Scenedesmus sp. EMC7 from stony desertification region and application of scenedesmus sp. EMC7

Abstract

The invention belongs to the field of microorganisms, and discloses a strain of Scenedesmus sp. EMC7 separated from biological crust soil in a karst rocky desertification region, and the preservation number of the strain is CGMCC No.41200. The invention also discloses a preparation method of the Scenedesmus sp. The scenedesmus disclosed by the invention is high in growth and reproduction speed, and after the scenedesmus liquid prepared from the scenedesmus is applied to barren sandy soil and loam, the water-retaining property of the soil can be improved, the erosion resistance of the soil is enhanced, the nutrients of the soil are improved, and the growth and nutrient absorption of the cold-land bluegrass plants are promoted, so that the degraded land is effectively ecologically restored and treated, the cost is reduced, and the economic benefit is increased. Environmental pollution is reduced, and sustainable and healthy development of the ecological environment is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to the separation and screening of biological crust algae and ecological restoration of degraded soil, and in particular to a crust soil Scenedesmus and application thereof. Background Art

[0002] Soil ecological degradation refers to the deterioration of the soil environment under the combined effects of natural and human factors, which is specifically manifested in a series of soil ecological environment deterioration processes and phenomena, including damage to the ecosystem structure, reduction of regulatory functions, attenuation of biodiversity, decline in soil productivity, and soil desertification, aridification, compaction, acidification, salinization, nutrient deficiency and imbalance. Against the backdrop of global climate change and rapid national economic development, my country's land degradation problem is experiencing unprecedented challenges. my country attaches great importance to ecological protection and restoration, and has compiled the "Overall Plan for Major Projects for the Protection and Restoration of Important Ecosystems in the Country", and people's awareness of ecological protection and restoration is constantly increasing.

[0003] Scholars have conducted relevant research on the restoration of degraded soil from the perspectives of physics, chemistry and biotechnology. Among them, soil microorganisms, as the link between the aboveground and underground, directly drive and participate in the absorption and transformation of carbon, nitrogen, phosphorus and other elements by plants, regulate soil moisture and physical properties, and promote healthy plant growth while improving the soil. Therefore, the application of microorganisms in the restoration of degraded soil has received more and more attention.

[0004] There are many kinds of soil microorganisms. Bacteria, fungi, and even algae isolated and screened from soil have been reported to improve soil and promote plant growth. Therefore, applying them to the ecological restoration of degraded soil will have potential effects. However, degraded soil is a harsh environment for most microorganisms. Existing microbial remediation technologies often have problems such as difficulty in adapting to and colonizing bacteria, which affects their restoration effects. It is very necessary and urgent to explore microbial strain resources with strong stress resistance and wide adaptability for the restoration of degraded ecosystems. Biological crust is a composite biological soil layer composed of soil particles, algae, lichens, mosses and microorganisms. It is widely distributed and often exists in harsh desertification areas such as rocky desertification and desertification. The evolution of biological crusts often follows the development process from algal crusts to lichen crusts and then to moss crusts. The results of high-throughput sequencing show that biocrusts at different succession stages contain a large number of algae, which are pioneer species. They may play an indispensable and important role in biocrusts, and play a vital role in the initial vegetation succession and anti-erosion performance improvement of degraded soils in deserts and semi-desert areas. Therefore, the growth of biocrusts in desertified areas means that the coexisting microorganisms in them may have strong environmental adaptability. Using the biocrusts grown in this area as an important source of functional microorganism screening and screening algae from them is of great significance for enriching the algae resource library and obtaining microbial groups that can adapt to difficult site habitats and effectively restore the ecosystem. Summary of the invention

[0005] The purpose of the present invention is to provide a highly adaptable Scenedesmus and its application in degraded land. The Scenedesmus is separated from the biological crust soil in the karst rocky desertification area, has a fast growth and reproduction speed, has the functions of improving soil physical properties, increasing soil nutrients, and promoting plant growth, has strong adaptability to adverse environments such as dryness and nutrient deficiency, and better solves the problem of unstable and unsatisfactory ecological restoration effects caused by the poor environmental adaptability when existing microbial agents are used, and has the advantages of being environmentally friendly and low cost.

[0006] The invention provides a Scenedesmus sp. EMC7 isolated from biological crust soil in a karst rocky desertification area, characterized in that the preservation number of the strain is CGMCC No.41200.

[0007] The present invention further provides the use of the Scenedesmus EMC7 in promoting plant growth and / or promoting plant nutrient absorption.

[0008] Specifically, the plant is a cold-weather plant, such as Poa frigida;

[0009] The promoting of plant growth is to increase the total length, plant height, root length, total weight, above-ground biomass and / or underground biomass of the plant;

[0010] The promoting of plant nutrient absorption is to improve the absorption of nitrogen and / or phosphorus.

[0011] Preferably, the application is to apply algae solution cultured for 2-4 weeks to the plant growth substrate, and the concentration of the applied algae solution is 10 2 to 10 4 algal cell number / mL, applied once every 3-12 days, for a total of 1-5 times.

[0012] The present invention also provides the use of the Scenedesmus EMC7 in promoting soil improvement.

[0013] Specifically, the soil is poor sand and / or loam.

[0014] In another specific aspect, the improvement is to increase soil water retention, enhance soil anti-erosion capacity and / or improve soil nutrients.

[0015] In a specific embodiment, an algae solution cultured for 2-4 weeks is applied to soil in which plants are grown.

[0016] The present invention also provides a method for preparing the algae liquid of the Scenedesmus EMC7, which comprises inoculating an activated single algae colony into a BG11 liquid culture medium and culturing at 25-29° C. and 2500-2900 lx of light; preferably, shaking 1-4 times at a fixed time every day, each time for 0.5-2 minutes.

[0017] The present invention also provides a plant growth promoter or soil improver, which contains the algae liquid of Scenedesmus EMC7.

[0018] The Scenedesmus described in the present invention has a fast growth and reproduction speed. The algae liquid prepared by the algae can be applied to poor sandy soil and loam to improve soil water retention, enhance soil anti-erosion ability, improve soil nutrients, promote the growth of cold-land bluegrass plants and nutrient absorption, thereby effectively restoring and managing the ecology of degraded land, reducing costs, reducing environmental pollution, and achieving sustainable and healthy development of the ecological environment.

[0019] Biomaterial deposit information:

[0020] The Scenedesmus sp. EMC7 of the present invention was deposited on July 5, 2024 in the General Microbiological Center of the China Culture Collection Administration (unit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101), which is abbreviated as CGMCC, the preservation number is CGMCC No.41200, and the classification name is Scenedesmus sp. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1The growth rates of four algae strains are shown. Note: (a) CC1, (b) LC8, (c) EMC7, and (d) AMC5.

[0022] Figure 2 Shows the soluble protein content of 4 strains of crustacean algae.

[0023] Figure 3 Shows the exopolysaccharide content of 4 crustacean algae.

[0024] Figure 4 The mechanical composition of aggregates in sandy soil (1) and loam soil (2) under different treatments. Note: A is S treatment, B is PYJ treatment, C is EMC7 treatment, and D is AMC5 treatment. DETAILED DESCRIPTION

[0025] The present invention is described below through specific implementation modes in order to better understand the present invention, but it does not constitute a limitation of the present invention.

[0026] 1. Isolation of algae strains from biological crust soil in rocky desertification areas

[0027] 1.1 Sources of biocrust soil materials and sample collection

[0028] The biological crusts were collected in Jianshui County, Honghe Hani and Yi Autonomous Prefecture, Yunnan Province (103°57'E, 23°09'N). The study area is a karst desertification area with no obvious standing vegetation. The altitude is 1500m. It belongs to the typical southern subtropical monsoon climate area. The annual average temperature is maintained at 19.8℃, and the annual precipitation is about 805mm, mainly concentrated in July and August, and the total number of frost-free days throughout the year reaches 307d. This study selected areas with little external interference, similar terrain characteristics and high stability of biological crusts as the research objects. Five sampling points were arranged according to the principle of random distribution, and the spacing between each sampling point was more than 2 meters. The random sampling method was used to repeatedly sample biological crusts at different development stages, mainly including algal crusts, lichen crusts, and moss crusts. The sampling area of ​​each sampling point is 15cm×15cm, 3-4cm thick. The lower crust layer is peeled off with a sterile small shovel, and the remaining is the soil sample. The collected soil samples are sieved through a 2mm sieve to remove roots and gravel. The collected samples are stored in a refrigerator at 4℃ for the separation of crust algae.

[0029] 1.2 Isolation and purification of crustacean algae

[0030] Prepare BG11 liquid culture medium (formula as follows: 1.5 g NaNO3, 0.04 g K2HPO3·3H2O, 0.075 g MgSO4·7H2O, 0.036 g CaCl2·2H2O, 0.006 g citric acid, 0.006 g ammonium ferric citrate, 0.001 g EDTA-Na2, 0.02 g Na2CO3, 1 mL trace elements, 1 L distilled water), adjust the pH range of BG11 liquid culture medium to 7.0 to 7.2, and sterilize at 121°C for 15 min. 30 mL of BG11 liquid culture medium was added to each 50 mL sterilized conical flask, and the collected biological crusts were evenly mixed into one sample according to the same developmental stage. 1 g of sieved crust soil sample was weighed and shaken in a shaker at 180 rpm for 30 min to fully mix the soil suspension, and then placed for enrichment culture at 27 ° C and 2700 lx of light. The sample was shaken twice a day at a fixed time for 1 minute each time. After culturing for 2 weeks, the sample was gradiently diluted to 1×10 3 -1×10 6 , transferred to 96-well plates, 200 μL per well, continued to culture for 2 weeks, selected 30% of the different wells in the 96-well plate to show visible green gradient dilution, stirred thoroughly in the green wells with a sterilized toothpick, then streaked in BG11 solid culture medium (agar was added to the above BG11 liquid culture medium, the addition amount was 18 g / L), placed in a light incubator at 27°C and 2700 lx for 2-3 weeks, observed the purity of the culture, and cultured the single clone by picking Transfer the sample to new BG11 solid culture medium and continue streaking purification. Observe the morphology of the algae on the plate until the green algae strains on the plate have consistent morphology and are not contaminated by foreign bacteria. They can be preliminarily determined to be pure algae, and further confirmed to be pure algae strains through optical microscope observation. Then use a sterile toothpick to pick up a single algae drop on the plate and culture it in BG11 liquid culture medium for 2 weeks. During this period, shake it twice at a fixed time every day for 1 minute each time to obtain pure algae liquid, mix it with 30% glycerol in a 1:1 ratio, and put it in a -80℃ ultra-low temperature refrigerator for preserving the algae strains.

[0031] Through the above steps, a total of 25 pure algae strains were isolated, including 10 pure algae strains isolated from algae crust soil, 8 pure algae strains isolated from lichen crust soil, and 7 pure algae strains isolated from moss crust soil.

[0032] 2. Biological characteristics of crustacean algae

[0033] 2.1 Cultivation and growth determination of crustacean algae

[0034] The 25 pure algae strains preserved at low temperature were streaked onto BG11 solid culture medium and cultured in a light incubator at 27°C and 2700lx for 3 weeks. Then, a single algae colony was picked with a sterile toothpick and inoculated into 30mL BG11 liquid culture medium and cultured at 27°C and 2700lx. The culture medium was shaken twice at a fixed time every day for 1 minute each time. After 4 weeks of culture, the OD680 value was measured to preliminarily judge the growth status of the algae strains.

[0035] The top four algae strains with the highest OD680 values ​​were selected for growth rate monitoring. The algae liquid fermented for 28 days was selected from the above four strains respectively, 1 mL was inoculated into 30 mL BG11 liquid culture medium, and cultured in a light incubator at 27°C and 2700 lx. The OD680 was first measured at the beginning of the culture, which was recorded as day 0. Thereafter, the OD680 was measured every 24 h until the OD value of the algae fermentation liquid was stable and showed a downward trend. The growth curve of the selected algae strains was plotted with the culture time as the horizontal axis and the OD680 value as the vertical axis.

[0036] The results showed that after 4 weeks of liquid culture, the top 4 growth conditions of the 25 pure algae strains were 1 strain from algal crust, 1 strain from lichen crust, and 2 strains from moss crust. The strain numbers were CC1, LC8, EMC7, and AMC5. In order to further understand the growth rate of the algae strains, the rate of the entire growth cycle was monitored. The results are as follows Figure 1 The OD680 values ​​of the four algae strains reached their maximum on the 29th day of culture, and the OD680 value of CC1 strain on the 29th day was

[0037] 1.182, and the OD680 values ​​of the other three algae strains were all over 2. The OD680 values ​​of LC8, AMC5 and EMC7 strains were 2.647, 2.4716 and 2.5289 at 29 days.

[0038] 2.2 Determination of soluble protein in different crustacean algae

[0039] Take 50 mL of algae solution after 28 days of culture, centrifuge at 4000 r / min for 10 min to obtain algae precipitate, place the obtained algae precipitate in a freeze dryer and dry overnight to harvest algae powder. Weigh 0.05 g of algae powder in a centrifuge tube, add distilled water to dilute 40 times, and use the BCA protein concentration assay kit to determine the soluble protein content.

[0040] Soluble protein contains nitrogen, which can serve as a nitrogen source for microbial growth and metabolism and provide sufficient material basis for soil nitrogen nutrient conversion. Figure 2The soluble protein content of the four algae strains exceeded 900 μg / mL, among which EMC7 and LC8 had higher soluble protein content, reaching 1216.92 μg / mL and 1123.65 μg / mL respectively.

[0041] 2.3 Determination of extracellular polysaccharides of different crustacean algae

[0042] The extracellular soluble polysaccharide content was determined using the anthrone sulfuric acid colorimetric method. Prepare a glucose standard solution (0.1 g / L), take several stoppered test tubes, and add different proportions of glucose standard solution and prepared anthrone sulfuric acid solution into the test tubes in the order and volume in the table. After adding the reagents, shake well and place in a boiling water bath to heat for 15 minutes. After heating, take out and cool to room temperature. Use an enzyme marker to measure the absorbance of each tube of reaction solution at 620 nm, and draw a standard curve according to the measured absorbance. Take 1 mL of the algae solution to be tested, measure the absorbance at 620 nm according to the same method as above, and calculate the extracellular soluble polysaccharide content according to the standard curve.

[0043] On the one hand, extracellular polysaccharides can serve as a carbon source for the growth and metabolism of soil microorganisms. On the other hand, extracellular polysaccharides can promote the formation of soil macroaggregates, thereby enhancing the soil's ability to resist erosion and helping to improve soil water retention. Figure 3 As shown, the extracellular polysaccharide contents of the four algae strains exceeded 400 μg / mL, among which EMC7 and LC8 had higher extracellular polysaccharide contents, reaching 603.69 μg / mL and 651.32 μg / mL, respectively.

[0044] 3. Molecular Biological Identification of EMC7 Strain Isolated from Moss Crust

[0045] Moss crust is the highest stage of biological crust development. The algae strain EMC7 isolated from it not only grows and reproduces quickly, but also contains high levels of soluble protein and extracellular polysaccharides. These characteristics indicate that the algae strain has excellent comprehensive performance and may play an important role in the subsequent application of the algae strain. Therefore, molecular biological identification of the algae strain was carried out, and the specific operations are as follows.

[0046] (1) DNA extraction of algae strains: 500 mL of algae cultured for 2 weeks was used as the extraction sample. It was centrifuged at 8000 rpm for 5 min. The algae precipitate was retained and transferred to a 2 mL centrifuge tube. A small spoonful of quartz sand was added. The tube was shaken on a high-speed wall breaker for 15 min. 800 μL of 2×CTAB extraction solution was added. The tube was placed in a 65°C water bath for 1 h. The tube was centrifuged at 12000 rpm for 10 min. The supernatant was removed and an equal amount of chloroform-isoamyl alcohol mixture was added. The mixture was shaken thoroughly and mixed evenly. After standing for 20 min, the tube was centrifuged at 12000 rpm for 10 min. The supernatant was retained and double the volume of pre-cooled anhydrous ethanol was added to the tube. After mixing, the tube was transferred to -20°C and allowed to stand for 30 min. The tube was centrifuged at 12000 rpm for 10 min. The supernatant was removed and 800 μL of ethanol was added to the precipitate. 75% ethanol, centrifuge at 12000rpm for 5 minutes and remove the supernatant. After the precipitate is naturally air-dried, add 50μL of distilled water and incubate at 37℃ for 30 minutes to dissolve. The obtained result is the extracted algae DNA sample.

[0047] (2) DNA amplification and identification: Three pairs of primers were selected to amplify the ITS, tufA, and psbA genes, respectively, to accurately identify the algae species and determine its taxonomic status. The first pair of primers was ITS-F (5'-GGAAGTAAAAGTCGTAACAAGG-3') and ITS-R (5'-TCCTCCGCTTATTGATATGC-3'), PCR system, primer 2.5 μL, DNA template 5 μL, 2×PCR mix 25 μL, ddH2O 15 μL, amplification conditions: 94°C pre-denaturation for 10 min, 30 cycles including 94°C denaturation for 1 min, 58°C annealing for 30 s, 72°C extension for 2 min, and final extension at 72°C for 10 min, for ITS sequence amplification; the second pair of primers was tufAF (5'-TGAAACAGAAMAWCGTCATTATGC-3') and tufAR (5'-CCTTCNCGAATMGCRAAWCGC-3'), PCR system, primer 2.5 μL, DNA template 5 μL, 2×PCR mix 25 μL, ddH2O The amplification conditions were as follows: 94°C pre-denaturation for 10 min, 94°C denaturation, 52°C annealing and 72°C extension for 1 min each, 35 cycles, and final extension at 72°C for 5 min, which was used for tuf A gene amplification; the third pair of primers was psbA-F (5'-GTNGAYATHGAYGGNATHMGNGARCC-3') and psb AR (5'-GGRAARTTRTGNGCRTTNCKYTCRTGC-3'), PCR system, 1 μL of each primer, 2 μL of DNA template, 12.5 μL of 2×PCR mix, 8.5 μL of ddH2O, and the amplification conditions were as follows: 94°C pre-denaturation for 5 min, 35 cycles including 92°C denaturation, 57°C annealing and 68°C extension for 1 min each, and final extension at 72°C for 10 min, which was used for psbA gene amplification. The three PCR products with clear bands were sent to Shanghai Bioengineering for sequencing, and the returned sequences were compared with the blast sequence on the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to obtain the identification results.

[0048] Three pairs of specific primers were used to amplify the ITS, tufA and psb genes of crust algae EMC7. The amplified products were sent to the company for sequencing. The gene sequences amplified by these three pairs of primers were blasted on the NCBI website. The website sorting is generally arranged by the size of the E Value value by default. The smaller the value, the higher the credibility of the sequence comparison. And further based on the comparison results, the sequence with the highest sequence length (Length) and sequence comparison consistency (Per.Identities) can be determined as its similar sequence. The results of the three pairs of gene comparisons all showed that the similarity between the EMC7 algae strain and the reference related bacteria was 99.42%, 98.02%, and 89.10%, respectively, indicating that the EMC7 algae strain is Scenedesmus vacuolatus.

[0049] Table 1 Blast alignment results of different gene sequence amplifications of crustacean EMC7

[0050]

[0051] 4. Effects of application of EMC7 on plant growth

[0052] 4.1 Planting of P. frigida and application of algae solution

[0053] The plant material selected was cold-land bluegrass, which is suitable for growing on the Qinghai-Tibet Plateau. 100 seeds with full grains were selected and evenly spread in flower pots filled with different growth substrates. Each flower pot was filled with 3L of substrate, including nutrient-poor sand and loam. Algae solution that had been cultivated for 3 weeks was applied to each pot. The algae solution was adjusted to 10% by dilution before application. 3 The number of algal cells / mL, 100 mL each time, applied once every week, for a total of 3 times. At the same time, the treatment of applying equal volumes of water (S) and BG11 liquid culture medium (PYJ) was set as a control, and another algae strain AMC5 isolated from moss crust was applied as an algae treatment control. Therefore, the experiment had a total of 4 treatments, namely CK, PYJ, EMC7, and AMC5, with 5 pots set up for each group, and watered in a timely and appropriate amount in the later stage.

[0054] 4.2 Collection of plant and soil samples

[0055] The experiment was terminated 6 weeks after the cold-land bluegrass was planted. Before harvesting the plants and soil, the plant height of bluegrass was measured first, and then the complete soil and plants in the pot were taken out together, the plants were carefully and completely peeled off from the soil, and then the root soil was carefully crushed to expose the plant roots, and the roots were cleaned with a small brush to fully scatter the soil on the roots. The collected plant samples were washed and sterilized at 105℃ for 30 minutes, and their above-ground and underground biomass and root length were measured. A part of the original soil of each pot soil sample was kept without destroying its structure, which was used for the determination of soil aggregate stability, and the other part of the soil was fully crushed and mixed, air-dried and sieved for the determination of soil physical and chemical properties.

[0056] 4.3 Determination of plant indicators

[0057] Use an electronic balance to measure the total weight, aboveground biomass and underground biomass of the plants, and use a ruler to measure the total length, plant height and root length of the plants. In addition, the nitrogen and phosphorus content of plant leaves was determined. The plant leaves were ground into powder using a high-speed grinder. 0.2g of plant sample was taken, 1.5g of anhydrous sodium sulfate and 5mL of selenium-saturated sulfuric acid were added to the digestion tube. It was best to digest overnight at 390℃ for 70 to 80min. Some samples that were difficult to digest may take a longer time, and the clarity of the liquid was used as the standard. After digestion, the liquid was cooled and diluted to 100mL. The total nitrogen and total phosphorus of the liquid after dilution were determined on a continuous flow analyzer.

[0058] The results showed (see Table 2) that the total length, plant height, root length, total weight, aboveground biomass, underground biomass, leaf nitrogen content, and leaf phosphorus content of the cold-land bluegrass planted in sandy soil were measured 6 weeks after sowing. It was found that the two treatments with crusted algae EMC7 and AMC5 were significantly higher than those with water (S) and BG11 liquid culture medium (PYJ), among which EMC7 had the best effect. There was no significant difference between the BG11 liquid culture medium (PYJ) treatment and the clear water (S) treatment, indicating that the algae and the extracellular substances secreted by them after the fermentation of the algae liquid are the main reasons for promoting plant growth. Compared with the PYJ treatment, the total length, plant height, root length, total weight, aboveground biomass, underground biomass, leaf nitrogen and phosphorus contents of plants treated with EMC7 algae solution increased by 71.91%, 70.21%, 85.71%, 126.59%, 106.86%, 154.93%, 11.16% and 265.38%, respectively.

[0059] Table 2 Effects of different treatments on the growth and leaf nitrogen and phosphorus content of Kentucky bluegrass in sandy soil

[0060]

[0061] The total length, plant height, root length, total weight, aboveground biomass, underground biomass, leaf nitrogen content, and leaf phosphorus content of cold-land bluegrass planted in loam substrate were measured 6 weeks after sowing. The results showed (see Table 3) that the two treatments of applying crust algae EMC7 and AMC5 were similar to those of sandy soil, and the indicators were significantly improved compared with the treatments of adding water (S) and BG11 liquid medium (PYJ). Among them, EMC7 still had the best effect, and there was no significant difference between the BG11 liquid medium (PYJ) treatment and the water (S) treatment. Using loam as the cultivation matrix, the total length, plant height, root length, total weight, aboveground biomass, underground biomass, leaf nitrogen and phosphorus content of the treatment with EMC7 algae solution increased by 20.53%, 23.75%, 16.90%, 37.58%, 42.77%, 30.83%, 18.59%, and 247.06% respectively compared with the PYJ treatment.

[0062] Table 3 Effects of different treatments in loam matrix on growth and leaf nitrogen and phosphorus content of Kentucky bluegrass

[0063]

[0064] 5. Improvement of soil matrix by application of crustacean EMC7

[0065] 5.1 Potting medium and treatment of cold-weather bluegrass plants

[0066] Two substrates, poor sandy soil and loam, were prepared. The two substrates were sieved through a 0.5 cm sieve to remove debris as potting soil. The amount of soil in each flower pot, the application of algae solution, and the collection of soil samples are described in Sections 4.1 and 4.2.

[0067] 5.2 Determination of soil physical and chemical properties and aggregate stability

[0068] The soil samples collected for measuring soil physical and chemical properties were sieved with a 1-2 mm sieve to remove impurities. The sieved soil was used to measure indicators related to soil nutrients and soil structure, including available phosphorus (AP), available potassium (AK), alkaline nitrogen (AN), organic matter (OM), soil extracellular polysaccharides (EPS), etc. The original state was used to determine the stability of soil aggregates.

[0069] Determination of available phosphorus (AP): weigh 2.5 g of air-dried sample passed through a 1 mm sieve into a 100 mL plastic bottle, add a small spoonful of phosphorus-free activated carbon and 100 mL of 0.5 mol / L NaHCO3 extract, and shake in a shaker for 30 minutes. After taking out, immediately filter with a drying funnel and phosphorus-free filter paper to collect the filtrate; aspirate 10 ml of the filtrate into a 50 ml volumetric flask, add 5 mL of molybdenum antimony anti-mixed color developer, and shake carefully. After 30 minutes, measure the OD880 value on the microplate reader, and calculate the available phosphorus content of the sample according to the phosphorus standard curve.

[0070] Determination of available potassium (AK): weigh 5 g of air-dried sample that has passed a 1 mm pore size sieve into a 100 mL plastic bottle, add 50 mL of 1 mol / L neutral ammonium acetate solution, shake in a shaker for 30 min, filter, and determine potassium directly in the filtrate on a flame photometer, and calculate the available potassium content of the sample based on the potassium standard curve.

[0071] Determination of alkaline nitrogen (AN): weigh 2g of air-dried sample that has passed through a 0.25mm sieve, place it in the outer chamber of a clean diffusion dish, gently rotate the diffusion dish to evenly spread the sample, and add H3BO4 into the inner chamber of the diffusion dish. - Add 2mL of methyl red-bromocresol green mixed indicator solution, then apply alkaline glue on the edge of the outer chamber of the diffusion dish, cover with frosted glass to leave a gap in the outer chamber of the diffusion dish, and quickly add 10mL of NaOH solution. Cover tightly immediately, gently rotate the diffusion dish to make the alkaline solution cover all the samples, place it in a constant temperature box at 40℃ for 48h, then take it out, titrate with 0.01mol / L (1 / 2H2SO4) standard solution, record the volume of standard solution used during the titration, and calculate the alkaline nitrogen content in the sample according to the formula.

[0072] Determination of organic matter (OM): Pass the sample through a 0.25mm sieve, weigh 0.1-0.5g and put it into a test tube, add 5mL of potassium dichromate solution (0.8M) and 5mL of concentrated sulfuric acid, shake well and put it into an oil bath, adjust the temperature to 175℃, boil for 5 minutes and take it out, after the test tube cools, pour it into a 200mL beaker, add water to keep the total volume at 60-70mL, add about 3-4 drops of o-phenanthroline indicator, and titrate with 0.2M ferrous sulfate solution. The solution changes from yellow to green, emerald green and then suddenly to brownish red, which means the titration is complete. Record the volume of ferrous sulfate used to titrate the sample, and calculate the organic matter content according to the formula.

[0073] Determination of extracellular polysaccharide (EPS): After the sample is passed through a 0.25mm sieve, weigh 1g, add 10mL 2mol / L H2SO4, and filter while hot after boiling in a water bath for 2h. Wash the digestion tube several times with hot deionized water, and collect the filtrate and washing liquid into a new digestion tube. Add 1g CaCO3, filter after no more gas is generated, collect the filtrate and make up the volume to 20mL to obtain the sample solution to be tested. Pipette 2mL of the test solution into a dry and clean 10mL centrifuge tube, immediately add 6mL of anthrone reagent, shake and mix, place in a boiling water bath and heat for 15min, quickly take out and immerse in an ice water bath to cool for 15min, and measure the absorbance at a wavelength of 620nm. According to the calibration curve of glucose content, the concentration of sugar in each sample solution is calculated from the absorbance of the sample solution, and its extracellular polysaccharide content is calculated.

[0074] Determination of soil aggregate composition and stability: Weigh 200 g of soil sample and place it on a sieve with apertures of 2 mm, 1 mm, and 0.25 mm to separate aggregates of >2 mm, 1-2 mm, 0.25-1 mm, and <0.25 mm. Weigh the mass of soil aggregates of each particle size and calculate the mass percentage of soil aggregates of each particle size. The mean weight diameter (MWD) and geometric mean diameter (GMD) are used to indicate the stability of soil aggregates. The higher the MWD and GMD values, the greater the aggregate stability. Therefore, these two indicators are calculated at the same time. The specific formula is as follows:

[0075]

[0076] Where: MWD is the mean weight diameter; w i is the mass percentage of the i-th particle size; x i Refers to the average diameter of the agglomerates.

[0077]

[0078] Where: GMD is the geometric mean diameter; m i is the mass of aggregates of different particle sizes (g); m ’ is the average diameter of the aggregates.

[0079] The results showed that for sandy soil, after 6 weeks of planting Poa frostii, the soil was collected and processed to determine its physical and chemical properties. It was found that the two treatments with crusted algae had significant effects on soil water retention and soil nutrients (see Table 4), soil structure improvement ( Figure 4(1)) and other aspects were significantly improved, among which EMC7 had the best effect. There was no significant difference between the BG11 liquid medium (PYJ) treatment and the clean water (S) treatment, indicating that the algae and its extracellular secretions after algae liquid fermentation were the main reasons for the soil improvement effect of this treatment. In addition, after the algae liquid was applied, it may also promote plant growth and improve soil quality, thereby creating a favorable living environment for the growth and metabolism of other microorganisms in the soil, so that they can synergize with algae strains and play a positive role in land ecological restoration. The analysis showed that the water content (SWC) of the sandy soil treated with EMC7 algae liquid was 41.26% higher than that of the treatment with BG11 liquid medium (PYJ), and the contents of organic matter (OM), total nitrogen (TN), total phosphorus (TP), alkaline nitrogen (AN), available phosphorus (AP), and available potassium (AK) were increased by 12.90%, 42.86%, 114.26%, 34.26%, 53.42%, and 2.8%, respectively. In addition, extracellular polysaccharides (EPS) are polysaccharide substances secreted outside the cells by microorganisms during their growth and metabolism. They play an important role in the formation and stability of soil aggregates, and can increase soil cohesion and improve its physical structure. From the results in Table 4, the EPS content in the sandy soil treated with EMC7 crust algae increased by 618.18% compared with the PYJ treatment. Soil structure is a key factor affecting soil fertility and material migration and transformation. Soil is composed of aggregates of different particle sizes formed by the interaction between solid particles. A good aggregate structure is of great significance to the transformation of soil nutrients. The two main indicators for evaluating soil structural stability are mean weight diameter (MWD) and geometric mean diameter (GMD). The higher the MWD and GMD values, the greater the stability of the aggregates. Studies have shown (see Figure 4 (1), Table 6), compared with the treatments of applying water (S) or BG11 liquid culture medium (PYJ), the application of EMC7 crust algae liquid increased the content of super-large aggregates > 2 mm in sandy soil and reduced the content of micro-aggregates (< 0.25 mm). The MWD and GMD values ​​were 0.96 and 0.72, respectively, which were significantly higher than those of the PYJ treatment and better than those of the AMC5 treatment.

[0080] For loam soil, after 6 weeks of planting P. frostii, the two treatments with crusted algae also showed significant improvements in soil water retention, soil nutrients, and soil structure compared with the treatments with clear water (S) and BG11 liquid medium (PYJ) (see Table 5, Figure 4(2), Table 7), among which EMC7 had the best effect. Specific data analysis showed that the water content (SWC), extracellular polysaccharides (EPS), organic matter (OM), total nitrogen (TN), total phosphorus (TP), alkaline nitrogen (AN), available phosphorus (AP), and available potassium (AK) contents of loam treated with EMC7 algae solution increased by 43.61%, 693.33%, 48.89%, 46.88%, 88.24%, 29.66%, 56.48%, and 9.60% respectively compared with those treated with PYJ. Regarding the effects on soil aggregate composition and soil stability, the results showed that compared with the treatments of applying water (S) or BG11 liquid culture medium (PYJ), the treatments of applying the two crust algae mainly increased the aggregates > 2 mm, and had no significant effect on the aggregate stability indicators MWD and GMD values. The effects of EMC7 treatment and AMC5 treatment were similar.

[0081] It can be seen that the application of crustacean EMC7 plays an important role in improving the water retention capacity of poor sandy soil and loam, increasing the organic matter and nutrients in sandy soil, and stabilizing the physical structure of sandy soil. It provides excellent microbial resources for the implementation of ecological restoration projects in desertified land and is of great significance.

[0082] Table 4 Effects of different treatments on the physicochemical properties of sandy soil matrix

[0083]

[0084] Table 5 Effects of different treatments on the physical and chemical properties of loam matrix

[0085]

[0086] Table 6 Mechanical stability of sand aggregates under different treatments

[0087]

[0088] Table 7 Mechanical stability of loam aggregates under different treatments

[0089]

Claims

1. A strain of Scenedesmus isolated from biological crust soil in karst rocky desertification area ( Scenedesmus sp.) EMC7, characterized in that Its accession number is CGMCC No. 41200.

2. Use of the Scenedesmus EMC7 as claimed in claim 1 in promoting plant growth and / or promoting plant nutrient absorption.

3. The use according to claim 2, characterized in that The plant is a cold-weather plant, such as Poa frigida; The promoting of plant growth is to increase the total length, plant height, root length, total weight, above-ground biomass and / or underground biomass of the plant; The promoting of plant nutrient absorption is to improve the absorption of nitrogen and / or phosphorus.

4. The use according to claim 3, characterized in that The application is to apply algae solution cultured for 2-4 weeks to the plant growth substrate, and the concentration of the applied algae solution is 10 2 to 10 4 algal cell number / mL, applied once every 3-12 days, for a total of 1-5 times.

5. Use of the Scenedesmus EMC7 as claimed in claim 1 in promoting soil improvement.

6. The use according to claim 5, characterized in that The soil may be poor sand and / or loam.

7. The use according to claim 5, characterized in that The improvement is to increase soil water retention, enhance soil anti-erosion capacity and / or improve soil nutrients.

8. The use according to claim 7, characterized in that The algae solution cultured for 2-4 weeks is applied to the soil where the plants are planted.

9. A method for preparing the algae liquid of Scenedesmus EMC7 according to claim 1, characterized in that: The activated single algae colony is inoculated into BG11 liquid culture medium and cultured at 25-29°C and 2500-2900 lx of light; preferably, the culture is shaken 1-4 times at a fixed time every day, each time for 0.5-2 minutes.

10. A plant growth promoter or soil conditioner, characterized in that: It contains the algae liquid of Scenedesmus EMC7 as claimed in claim 1.

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