum odoratum, Betula platyphylla, Betula platyphylla, and modified moss. Quanlin Forest Farm of Xinqing Forestry Bureau in Xiaoxing'anling is located in the north of Yichun and the hinterland of Xiaoxing'anling (48°21′12.4″N; 129°36′23.1″E, 351m above sea level), with a thawing period of 6 months and a frost-free period of 107 days. It has an area of ​​14 hectares of typical peat moss swamp wetlands with clear and well-preserved communities. The main vegetation includes Dahurian larch, spring elm, white birch, firewood birch, Vaccinium dusi, Carex truncatum, and small-leaved seal, etc. It is a priority area for biodiversity conservation in China.

[0039] The experiment was conducted in two sites with Vaccinium vitis viniferum communities and nearby plots without Vaccinium vitis viniferum communities. Three plots were set up under the same geological conditions. Each plot was approximately 100 m apart. Each plot consisted of 15 2m×2m plots in a row, separated by a 1m buffer zone. Within each plot, 15 plots were randomly assigned to the following treatments: (1) Degraded-compound substrate and single edible fungus waste were applied in the degraded area; (2) Similar-compound substrate and single edible fungus waste were applied in similar habitats.

[0040] 1.1 Determination of plant biomass

[0041] After applying composite matrix and single edible fungus waste in the degraded areas and plots with similar habitats at the two locations, all Vaccinium vitis-idaea plants in the entire plot were sampled in the fourth year. The sampled plants were then washed with water, and the surface moisture of the plants was then absorbed with filter paper. The fresh weight of the whole plant was determined using an analytical balance.

[0042] 1.2 Determination of mycorrhizal colonization rate of plant roots

[0043] Ericoid roots were separated from moss roots and soil, washed with water, and stored in formolybdate acetic alcohol until processing. All dead or damaged roots and all fine roots <1 mm without obvious lignification were removed. After staining with basin blue, they were mounted on semi-permanent slides in polyvinyl alcohol glycerol. There were 3 slides for each sample, and 15 root segments were placed on each slide. After observation and photography under a microscope, the root infection rate of endomycorrhizal fungi was calculated by the magnification cross method.

[0044] 1.3 Determination of plant nutrient content

[0045] The total nitrogen in the plant is digested with concentrated sulfuric acid and hydrogen peroxide, and the organic nitrogen is converted into ammonium salt, which is determined by flow analysis. Specific steps: weigh 0.1-0.2g of finely ground and dried (passed through a 0.25-0.5mm sieve) plant sample, put it in a digestion tube, add water to moisten it; add 5ml of concentrated sulfuric acid; shake well, put a small curved neck funnel at the mouth of the tube (left overnight), heat it on an electric furnace (initial temperature is about 190℃), after 10-15min (white smoke), gradually increase the temperature (about 380℃) for 20-30min; when the solution is all brown-black, remove it, cool it slightly for 20-30min, add 10 drops of 300g L -1 H2O2, shake the digestion tube, continue to digest on the digestion instrument, remove it after 10-15 minutes, add H2O2, repeat 2-3 times until the digestion liquid is colorless or clear, and then heat it for 5-10 minutes to remove the remaining H2O2. After cooling, adjust the volume to 100ml, and the digestion liquid is used to determine the nitrogen concentration of the plants; the nitrogen concentration of the plants is determined by a flow analyzer.

[0046] 1.4 Extraction of soil DNA and real-time quantitative PCR (Q-PCR)

[0047] Fast Spin Kit for Soil (MP Biomedicals, USA) was used to extract total DNA from soil microorganisms. Accurately weigh 0.5 g of thawed soil stored at -80 °C for each sample, and extract total soil DNA in strict accordance with the kit instructions. The obtained DNA was dissolved in DES buffer. After the extraction, the total DNA of soil microorganisms was detected by 1% agarose electrophoresis, and the concentration was determined using Nano Drop 2000 nucleic acid analyzer (Thermo Scientific, USA). After the detection, the DNA was stored at -20 °C for future use.

[0048] Using the data publicly available from the GenBank, specific PCR primers targeting the functional mycorrhizal-associated fungi Meliniomyces variabilis, Oidiodendron maius, and Phialocephala fortinii were constructed using Primer Premier5.0 software ( Table 1 ).

[0049] Table 1. Specific molecular marker primers for qPCR quantitative analysis of related fungi developed based on the sequence database (http: / / blast.ncbi.nlm.nih.gov / Blast.cg)

[0050]

[0051]

[0052] Quantitative PCR uses plasmid DNA with known copy number as standard template, and makes a standard curve by 10-fold gradient dilution. The standard sample and the test sample are set to be repeated three times. After amplification, the copy number of the test sample is calculated based on the standard curve drawn according to the standard sample data.

[0053] like Figure 1 As shown in the figure, in the degradation area of ​​Vaccinium vitis-idaeae, the application of composite cultivation substrates was compared with the application of single application waste materials. The plant biomass per unit area (a), total nitrogen content (b) and mycorrhizal colonization rate (c) of the two sites of Hanjiayuan Forestry Bureau in Daxing'anling and Xinqing Forestry Bureau in Xiaoxing'anling were significantly higher than those of the single application waste materials; in similar habitats, the plant biomass per unit area of ​​Hanjiayuan Forestry Bureau in Daxing'anling and Xinqing Forestry Bureau in Xiaoxing'anling was not significantly different, but the plant total nitrogen content and mycorrhizal colonization rate were significantly higher than those of the single application waste materials. The above results show that the application of composite cultivation substrates can promote the increase of plant biomass, total nitrogen content and mycorrhizal colonization rate, whether in population degradation or in similar habitats, and can promote plant growth and improve mineral nutrition.

[0054] The abundance of the main ericoid mycorrhizal fungi Oidiodendron maius, Meliniomyces variabilis, and Phialocephala fortinii in the rhizosphere matrix of Vaccinium vitis-idaea cultivated in different cultivation sites and different matrices was amplified by Q-PCR. Figure 2 The results showed that the gene copy numbers of different mycorrhizal fungi were different, with Oidiodendron maius having the highest number (0.38×10 5 ~28.36×10 5), followed by Meliniomyces variabilis (0.72×10 3 ~21.65×10 3 ), Phialocephala fortinii is the smallest (0.63×10 2 ~44.84×10 2 ). Whether in the degradation area or in the habitat similarity area, the abundance of mycorrhizal fungi Phialocephala fortinii and Oidiodendron maius in the composite cultivation medium was significantly higher than that in the single application of waste materials. This indicates that the composite cultivation medium is suitable for the growth of symbiotic mycorrhizal fungi and promotes the nutrient absorption of Vaccinium vitis-idaea, which has a promoting effect on the population recovery of Vaccinium vitis-idaea.

[0055] Example 4.

[0056] In order to further clarify the effect of composite cultivation matrix on the growth of Vaccinium vitis-idaea plants, we measured the physicochemical properties of rhizosphere matrix nutrients and related enzyme activities after three consecutive years of application, so that we can understand the utilization efficiency and chemical processes of each nutrient in the matrix.

[0057] 1. Determination of the physicochemical properties of the matrix

[0058] Soil pH was determined by a soil suspension of soil and water (1:2.5 W / V) for 30 min and then measured with a pH meter. Soil moisture content was determined by drying at 105 °C. Soil density was determined by gravimetric analysis. Soil organic carbon was determined by the oil bath-potassium dichromate method. Soluble total nitrogen (TSN) was digested by the Kjeldahl method and determined by a continuous flow chemical analysis system (SKALAR SAN, The Netherlands). Soil ammonium nitrogen (NH 4+ -N) and nitrate nitrogen (NO3 - -N) was determined using a continuous flow chemical analysis system (SKALAR SAN, The Netherlands). Soil soluble organic nitrogen (SON) was determined by subtracting (ammonia nitrogen + nitrate nitrogen) from soluble total nitrogen. Soil total phenol (Phenol) was determined by extraction with sulfuric acid solution and color development with Folin reagent.

[0059] 2. Determination of related enzyme activities

[0060] The detection of related enzyme activities mainly refers to the method of Franz Schinner. Protease is determined by ninhydrin colorimetry after 24 hours of incubation using casein as the substrate. Urease is determined by phenol-sodium hypochlorite colorimetry. L-asparaginase is determined by the method described by Frankenberger and Tabatabai. Arginine deaminase is determined by the method described by Alef and Kleiner. β-glucosidase and cellulase refer to Guan Songyin's soil enzymes and their research methods. Polyphenol oxidase refers to Lin Xiangui's principles and methods of soil microbial research.

[0061] The results are shown in Table 2. The contents of Phenol, SOC, and SON in the rhizosphere matrix of Vaccinium vitis-idaea treated with the composite cultivation matrix were significantly higher than those treated with the single waste material. + -N, NO3 - -N, Bulkdensity content was significantly lower than that of the treatment with single waste material. At the same time, Phenol and SON in the matrix were significantly positively correlated with mycorrhizal infection rate and plant biomass; Bulk density, NH4+-N, NO3 - -N was significantly negatively correlated with mycorrhizal infection rate and plant biomass; pH and SOC were not correlated with mycorrhizal infection rate (as shown in Table 3). Since Vaccinium vitis-idaea is a mycorrhizal plant, 80% of its nitrogen source comes from soluble organic nitrogen sources, and the soluble organic nitrogen SON content in the composite cultivation matrix ranged from 161.95 to 195.52 mg·kg -1 Therefore, the composite cultivation matrix can have sufficient nitrogen source to ensure the absorption and utilization of plants. + -N, NO3 - -N and Bulk density contents were significantly lower than those in the single waste treatment, probably because the higher Phenol content further inhibited the mineralization of soluble organic nitrogen, which was more conducive to the accumulation of soluble organic nitrogen.

[0062] Table 2 Effects of different cultivation substrates on the physicochemical properties of the rhizosphere of Vaccinium vitis-idaea

[0063]

[0064]

[0065] Note: SOC, TSN and SON represent organic carbon, soluble total nitrogen and soluble organic nitrogen in the rhizosphere matrix of Vaccinium vitis-idaea. Letters in the same column (a and b) indicate significant differences in the rhizosphere soil of different treatments at the same location (n=3, P<0.05), the same below.

[0066] Table 3 Correlation analysis between plant nitrogen content and plant biomass and environmental variables

[0067]

[0068] Note: * is significantly different at the 0.05 level, ** is significantly different at the 0.01 level, *** is significantly different at the 0.001 level, the same below.

[0069] In order to further understand the utilization efficiency and chemical process of each nutrient in the matrix, we measured the activities of enzymes related to carbon and nitrogen metabolism in the rhizosphere matrix. Figure 3 As shown. The protease in the rhizosphere matrix of Vaccinium vitis-idaea treated with composite cultivation matrix ( Figure 3 a), polyphenol oxidase ( Figure 3 g) activity was significantly higher than that in the treatment with a single application of waste, while urease ( Figure 3 b) Arginine deaminase ( Figure 3 c), L-asparaginase ( Figure 3 d), β-glucosidase ( Figure 3 e), cellulase ( Figure 3 f) activity was significantly lower than that of the single application of waste material treatment. We found that with the decrease of Phenol content, in addition to the significant decrease in polyphenol oxidase activity, the activities of urease, β-glucosidase and cellulase were significantly increased, further confirming the regulatory effect of polyphenol compounds on matrix nutrients. At the same time, the Phenol content in the matrix was significantly positively correlated with the total nitrogen content of the plant (Table 3). This shows that the Phenol content can affect the matrix nitrogen cycle and thus affect plant growth and nitrogen nutrition. In addition, Phenol inhibited the activity of carbon metabolism-related enzymes (glucosidase, cellulase) and reduced the bulk density of the matrix. The activity of polyphenol oxidase and extracellular protease in the composite cultivation matrix was significantly higher than that in the single application of waste material treatment. It may be that in a nitrogen-limited environment, the competition between plants and microorganisms for available nitrogen will be more intense, which will promote microorganisms to increase the secretion of extracellular enzymes and increase the availability of nitrogen, thereby improving the absorption and utilization of nitrogen nutrition in the soil by plants.

[0070] Although the phenol-rich composite cultivation matrix inhibits the mineralization of soil organic nutrients, phenol can regulate the formation of humus litter layer by inhibiting the decomposition of organic matter, preserve nutrients, and create more favorable environmental conditions for root growth. At the same time, polyphenols shift the dominant pathway of nitrogen cycle from mineral form to organic form to reduce potential nitrogen loss in the ecosystem and maximize the use of nitrogen fixed in the matrix by mycorrhizal symbionts, thus giving the host vaccinium plant a competitive advantage. Ericoid mycorrhizal fungi can not only mobilize nitrogen in the protein-polyphenol complex, but also use phenol as the sole carbon source in the presence of high concentrations of phenol to promote the growth of hyphae without affecting the activity of extracellular carboxyl proteases. There is a lot of evidence that ericoid fungi produce and release extracellular tyrosinase (polyphenol oxidase) and carboxyl proteases into the mycelium. Therefore, tyrosinase activity in the mycelium will reduce the binding of Phenol to proteins, thereby promoting the action of extracellular carboxyl proteases on complex proteins. This also explains why high concentrations of Phenol do not affect protease activity.

[0071] Example 5.

[0072] Peat moss is a key species for creating a low-temperature, high-organic-matter, acidic habitat for Vaccinium vitis-idaea. At the same time, peat moss can coexist with cyanobacteria and nitrogen-fixing bacteria to promote nitrogen accumulation, so peat moss is also the best substrate for promoting the growth of Vaccinium vitis-idaea. In the experiment, we found that the decomposition of the composite cultivation substrate will also promote the growth and reproduction of peat moss, and reduce the surface temperature below 20 cm during the high temperature period in summer, inhibit the melting of permafrost, and promote the recovery of permafrost.

[0073] In the fourth year of the experiment, we used a thermometer to measure the temperature at a depth of 20 cm from the surface every 15 days from May to September, and determined the fresh weight of peat moss per unit area.

[0074] The results are as follows Figure 4 As shown in the figure, the fresh weight of peat moss in the two sites treated with composite cultivation substrates was significantly higher than that in the treatment with single waste material, and the fresh weight of peat moss in the degradation area was 25.17-32.56 kg / m 2 The fresh weight of peat moss in similar habitats is 13.28-20.28 kg / m 2 The higher fresh weight of peat moss in the degraded area than in similar habitats may be related to the base number of peat moss reproduction. There is only a small amount of peat moss or no peat moss in similar habitats. At the same time, we found that after applying the composite cultivation substrate, the temperature at a depth of -20 cm from the surface of the degraded area was the lowest and most stable, and the temperature was always below -4°C throughout the year ( Figure 5Similarly, the temperature change of the treatment with single edible fungus waste was the most dramatic, which may be related to the weak ability of the substrate to insulate and retain water and the small amount of peat moss produced. Therefore, the continuous application of composite cultivation substrate can not only create a low temperature, acidic and high organic matter suitable environment for Vaccinium vitis-idaea, but also promote the production of peat moss and the recovery of permafrost, which is a technical model worthy of promotion.

[0075] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A composite cultivation matrix formula for restoring Vaccinium vitis-idaea colonies, characterized in that: The formula is composed of the following ingredients: edible fungus waste containing hardwood components: fallen leaves and litter in a volume ratio of 3:2, and 1 kg / m3 of agricultural and forestry water retaining agent gel after 150 times water absorption. 3 , the conidia concentration reached 10 8 The Ericoid mixed bacterial solution containing 100 spores / mL was diluted to a minimum of 1000 times at 0.2 L / mL. 3 Spray inoculate into the composite cultivation matrix, 2000 times of phoxim 50% emulsifiable concentrate or 300 times of trichlorfon 30% emulsifiable concentrate at 2.5L / m 3 Mix thoroughly and add water to make the moisture content of the composite cultivation medium reach 70%.

2. The formulation according to claim 1, characterized in that The content of hardwood components is 30% crude fiber, 10% crude protein and 7% crude fat.

3. The formulation according to claim 1, characterized in that Leaf litter refers to the fallen leaves of pine needles on the ground of larch or mixed coniferous and broad-leaved forests.

4. The formulation according to claim 1, characterized in that High molecular weight water-absorbing resin as the gel component of agricultural and forestry water-retaining agent.

5. The formulation according to claim 1, characterized in that The composition of the Ericoid mixed bacterial solution is Phialocephalafortinii: Meliniomyces variabilis: Oidiodendron maius in a volume ratio of 1:1:

1.

6. The formulation according to claim 1, characterized in that The dosage of the Ericoid mixed bacterial solution inoculated in the formula is 0.2L / m 3 .

7. A method for restoring a Vaccinium vitis-idaea colony, characterized in that: The specific steps of the method are as follows: Step 1: In the degraded area of ​​​​the blueberry community, before applying the substrate, use a brush cutter to cut off the competitive shrubs in the blueberry community, and spread the shrubs on the blueberry plants after cutting. Before freezing and snowing in November, spread the composite cultivation substrate formula described in any one of claims 1 to 6 on the blueberry community, with a minimum thickness of 20 cm, and apply it continuously for 3 years; Step 2: In a forest clearing with a snow thickness of more than 40 cm in winter, after clearing the forest and before the snow falls in winter, spread the composite cultivation matrix formula of any one of claims 1 to 6 on the plot to be planted with a thickness of 20 cm, use a micro rotary tiller to rotary till the land to make a 1m wide × 0.2m high bed, cover and fix it with a 3-needle shade net after the snow melts in the spring of the second year, and plant the vaccinium nutritious pot seedlings in the middle of the planting bed at a spacing of 50 cm from late May to early mid-June. When planting, cut the shade net crosswise with a wallpaper knife, dig holes with a small shovel, the depth of which is equivalent to the height of the nutritious pot, and then cover and tamp it down, and cover the cut shade net tightly with surrounding soil or matrix.

8. The method according to claim 7, characterized in that The competing shrubs in step 1 are Betula serrata, Salix vaccinium and Rhododendron.

9. Use of the formulation according to any one of claims 1 to 6 in restoring breeding of Vaccinium vitis-idaea populations.

10. The use according to claim 9, characterized in that: Applications also include the generation of peat moss and the promotion of permafrost applications.

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