CN114303811A

CN114303811A - Method for reducing non-point source pollution of phosphorus in navel orange garden based on AM fungus

Info

Publication number: CN114303811A
Application number: CN202111439319.0A
Authority: CN
Inventors: 刘洪光; 孔召玉; 张利超
Original assignee: Jiangxi Academy of Water Resources
Current assignee: Jiangxi Academy of Water Resources
Priority date: 2021-11-30
Filing date: 2021-11-30
Publication date: 2022-04-12

Abstract

The invention discloses a navel orange garden phosphorus non-point source pollution abatement method based on AM fungi, which comprises the following steps of (1) investigating the AM fungi in the navel orange garden to determine an AM fungus regulation and control strategy in the navel orange garden; (2) the table top and the ladder wall of the navel orange garden are realized by using the AM fungus-plant combination, so that the loss of source phosphorus is reduced; (3) based on the key effect of the AM fungus in the grass ditch phosphorus loss, the method for applying the navel orange garden bactericide is optimized, and the process of intercepting phosphorus is strengthened. The method can provide an effective solution for ecological promotion and clean production of the navel orange garden.

Description

Method for reducing non-point source pollution of phosphorus in navel orange garden based on AM fungus

Technical Field

The invention relates to the technical field of environmental management, in particular to a navel orange garden phosphorus non-point source pollution abatement method based on AM fungi.

Background

The lower limit of the pollution of the phosphorus is one tenth of that of the nitrogen, so the phosphorus is a main limiting factor of water eutrophication, and the phosphorus loss is relatively easy to control. If the phosphorus can be controlled to migrate to the water body, the problem of water body eutrophication can be solved. The history of fruit planting in Jiangxi province is long, and the fruit tree cultivation is second to rice in the planting industry, so that the method is an important direction for improving the agricultural economic benefit and accurately controlling poverty in our province. The fruit industry in our province is dominated by subtropical fruits, wherein the fruit brands of Gannan navel orange, Nanfeng honey orange, Fengxin kiwi fruit, Xinfeng sweet pomelo and the like are famous nationwide. However, dry soil in southern red soil regions is generally deficient in phosphorus, with areas with severely deficient total phosphorus (<0.04g/kg) reaching 28.5% and areas with severely deficient available phosphorus (<5mg/kg) reaching 77.8%. Therefore, at the beginning of orchard establishment, fruit growers apply a large amount of phosphate fertilizer. However, the current utilization rate of the phosphate fertilizer is only 10% -15%, the surface coverage is insufficient, and the excessive phosphate fertilizer is easy to migrate to a water body along with silt, so that non-point source pollution is caused. Therefore, the reduction of the phosphorus loss at the initial stage of orchard establishment is the important factor in controlling the non-point source pollution of orchards in south China.

Disclosure of Invention

The invention aims to solve the problems that: the AM fungus-based navel orange garden phosphorus non-point source pollution reduction method is provided, and an effective solution can be provided for ecological promotion and clean production of the navel orange garden.

The technical scheme provided by the invention for solving the problems is as follows: a method for reducing non-point source pollution of phosphorus in a navel orange garden based on AM fungi comprises the following steps

(1) Investigating AM fungi in the navel orange garden to determine an AM fungus regulation strategy in the navel orange garden;

(2) the table top and the ladder wall of the navel orange garden are realized by using the AM fungus-plant combination, so that the loss of source phosphorus is reduced;

(3) based on the key effect of the AM fungus in the grass ditch phosphorus loss, the method for applying the navel orange garden bactericide is optimized, and the process of intercepting phosphorus is strengthened.

Preferably, the step (1) specifically comprises

(1.1) sample collection:

selecting three platforms with a distance of 200m in a navel orange garden, and selecting 3 navel orange trees on each platform; collecting surface soil of more than 30cm at a position 50cm away from each tree by a five-point sampling method, and mixing to obtain a sample; transporting to a laboratory through an ice box, screening off root systems and gravels after sieving by 2mm, and storing in a refrigerator at minus 80 ℃;

(1.2) extracting and detecting total DNA:

extracting total DNA of soil by using the kit, carrying out 0.8% agarose gel electrophoresis for molecular size judgment, and quantifying the DNA by using an ultraviolet spectrophotometer; whether the quality of the sample is qualified or not is determined by whether the effective target band can be amplified by the subsequent PCR;

(1.3) DNA amplification:

after components required by PCR reaction are prepared, performing pre-denaturation on a PCR instrument at 98 ℃ for 30s to ensure that template DNA is fully denatured, and then entering an amplification cycle; in each cycle, the template is denatured by maintaining the temperature at 98 ℃ for 15 seconds, then the temperature is reduced to 50 ℃ and maintained for 30 seconds, so that the primer and the template are fully annealed; maintaining at 72 deg.C for 30s, extending the primer on the template, and synthesizing DNA to complete one cycle; repeating the cycle 25-27 times to allow the amplified DNA fragments to accumulate in large quantities; finally, keeping the temperature at 72 ℃ for 5min to ensure that the product is completely extended and stored at 4 ℃;

(1.4) building a library;

and (1.5) performing quality inspection and sequencing on the library.

Preferably, the step (1.4) specifically comprises

(1.4.1) cutting the base protruded from the 5 ' End of the DNA by using End Repair Mix2 in the kit, completing the base deleted from the 3 ' End, and adding a phosphate group on the 5 ' End, wherein the method comprises the following specific steps:

a. 30ng of the mixed DNA fragment is taken to be supplemented with water to 60 mu L, and 40 mu L of End Repair Mix2 is added;

b. blowing and uniformly mixing by using a gun, and incubating for 30min at the temperature of 30 ℃ on a PCR instrument;

c. purifying the end repairing system by using BECKMAN AMPure XP beads, and finally eluting by using 17.5 mu L of Resuspension buffer;

(1.4.2) adding A to the 3 'end, wherein in the process, the 3' end of the DNA is independently added with an A base, and the specific steps are as follows:

a. adding 12.5 mu L A-Tailing Mix into the DNA after fragment selection;

b. blowing and mixing uniformly by using a gun, and placing on a PCR instrument for incubation, wherein the procedure is as follows: 30min at 37 ℃; 5min at 70 ℃; 4 ℃, 5 min; infinity at 4 ℃;

(1.4.3) adding a linker with a specific tag, and the process is to allow the DNA to finally hybridize to the Flow Cell, and the specific steps are as follows:

a. adding 2.5. mu.L of Resuspension buffer, 2.5. mu.L of Ligation Mix and 2.5. mu.L of DNA adapter Index into the system with the A added;

b. blowing and uniformly mixing by using a gun, placing on a PCR instrument, and incubating for 10min at 30 ℃;

c. adding 5 mu of LStopLigaptionbuffer;

d. purifying the system with the joint by using BECKMAN AMPure XP beads;

(1.4.4) amplifying the DNA fragment with the adaptor by PCR, and then purifying the PCR system by using BECKMAN AMPure XP beads;

(1.4.5) final fragment selection and purification of the library by 2% agarose gel electrophoresis.

Preferably, the step (1.5) specifically comprises

(1.5.1) library quality control and quantitation

Taking 1 mu L of library, using Agilent High Sensitivity DNA Kit to perform 2100 quality inspection on the library on an Agilent Bioanalyzer machine, and using Quant-iT PicoGreen dsDNA Assay Kit to quantify the library on Promega QuantiFluor, wherein the concentration of the qualified library is more than 2nM after calculation;

(1.5.2), sequencing

For the qualified library, paired-end sequencing of 2 × 250bp was performed on a MiSeq machine using MiSeq Reagent Kit V3(600 cycles); firstly, diluting a library needing to be loaded on a computer to 2nM in a gradient manner, and then mixing the sample according to the proportion of the required data amount; denaturing the mixed library into single chains by 0.1N NaOH and carrying out on-machine sequencing;

(1.5.3), sequence annotation

After removing barcode and primers, obtaining effective data by using FLASH, QIIME or MOTHUR, dividing ASV by DADA2 software and selecting representative sequences, and simultaneously comparing and annotating ASV representative sequences with the fungus ITS database Unite _ 8.

Preferably, the step (2) specifically comprises the steps of uniformly paving the microbial inoculum on the table top and the surface layer of the wall soil of the navel orange garden, disinfecting seeds for 2min by using 75% alcohol, and then sowing the seeds after washing the seeds by using distilled water.

Preferably, the step (3) specifically comprises the steps of opening a grass ditch in the navel orange garden, planting herbaceous plants in the grass ditch, and inoculating the AM fungi.

Compared with the prior art, the invention has the advantages that: the AM fungus can infect typical grass-growing plants under natural conditions, can obviously improve the stem length, tillering number, leaf number, stem node number and biomass of the overground parts of the trifolium repens, the bermuda grass and the paspalum, and is beneficial to quickly covering the land surface of the navel orange garden which is initially built; the AM fungus can obviously increase the indexes of the root length, the root volume, the root surface area and the like of the trifolium repens, the bermuda grass and the paspalum vaginatum, strengthen the fixing effect of grass-growing measures on the soil, and provide an effective solution for ecological promotion and clean production of the navel orange garden.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

Fig. 1 is a graph of the infection rate, wherein Control, FM and RI represent no inoculation, inoculation f. BSY, BXC and QB represent trifolium repens, paspalum and paspalum, respectively.

FIG. 2 is a graph comparing AM fungus with Control plants, wherein Control, FM and RI represent no inoculation, inoculation of F. BSY, BXC, GYG and QB represent Trifolium repens, paspalum, Cynodon dactylon and Paspalum vaginatum, respectively (the same below).

FIG. 3 is a graph of the effect of inoculation with the AM fungus on the grass stem length of four plants.

FIG. 4 is a graph showing the effect of inoculation with AM fungus on the number of tillers in four plants.

FIG. 5 is a graph showing the effect of AM inoculation on the leaf number of three plants, which is the average leaf number per stem and is not shown since three white leaves are 3 leaves per stem.

FIG. 6 is a graph showing the effect of AM inoculation on the number of rhizomes of dog teeth.

FIG. 7 is a graph showing the effect of AM inoculation on the aerial biomass of four plants.

FIG. 8 is a plot comparing the inoculated AM fungus with control plant roots.

FIG. 9 is a graph showing the effect of AM inoculation on the biomass of the underground parts of four plants.

FIG. 10 is a graph of the effect of inoculation with the AM fungus on the root-cap ratio of four plants.

FIG. 11 is a graph showing mycorrhizal dependence of four plants on Rhizophagus Intraradics (RI).

FIG. 12 is a graph of mycorrhizal dependence of four plants on Funneliformis Mossseae (FM).

FIG. 13 is a graph of the effect of natural rainfall herbivory measures (with AM fungus) on the erosion modulus of orchards.

FIG. 14 is a diagram of a pot for raising seedlings after sowing.

Figure 15 is a schematic view of a soil box design.

FIG. 16 is a schematic view of the grassland before being flushed.

FIG. 17 is a schematic view of the grassed ditch after washing.

FIG. 18 is a graph of the effect of AM fungus on mean flow rate in furrows under water flow.

FIG. 19 is a graph of the effect of AM fungus on furrow flow rate under water flow brushing.

FIG. 20 is a graph showing the effect of AM fungus on the depth of runoff from grass furrows under water current scouring.

FIG. 21 is a graph of the effect of AM fungus on the width of the runoff in a raceway under water flow brushing.

FIG. 22 is a graph showing the effect of AM fungus on grass furrow diameter flow under water flow scouring.

FIG. 23 is a graph of the effect of AM fungus on overall runoff of a raceway under water flow scouring.

FIG. 24 is a graph of the effect of AM fungus on the power of grass furrow irrigation runoff.

FIG. 25 is a graph of the effect of AM fungus on mean runoff power from grass furrow erosion.

FIG. 26 is a graph showing the effect of AM fungus on the sand production by furrow erosion.

Fig. 27 is a graph of the effect of AM fungus on overall sand production by grasspit scour and sand reduction rate, where the percentage in the graph is the reduced ratio of AM treatment to control (ratio (CK-AM) × 100%/CK), the same below.

FIG. 28 is a composition diagram of the erosion loss of phosphorus in the furrow of four plant plants.

FIG. 29 is a line fit plot of phosphorus in the eluted granular state versus total phosphorus.

FIG. 30 is a graph showing the effect of AM fungus on total phosphorus loss by grassland erosion and phosphorus reduction rate.

FIG. 31 is a graph showing the effect of AM fungus on the runoff of grassland scoured particulate phosphorus.

FIG. 32 is a graph showing the effect of AM fungus on the runoff of grass furrow washed particulate phosphorus and the phosphorus reduction rate.

FIG. 33 is a graph of the effect of AM fungus on the amount of scoured soluble phosphorus runoff from grassfurrows.

FIG. 34 is a graph of the effect of AM fungus on the amount of scoured soluble phosphorus runoff from a grassland.

FIG. 35 is a non-linear fit of particulate phosphorus loss and runoff power.

Figure 36 is a non-linear fit plot of total phosphorus loss and runoff power.

FIG. 37 is a photograph of the microrelief of a grass furrow after washing.

Detailed Description

The embodiments of the present invention will be described in detail with reference to the drawings and examples, so that how to implement the technical means of the present invention to solve the technical problems and achieve the technical effects can be fully understood and implemented.

It will be understood that the terms "comprises" and/or "comprising," when used in this specification and the appended claims, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

It is also to be understood that the terminology used in the description of the embodiments of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the embodiments of the invention. As used in the description of embodiments of the present invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Example 1

Survey of AM fungi in Gannan navel orange garden

Collecting samples: in 2019, soil samples were collected from the Jinqiao navel orange base (E115.29, N26.24) in the Tungkou town of Shangxiang county of Gangzhou city in 11 months. Planting Neholl navel orange in garden, the tree age is 15 years, and the table top and ladder wall are covered with bermudagrass. Three benches 200m apart were selected in the garden, with 3 navel orange trees selected for each bench. Surface soil of more than 30cm is collected at a position 50cm away from each tree by a five-point sampling method, and the surface soil is mixed to be used as a sample. Transporting to a laboratory through an ice box, screening off root systems and gravels after sieving by 2mm, and storing in a refrigerator at minus 80 ℃.

Extraction and detection of total DNA: the kit is used for extracting total DNA of soil, 0.8% agarose gel electrophoresis is carried out for molecular size judgment, and an ultraviolet spectrophotometer is used for quantifying the DNA. Whether the sample quality is qualified or not is determined by whether the effective target band can be amplified by the subsequent PCR.

③ DNA amplification:

ribosomal DNA contains multiple conserved regions and highly variable regions, and primers are designed using the conserved regions to amplify single or multiple variable regions of the rRNA gene, which are then sequenced to analyze microbial diversity. Due to the restriction of MiSeq sequencing read length, and also to ensure the sequencing quality, the insert range for optimal sequencing was 200-520 bp. To analyze the AM fungal and overall fungal status, the assay selects a fungal ITS region of approximately 250bp in length for sequencing. Fungal ITS specific primers, ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') and ITS2 (5'-GCTGCGTTCTTCATCGATGC-3') were selected for PCR amplification. The barcode in the pre-primer is a 7 base oligonucleotide sequence used to distinguish different samples in the same library. The PCR used NEB Q5 DNA high fidelity polymerase, and the system is shown in the following table:

TABLE 1-1 navel orange garden fungus ITS gene propagation system

The procedure is as follows:

after the components required for PCR reaction are prepared, the template DNA is fully denatured by pre-denaturing at 98 ℃ for 30s on a PCR instrument, and then the amplification cycle is carried out. In each cycle, the template is denatured by holding at 98 ℃ for 15 seconds, then the temperature is reduced to 50 ℃ and held for 30 seconds, so that the primer and the template are fully annealed; the primers were extended on the template and DNA synthesized by holding at 72 ℃ for 30s, completing one cycle. This cycle was repeated 25-27 times to allow the amplified DNA fragments to accumulate in large amounts. Finally, the product was kept at 72 ℃ for 5min to allow complete extension and stored at 4 ℃.

The amplification result was subjected to 2% agarose gel electrophoresis, and the objective fragment was excised and recovered using an Axygen gel recovery kit.

PCR products were quantified on a Microplate reader (BioTek, FLx800) using the Quant-iT PicoGreen dsDNA Assay Kit, and then mixed according to the amount of data required for each sample.

The process is to use TruSeq Nano DNA LT Library Prep Kit of Illumina company to establish a Library. The End repairing process is firstly carried out by utilizing End Repair Mix2 in the kit to cut the base protruding from the 5 ' End of the DNA, completing the base missing from the 3 ' End and adding a phosphate group on the 5 ' End, and the concrete steps are as follows:

c. the end repair system was purified using BECKMAN AMPure XP beads and finally eluted with 17.5. mu.L of Resuspension buffer.

The second step is adding A at the 3 ' end, in the process, a single A base is added at the 3 ' end of the DNA to prevent the self-connection of the DNA fragments and ensure the connection of the DNA and a sequencing joint with a protruding T base at the 3 ' end, and the specific steps are as follows:

a. adding 12.5 mu L A-Tailing Mix into the DNA after fragment selection;

b. blowing and mixing uniformly by using a gun, and placing on a PCR instrument for incubation, wherein the procedure is as follows: 30min at 37 ℃; 5min at 70 ℃; 4 ℃, 5 min; 4 ℃ and infinity.

The third step is to add a linker with a specific tag, which is to allow the DNA to finally hybridize to the Flow Cell, and the specific steps are as follows:

c. adding 5 mu L of Stop Ligation buffer;

d. the linker-added system was purified using BECKMAN AMPure XP beads.

The fourth step is to amplify the DNA fragment to which the linker has been added by PCR, and then purify the PCR system using BECKMAN AMPure XP beads.

The fifth step is the final fragment selection and purification of the library by 2% agarose gel electrophoresis.

Quality control and sequencing of library

a. Library quality inspection and quantification

A2100 quality test was performed on 1. mu.L of the library using the Agilent High Sensitivity DNA Kit on an Agilent Bioanalyzer machine, and the qualified library should have a single peak without a linker. The library was quantified using the Quant-iT PicoGreen dsDNA Assay Kit on the Promega QuantiFluor, and the calculated concentration of the qualified library should be above 2 nM.

b. Sequencing

For the qualified libraries, 2 × 250bp paired-end sequencing was performed on a MiSeq machine using MiSeq Reagent Kit V3(600 cycles). The library to be programmed (Index non-reproducible) was first diluted in a gradient to 2nM and then mixed in the desired data size ratio. The pooled library was denatured into single strands with 0.1N NaOH for on-machine sequencing. The amount of the library can be controlled between 15 and 18pM according to the actual situation.

e. Sequence annotation

After removing barcode and primers, software such as FLASH, QIIME, MOTHUR and the like is used for obtaining effective data, DADA2 software is used for dividing ASV (amplification sequence variant) and selecting a representative sequence, and meanwhile, the ASV representative sequence is compared and annotated with the fungal ITS database Unite _ 8.

Results and analysis

AM fungal status in navel orange orchard

A total of 1608 fungi ASV were identified in navel orange garden soil, but only 4 AM fungi. The composition analysis of the navel orange garden soil fungal colony also shows that the content of AM fungi is extremely low. Therefore, the separation of the AM fungus from the navel orange garden is difficult. Project cohorts were decided to collect AM fungi to ensure that projects were propelled normally. The project group obtains AM fungus strain from the arbuscular mycorrhizal fungi germplasm resource library (Bank of Glomeromycota in China, BGC) of Beijing academy of agriculture and forestry for propagation and subsequent research.

Example 2

This example used a factorial design for a potted greenhouse test involving two factors, plant and AM fungi, namely 4 plants: clover (BSY), paspalum natatum (BXC), bermuda grass (GYG) and paspalum vaginatum (QB), 3 AM fungal treatments: fusanformis Mosseae (FM), Rhizophagus Irregularis (RI), no vaccination Control (Control), 5 replicates per treatment, for a total of 4 × 3 × 5 to 60 pots.

The soil used in the test is collected from abandoned farmland (28 degrees 92 'N, 117 degrees 48' E) of the Lean river basin in Jiangxi province, the pH of the soil is 4.65, each kg of the soil contains 8.21g of organic carbon, 1.15g of total nitrogen and 0.57g of total phosphorus, the soil is not sterilized before potting, and the soil is kept consistent with the actual condition of field soil to the maximum extent. 850g of soil is filled in each pot, 10g of corresponding microbial inoculum is respectively inoculated, and 10g of inactivated microbial inoculum and 10ml of microbial inoculum filtrate (10 mu m) are inoculated in a contrast manner. When in sowing, the microbial inoculum is evenly spread on the surface layer of soil, seeds are sown after being disinfected (distilled water is used for washing after 2min by 75 percent alcohol), 20 seeds are sown in each pot, and 15 seeds are reserved after thinning after growing for two weeks. The test run period was 118 days. And (3) respectively measuring the AM fungal infection rate, the above-ground and underground indexes of the plant during harvesting, and analyzing the soil aggregate condition. At the final harvest, 2 pots of bermudagrass die, so that the number of repetition of some indexes of bermudagrass in the follow-up process is 3, and the number of repetition of other 3 plants is 5.

Determination of fungal infection Rate

Carefully cleaning the root segments collected from the soil, using PThe method of hilllps and Hayman (1970) stains root segments as follows: placing the cleaned root segment in 10% KOH solution, heating in 90 deg.C water bath for 15min until the root segment is transparent, and treating with alkaline H₂O₂Softening for 20min, acidifying in 1% HCl, rinsing for 4 times, placing in trypan blue solution, and dyeing for 2h at normal temperature; the dyed roots were then placed in lactic acid glycerol and decolorized at room temperature. The next day, the decolorized root samples were sliced, and the structures of arbuscular, vesicular, spore and hyphae were observed under a microscope, and the AM fungal infection rate was counted by the cross method (Giovannetti and Mosse, 1980).

Infection rate (%) (number of mycorrhiza on cross point/total number of cross points) × 100% formula 2-1

Overground growth index analysis

Measuring the length of grass stem, tillering number, leaf number and stem node number. Placing the overground part of the plant into an envelope, putting the envelope into an oven, drying the envelope at 80 ℃ for 36h until the weight is constant, and weighing the biomass of the overground part.

Cleaning and flattening the plant root system in a root disc, obtaining a root system photo by a scanner (Epson), and analyzing by WinRhizo software to obtain the total root length (L), the root volume (V), the root surface area, the diameter (D), the root tip number and the bifurcation number. Then, the root system is wiped dry by absorbent paper, dried for 36 hours at 80 ℃ to constant weight, and the biomass of the root system is weighed. And calculating the root characteristic index through a formula 2-2 to a formula 2-4.

(ii) longer than root (Specific root length, SRL (m/g)) ═ L/W formula 2-2

Root mass density ratio (RMD (g/cm))³) W/V formula 2-3

Length of Root Density (RLD (cm/cm)³) L/V formula 2-4

Root-to-crown ratio and mycorrhiza dependence calculation

The root-cap ratio and mycorrhiza dependence were calculated by equations 2-5 and 2-6:

root cap ratio (root biomass/underground part biomass) 2-5

Mycorrhiza dependence (AM inoculated fungal plant biomass-control biomass) × 100%/control biomass formula 2-6

Soil aggregate stability analysis

Mean Weight Diameter (MWD) and Geometric Mean Diameter (GMD)

The soil aggregate stability is determined by wet sieving method, which comprises placing soil sample on top of a sleeve sieve (with aperture of 2mm, 1mm, 0.5mm, 0.25mm in sequence) placed in a water bucket, starting a motor after 2min to vibrate the sleeve sieve in water at frequency of up-down movement distance of 4cm and vibration speed of 30 times/min for 0.5 h. Then the sieve is taken off the water surface, after the water is slightly dried, the aggregates left on the sieve are washed by a washing bottle, washed into a beaker with known mass, dried and weighed, and the mass is accurate to 0.01g, and each treatment is repeated for 3 times.

The agglomerate water stability is expressed in terms of the mean mass diameter (MWD) and the Geometric Mean Diameter (GMD), calculated as follows from equations 2-7 and 2-8:

in the formula: x is the number of_iThe mean value of the aperture of the two sieves before and after screening the size fraction is mm; w is a_iMass fraction of the i-th particle size aggregate,%. Water Stable agglomerate content (Water Stable Aggregates)

After the soil aggregate is subjected to dry screening and wet screening, calculating the content of the water-stable aggregate through formulas 2-9:

effect of grass-growing measures on orchard soil erosion

The local AM fungi can be nurtured by growing grass in the orchard, and the AM fungi has the effects of reducing soil erosion and preventing and controlling phosphorus loss by combining grass growing measures. According to positioning observation data of two years of 2017 and 2018 of water and soil conservation ecological technology garden grass growing districts in Jiangxi province and orchard, analyzing the mud and sand amount of clover and astragalus sinicus full garden coverage, clover and astragalus sinicus strip coverage, bermuda grass full garden coverage and ploughing measures so as to evaluate the influence of grass growing measures (including AM fungi) on water and soil loss of the orchard. As the phosphorus exists in a granular state, the test can indirectly reflect the prevention and control effect of the AM fungus on the orchard phosphorus loss.

Statistical analysis

The effect of inoculated AM fungus on the plant aerial part characteristics, underground part characteristics and soil aggregate related indicators was analyzed by univariate analysis of variance (ANOVA) and Duncan multiple comparison (SPSS v 23.0).

Results and analysis

Infection rate

The experiment uses unsterilized soil to explore the effect of the AM fungus in natural soil. The AM fungal infection rate of Trifolium repens, paspalum and paspalum was significantly higher than the control. The FM-inoculated paspalum, trifolium repens and bermuda grass were 5.4-fold, 2.2-fold and 0.49-fold higher in infestation than the controls, respectively (Table 2-1 and FIG. 1). Paspalum, trifolium repens, bermuda grass and paspalum palustre inoculated with RI are 7.8 times, 1.3 times, 1.8 times and 1.1 times higher than those of the control respectively. The two AM fungi selected in the test can form a symbiotic relationship with the four herbs in natural soil, and the infection of RI is superior to that of FM.

TABLE 2-1 AM fungal infection (%)

Note: control, FM and RI represent no inoculation, inoculation f. BSY, BXC, GYG and QB represent Trifolium repens, paspalum, Cynodon dactylon and paspalum, respectively. The different letters contained after each column of data represent significant differences at the p-0.05 level by duncan's test. Effect of AM fungi on growth of aerial parts of plants

The study showed that inoculation with the AM fungus significantly promoted overground growth of trefoil, paspalum, bermuda grass and paspalum (fig. 2).

Inoculation of RI and FM significantly increased Trifolium repens (F) compared to controls_2,124.93, p 0.027) and paspalum natatum (F)_2,124.232, p 0.041). Inoculation of the two AM fungi on Bermuda grass (F)_2,103.207, p 0.084 and paspalum (F)_2,122.999, p 0.088) did not reach a statistically significant level (fig. 3). The stem lengths of trifolium repens, paspalum, bermuda grass and paspalum vaginatum inoculated with RI were 27%, 19%, 85% and 21% higher than the control, respectively. The stem lengths of FM-inoculated trifolium repens, paspalum, bermuda grass and paspalum were 27%, 22%, 37% and 31% higher than the control, respectively. The two AM fungi have the most remarkable promoting effect on the stem growth of bermuda grass, and RI is superior to FM.

Inoculation with AM fungus (RI and FM) significantly increased Trifolium repens (F) compared to controls_2,123.216, p 0.076) and bermuda grass (F)_2,10Tillering number 4.89, p 0.033) (fig. 4). The tillering number of the trifolium repens and the bermuda grass inoculated with RI is respectively increased by 31 percent and 49 percent compared with that of a control. Tillering numbers of FM-inoculated white clover and bermudagrass are respectively increased by 24% and 34% compared with those of the control. Inoculating AM fungus to paspalum (F)_2,120.353, p 0.705) and paspalum natatum (F)_2,121.493, p 0.263) tiller number did not reach a statistically significant level. However, the tillering of both paspalum and paspalum inoculated with RI increased 31% and 20% over the control. On the whole, the promotion effect of inoculating RI on the tiller number of four grass is better than that of FM, and the promotion effect of inoculating two AM fungi on the tiller number of bermudagrass is most obvious.

Inoculation with AM fungi (RI and FM) against paspalum (F)_2,120.168, p 0.847), cynodon dactylon (F)_2,102.219, p 0.17) and paspalum natatum (F)_2,12The effect of leaf number 0.911, p 0.428 did not reach a significant level (fig. 5). However, the numbers of leaves inoculated with RI and FM Bermuda grass were still 85% and 19% higher than the controls.

Inoculation of AM fungus significantly increased the average number of nodes per tillering of Bermuda grass (F)_2,102.657, p 0.119) (fig. 6). The number of stem nodes of vaccinated RI and FM Bermuda grass was increased by 1.3 times and 0.27 times, respectively, compared to the control. No stem nodes were found in the branches of Trifolium repens, paspalum and paspalum, and were not counted in this study.

Inoculation with AM fungi (RI and FM) compared to non-inoculated controlsWhite clover (F)_2,12＝51.665,p<0.0001) and Caragana carvi (F)_2,122.87, p 0.096) was significantly increased. The increasing range of inoculating RI and FM to the biomass on the overground part of the trifolium repens reaches 0.4 time and 2.4 times respectively; the range of increase in biomass in the aerial part of paspalum by inoculation of RI and FM reached 1.6 times and 0.67 times, respectively. Although the effect of inoculating AM fungus on the biomass of the aerial parts of Bermuda grass did not reach a significant level (F)_2,102.098, p 0.173), but the biomass of aerial parts of vaccinated RI and FM cynodon dactylon increased 1.4-fold and 0.81-fold, respectively, compared to the control. The biomass of the overground part of the paspalum natatum inoculated with AM fungus has no significant difference with the control (F)_2,12＝3.463,p＝0.065)。

Taken together, inoculation with these two AM fungi (RI and FM) significantly promoted aerial growth of Trifolium repens, paspalum, bermuda grass and paspalum by increasing stem length, tiller number, leaf number, stem node number and biomass, among others. However, there are differences in the effect of different plant and AM fungus combinations. The above-ground data are integrated, the effect of inoculating RI on promoting the herbage is better than that of FM, and Bermuda grass-RI is the optimal combination.

Effect of fungi on plant root characteristics

Root systems are the key for improving soil structure and fixing soil of plants, and four plant root systems are more developed after AM fungi are inoculated (figure 8).

Inoculation of AM fungus significantly increased the total root length of Trifolium repens (F)_2,12＝36.554,p<0.0001), total root length of inoculated RI and FM white three leaves increased 0.68-fold and 2.4-fold, respectively, over the control (table 2-2). Although the effect of inoculated AM fungus on the total root length of Bermuda grass and Paspalum did not reach a statistically significant level, the total root length of inoculated FM Bermuda grass increased 26% over the control, and the total root length of inoculated RI and FM Paspalum increased 14% and 27%, respectively, over the control. However, inoculation with both AM fungi had no significant effect on total root length of paspalum natatum, and inoculation with FM even showed some negative effect.

TABLE 2-2 Effect of inoculation with AM fungus on Total root Length of four plants

Note: control, FM and RI represent no inoculation, inoculation f. BSY, BXC, GYG and QB represent Trifolium repens, paspalum, Cynodon dactylon and paspalum, respectively. Values in parentheses are standard deviations, and each column of data is labeled with a different letter indicating significant differences at the p-0.05 level by duncan's test (same below).

Inoculation of AM fungus significantly increased the root volume of Trifolium repens (F)_2,12＝42.813,p<0.0001), inoculation of RI and FM white three root volumes increased 1.3-fold and 4.8-fold, respectively, over the control (tables 2-3). Although the effects of inoculated AM fungus on the volume of Bermuda grass and Paspalum were not statistically significant, the volume of inoculated RI and FM Bermuda grass was increased by 2.2-fold and 3.5-fold, respectively, and the volume of inoculated RI and FM Paspalum was increased by 0.94-fold and 1.8-fold, respectively, compared to the control. However, inoculation of these two species of M.did not significantly affect the size of the paspalum natatum roots.

TABLE 2-3 Effect of inoculation with AM fungus on root volume of four plants

Inoculation of AM fungus significantly increased the root surface area of Trifolium repens (F)_2,12＝44.884,p<0.0001), root surface area of inoculated RI and FM trifolium repens increased 1-fold and 3.5-fold, respectively, over the control (tables 2-4). The effect of inoculation of these two AM fungi on the root surface area of Cynodon dactylon and Caragana carvi did not reach a statistically significant level, but the root surface areas of the inoculated RI and FM Bermuda grass were increased by 0.49-fold and 1-fold respectively compared to the control, and the root surface areas of the inoculated RI and FM paspalum carvi were increased by 51% and 91% respectively compared to the control. However, inoculation of these two AM fungi did not significantly affect the surface area of the paspalum natatum, and inoculation of FM even served to reduce the surface area of the paspalum natatum.

Tables 2-4 Effect of inoculation with AM fungus on root surface area of four plants

Inoculation of AM trueThe fungus remarkably improves the diameter (F) of the white clover root_2,12＝18.008,p<0.0001), the root diameter of inoculated RI and FM trifolium repens increased 19% and 33% respectively over the control (tables 2-5). Although inoculation with these two AM fungi did not have a statistically significant effect on the root diameter of Cynodon dactylon and Paspalum vaginatum, the diameter of inoculated RI and FM Cynodon dactylon increased 1-fold and 69% respectively, and the root diameter of inoculated RI and FM Paspalum vaginatum increased 31% respectively. However, inoculation with both AM fungi had no significant effect on the diameter of the paspalum natatum roots and inoculation with FM showed some negative effect.

TABLE 2-5 Effect of inoculation with AM fungus on four plant diameters

Inoculation of AM fungus significantly increases the number of trefoil roots (F)_2,12＝25.945,p<0.0001), the number of root tips of the inoculated RI and FM white three leaves increased by 48% and 2 times, respectively, compared with the control (tables 2-6). Although the effect of inoculation of these two fungi on the number of bermudagrass and paspalum roots was not statistically significant, the FM-inoculated bermudagrass roots were increased 45% over the control, and the numbers of RI-and FM-inoculated paspalum roots were increased by 17% and 38%, respectively, over the control. However, inoculation with both AM fungi had no significant effect on paspalum natatum number, and inoculation with FM even slightly reduced paspalum natatum number.

TABLE 2-5 Effect of inoculation with AM fungus on four plant diameters

The inoculation of AM fungus remarkably improves the trifolium repens branching number (F)_2,12＝31.3,p<0.0001), inoculation of RI and FM trifoliate root bifurcations increased 1.1-fold and 3.8-fold, respectively, over the control (tables 2-7). Although inoculation with these two AM fungi had a statistically significant level of effect on root branching in Bermuda grass and Caragana carvi, the FM inoculation of Bermuda grass had a 62% increase in root branching over the control, and the inoculation of RI and FM Caragana carvi had a 31% and 85% increase in root branching over the control, respectively. But instead of the other end of the tubeThe inoculation of the two AM fungi had no significant effect on the number of bifurcations of the pennisetum setosum roots and showed negative effects.

TABLE 2-7 Effect of inoculation with AM fungus on root branching of four plants

Inoculation with AM fungus significantly reduced the specific root length of Trifolium repens (F)_2,129.208, p 0.004), the inoculated RI and FM trifolium root length decreased by 62% and 58% respectively compared to the control (tables 2-8). Although inoculation with these two AM fungi had a statistically significant level of effect on specific root length of Bermuda grass and Paspalum vaginatum, the specific root length of RI inoculated Bermuda grass was reduced by 64% compared to the control, and the specific root length of RI inoculated and FM paspalum was reduced by 41% and 30% compared to the control, respectively. However, inoculation with these two AM fungi had no significant effect on the specific root length of paspalum natatum, and the specific root length of FM paspalum natatum increased by 69% over the control.

TABLE 2-8 Effect of inoculation with AM fungus on the growth of four plants compared to root

The effect of inoculated AM fungus on the root mass density ratio of the four plants reaches a statistically significant level, but the inoculated RI and FM trilobate root mass density ratios are increased by 82% and 26% respectively compared with the control (tables 2-9). The mass density of the roots inoculated with RI-paspalum and bermuda grass was reduced by 26% and 29% respectively compared with the control, and the mass density of the roots inoculated with FM-paspalum and bermuda grass was reduced by 34% and 33% respectively compared with the control. The root mass density ratio of inoculated two AM fungi, ditch millet, and the control were not significantly different.

TABLE 2-9 Effect of inoculation with AM fungus on the root Mass Density ratio of four plants

Inoculation with AM fungus significantly reduced the root length density of Trifolium repens (F)_2,1210.929, p 0.002), the root length density of the inoculated RI and FM white clover leaves was reduced by 27% and 42%, respectively, compared to the control (tables 2-10). The effect of inoculation of these two AM fungi on the root length density of Bermuda grass and Caragana carvi did not reach a statistically significant level, but the root length density of the inoculated RI and FM Bermuda grass was reduced by 67% and 42% respectively compared to the control, and the root length density of the inoculated RI and FM Paspalum carvi was reduced by 40% and 37% respectively compared to the control. In contrast, the FM-inoculated paspalum natatum increased 20% over the control.

TABLE 2-10 Effect of inoculation with AM fungus on root length Density in four plants

Inoculation of AM fungus significantly increased Trifolium repens root biomass (F) compared to the uninoculated control_2,12＝39.216, p<0.0001), RI and FM inoculated trifolium repens root biomass increased 3-fold and 6-fold, respectively, over the control (fig. 9). The effect of inoculating these two AM fungi on the root biomass of bermuda grass and paspalum did not reach a statistically significant level, but the root biomass of inoculated RI and FM bermuda grass increased 1-fold and 1.5-fold respectively compared to the control, and the root biomass of inoculated RI and FM paspalum increased 1-fold and 1.4-fold respectively compared to the control. However, inoculation with AM fungus significantly reduced the biomass of the roots of paspalum natatum (F)_2,123.998, p 0.047), the root biomass of RI and FM paspalum was reduced by 22% and 46% respectively, showing strong parasitic effects (fig. 9).

Root-to-crown ratio and mycorrhiza dependence

Inoculation of AM fungus significantly increases the root-crown ratio of Trifolium pratense (F)_2,127.982, p 0.006), the root cap ratio of inoculated RI and FM white clover increased 1.75-fold and 1-fold, respectively, over the control (fig. 10). The effects of AM-inoculated fungi on the ratio of the root-crown of the other three plants did not reach a statistically significant level, but the root-crown of FM-inoculated paspalum, bermuda grass and paspalumIncreased by 22%, 46% and 16% compared to the control, respectively, and the root cap of paspalum, bermuda grass and paspalum inoculated with RI decreased by 15%, 15% and 11% compared to the control, respectively.

The mycorrhiza dependence of the root system of the trifolium repens on RI is obviously higher than that of the root system of the other three plants (F)_3,146.359, p 0.006), but the above-ground partial and total dependence was not significantly different from the other three plants (fig. 11). The dependence of trilobe, bermudagrass and paspalum on RI is positive, namely the profit is gained in symbiosis, and RI is expressed as symbiotic effect. However, the dependence of the paspalum natatum on RI is lower than that of the other three plants, and the root system even shows negative effect, which indicates that the symbiotic benefit of the paspalum natatum and RI is poor.

Root system of Bai clover (F)_3,167.122, p 0.003), aerial parts (F)_3,163.169, p 0.053) and total dependency (F)_3,164.607, p 0.017) were all significantly higher than the other three plants (fig. 12). The dependence of the trifolium repens, the cynodon dactylon and the paspalum vaginatum on FM is positive, which shows that the FM exerts symbiotic effect after infecting the root systems of the 3 plants. However, the dependence of paspalum on FM is negative, i.e. FM shows a parasitic effect on paspalum, and thus paspalum cannot benefit from symbiosis with FM. Bermudagrass and paspalum are relatively dependent on f.

Aggregate stability analysis

The effect of inoculation with the AM fungus on the MWD and GMD of 4 plant soils was significant (tables 2-11 and 2-12).

TABLE 2-11 Effect of inoculation with AM fungus on the mean weight diameter of the soil for four plants

TABLE 2-12 Effect of inoculation with AM fungus on the mean diameter of the soil pool of four plants

Inoculating AM fungus to 4 plants>0.05mm water-stable agglomerates (WSA)_0.05) The effect of content did not reach a statistically significant level, but WSA inoculated with RI and FM trifolium repens treatment_0.05The content was increased by 25% and 11% respectively from the control (tables 2 to 13). Inoculation of AM fungus WSA treated by 3 other plants_0.05The content is not significantly different from the control.

Tables 2-13 Effect of inoculation with AM fungus on the Water-stable agglomerate (>0.05mm) content of four plants

Effect of fungus in combination with plants on orchard soil erosion

Under the condition of natural rainfall, the erosion modulus of the orchard adopting different measures is calculated. The calculation results of 2 continuous years (2017 and 2018) show that compared with clear ploughing, the grass-growing measure (containing AM fungus) greatly reduces the erosion modulus of the orchard, and the reduction amplitude is more than 98% (fig. 13). The particulate phosphorus lost in red soil accounts for more than 72% of the total phosphorus (see chapter 3), so that the proportion of phosphorus output in orchards can be reduced by grass-growing measures containing AM fungi by more than 71%.

Example 3

Influence and mechanism of AM fungus on interception of phosphorus in drainage ditches of navel orange gardens

Soil and biological materials

The soil is from Shangzhen of Ningdu county of Ganzhou city, the matrix is red sandstone, the pH of the soil is 5.17, each kg of the soil comprises 2.95g of organic matter, 0.18g of total nitrogen, 0.30g of total phosphorus, 45.49g of total potassium, 1.90mg of quick-acting phosphorus, 99.60mg of quick-acting potassium and 72.46mg of alkaline hydrolysis nitrogen, and the proportion of clay grains, powder grains and sand grains is 7.07%, 16.16% and 76.77% respectively.

Astragalus sinicus (ZYY), Lotus japonicus (BMG), zoysia Japonica (JLC) and Cynodon dactylon (GYG) seeds were purchased from flower Co., Ltd, Jiangsu Shuyang. Culturing with seedling tray (53cm × 27cm × 6cm) to obtain leguminous plantThe seeding density of the astragalus sinicus and the crowtoe is set to be 12g/m²The grass family (zoysia japonica and bermuda grass) was set at 10g/m². The seeds were sterilized with 75% alcohol for 3min, sowed, 24 seedling pots were planted for each grass seed, covered with nonwoven fabric, and watered thoroughly with a watering can (fig. 14).

After the four plants grow for 57 days, benomyl (purchased from Jiangsu Lanfeng biochemical engineering Co., Ltd., 50% of effective components, 100g of the agent is mixed with 50L of water and is sprayed by a spray can till the solution is completely poured) is used for inhibiting AM fungi in soil of half seedling pots of each plant, and the Control (CK) is used; the remaining nursery pots treated with equal amount of water were used as AM. The benomyl treatment is carried out for 10 times totally, and the benomyl treatment is finished after the plants grow for 117 days.

Grass ditch arrangement

Grass furrows with a length of 2000mm, a width of 500mm and a height of 500mm were arranged by using a custom soil trough (fig. 15).

The bottom part of the soil is 200mm and is filled with stones, and the soil for planting plants is covered on the soil. After the plants grow for 117 days, the plants are laid on the grass ditches. The gaps are reduced by fitting as much as possible during splicing, and the plants are fully covered in the ditch (figure 16). The grass furrows before scouring are U-shaped furrows with the maximum depth of 10cm and the width of 50 cm. And after the plants are stabilized in the tank for 19 days and the root systems of the plants are completely attached to the soil in the tank, starting a phosphorus-containing water flow scouring test.

Scouring test and index determination

KH-containing water for scouring₂PO₄1.5mg/L, flow rate set to 15L/min, and ramp set to 5. The washing is carried out once a day for 30min and continuously for 5 days.

Flow rate: in the scouring process, the potassium permanganate method is adopted to record the time of red liquid passing through three slope marks of the upper part, the middle part and the lower part of the grass furrow after the scouring starts for 6min, 12min, 18min, 24min and 28min respectively, the flow rate is calculated by using the distance between the slope marks and the flowing time, and the average flow rate is calculated by using the three slope flow rates.

The runoff depth: in the scouring process, the runoff depth of the upper, middle and lower slopes of the grass gully is measured at the 6 th min, 12 th min, 18 th min, 24 th min and 28 th min after the scouring is started respectively.

③ the runoff is wide: in the washing process, the runoff widths of the upper, middle and lower slopes of the grass ditches are measured at 6min, 12min, 18min, 24min and 28min after the washing is started respectively.

Yield: collecting all the produced flow sand every 3min, collecting 10 samples in total, and calculating the flow rate and total flow rate of each scouring.

Fifth, runoff power: referring to the method of Wangwei et al (2021), the runoff power is calculated according to the formula 3-1:

in the formula: τ is radial flow shear force (Pa); v is the average flow velocity (m/s); gamma is water flow volume weight (kg/m)³) (ii) a h is the average water depth (m) of the water passing section; s is a hydraulic gradient (m/m), and can be approximately replaced by a sine value of the gradient; g is gravity acceleration, and is 9.8m/s²。

Yield of sand: 400mL of each of the collected 10 samples was filtered, and the amount of sand produced was measured by a drying method.

Loss of phosphorus: and respectively taking 100mL of the 2 nd, 6 th and 9 th samples in the collected samples, determining the content of granular phosphorus and soluble phosphorus by using an alkali-molybdenum-antimony colorimetric resistance method, and calculating the total phosphorus loss.

Eighthly, analyzing the surface morphology of the grass furrow after scouring: after the scouring test is finished, the shape of the scoured grass ditches is shot frame by utilizing the height of an unmanned aerial vehicle 2m away from the grass ditches, information extraction and operation of Cloud match (version 2.1.2) post-processing software are utilized, a plane formed by 4 corner points of an outer frame at the top of the soil tank is taken as a reference plane, the erosion terrain of the grass ditches is obtained, and the micro-landform characteristic analysis of the grass ditches under different AM processing is carried out.

Statistical analysis

The interception efficiency of the AM fungi on the sand reduction rate, the total phosphorus and the granular phosphorus is calculated by adopting a formula 3-2 to a formula 3-4:

and carrying out linear fitting on the total phosphorus-particle phosphorus by utilizing originPro2016, carrying out nonlinear fitting on the total phosphorus-runoff power and the particle phosphorus-runoff power, and carrying out square root conversion on the total phosphorus data before fitting. All pictures were prepared from OriginPro 2016.

Results and analysis

The influence of water flow scouring on grass-planting ditches built by different plants is different, and the AM fungus has obvious influence on landforms after scouring. As can be seen from fig. 17, the AM fungus significantly reduced the extent of erosion of the grass furrow compared to the control. The contribution of the AM fungus to the resistance of the grassland to water flow scouring and the retention of the phosphorus is analyzed from the angles of hydraulic parameters, sand production characteristics, loss phosphorus form, phosphorus retention and the like, and the action mechanism of the AM fungus on the phosphorus retention is revealed by combining with micro-topography analysis.

Influence of fungi on hydraulic parameters of grass furrow washing

Flow rate: the AM fungus reduced the average flow rate of milk vetch and bermudagrass (fig. 15). The average flow rate of the AM milk vetch furrows was reduced by 16%, 23% and 11% from the control at

day

1, 3 and 4, respectively. In the flushing process of the first 4 days, the average flow rate of the AM bermudagrass is respectively reduced by 7%, 16%, 14% and 12% compared with the control, and the flow rate of the AM fungus bermudagrass in the 5 th day has no obvious difference from the control. However, the AM fungus had no significant effect on the flow rate of crowtoe and zoysia.

Analysis of the flow rates of the different slopes revealed that the flow rates of the AM astragalus sinicus furrows of the different slopes were lower than the control (fig. 19). The flow rate of bermudagrass is expressed as downhill grade > moderate grade > uphill grade, and the flow rate of AM grassland is lower than that of control. However, the AM fungus has no significant effect on the flow rate of different slopes of the crowtoe and zoysia japonica furrows.

The runoff depth: as can be seen from the runoff depth data, the runoff depth of the middle slope of the astragalus sinicus furrow was greater, and the runoff depth of AM treatment was lower than the control (fig. 20). The influence of the slope and the AM fungi on the furrow diameter flow depth of the crowtoe grass is not obvious. The radial flow depth of the zoysia japonica ditch is represented as follows: the middle slope position, the ascending slope position and the descending slope position, and the radial flow depth of the AM treatment descending slope position is the minimum. The runoff depth was greater in the ascending and middle slopes of bermuda grass furrows and was shown to be lower in AM treatment than in controls.

③ the runoff is wide: the runoff width of the AM treated astragalus sinicus ditch is obviously higher than that of a control (figure 21), especially the washout on 2 nd, 3 rd and 4 th days is higher than that of the control by more than 50 percent, and the runoff widths of different slopes are relatively close. The runoff width of the downward slope of the crowtoe grass furrow treated by the AM is the largest, and the runoff width of the upward slope treated by the AM is higher than that of the contrast, but the runoff width of the upward slope is not obviously influenced. For the zoysia japonica ditch, the runoff width of the ascending slope position treated by the AM is obviously higher than that of the contrast, the difference between the AM treatment and the contrast is not obvious between the middle slope position and the descending slope position, but the runoff width treated by the AM has a tendency higher than that of the contrast, and the runoff width of the descending slope position is higher than that of the middle slope position and the ascending slope position. The runoff width of the bermudagrass trench is represented by that the downhill position is greater than the middle slope position and the uphill position, and the runoff width of the AM treatment grass trench is obviously higher than that of the contrast at the three slope positions.

Yield: the AM-treated production rate was lower than the control at two days of the pre-gutter wash, but significantly higher during the subsequent wash (fig. 22). The yield of the radix bustae treated by AM is higher than that of the control, but the amplitude is not large. The production flow was higher on the first two days and 5 days of AM treatment of zoysia japonica furrows, but lower on the third four days of washout than control. There was no significant difference between the flux in the bermudagrass treated with different AM. Overall, the runoff of AM-treated milk vetch, Lotus japonicus and zoysia japonica furrows was slightly higher than the control, but AM-treatment had no significant effect on bermudagrass furrow runoff (fig. 23).

Water flow power: the presence of the AM fungus significantly reduced the grass furrow runoff power, and this effect was consistent in four plant established grass furrows (fig. 24). The AM fungus reduces the runoff power of astragalus sinicus, bermuda grass, crowtoe and zoysia japonica by 36.75%, 26.96%, 9.84% and 6.7% respectively (figure 25).

Influence of AM fungus on characteristics of grass ditch scouring sand production

The AM fungus remarkably reduces the sand yield of four plant grass-planting ditches (figure 26), and the sand reduction rates of astragalus sinicus, crowtoe, zoysia japonica and bermudagrass respectively reach 88.5%, 69.9%, 79.3% and 10.4% (figure 27).

Morphometric analysis of lost phosphorus

After the phosphorus-containing water flow washing, the phosphorus loss form is mainly in a granular state, the granular phosphorus loss amounts of the astragalus sinicus, the zoysia palustris and the bermudagrass respectively account for 85%, 90%, 72% and 90% of the total flow loss amount (figure 28), and the main form of the phosphorus loss of the bermudake washing is granular phosphorus which migrates along with sediment loaded with the phosphorus.

Linear fit analysis showed that the fit coefficient and correlation coefficient of total phosphorus and granular phosphorus were as high as 0.988 and 0.995 (FIG. 29), respectively, and that total phosphorus loss increased linearly with granular phosphorus loss, indicating that granular phosphorus dominates the phosphorus loss.

Influence of AM fungus on grass furrow to intercept phosphorus loss

The AM fungus remarkably reduces the phosphorus loss of four plant grass ditches, improves the interception efficiency of the grass ditches to the phosphorus, and reduces the total phosphorus loss of astragalus sinicus, crowtoe, zoysia japonica and bermudagrass by 71%, 55% and 22% respectively (figure 30). Moreover, the increase of the interception rate of the AM fungus to the glyphosate is mainly embodied in granular phosphorus, but not soluble phosphorus. Particulate phosphorus loss by AM fungus was reduced by 82%, 63%, 77% and 23% for astragalus sinicus, crowtoe, zoysia japonica and bermudagrass, respectively (fig. 31 and 32). In contrast, the AM fungus instead showed a tendency to increase soluble phosphorus loss (fig. 33 and 34), consistent with the runoff change.

Mechanism analysis of AM fungus interception grass ditch phosphorus element

Nonlinear fitting analysis shows that both granular phosphorus and total phosphorus loss form a quadratic relation with runoff power, and the runoff power is obviously increased along with the increase of the runoff power (fig. 35 and fig. 36), which indicates that the runoff power is a key factor for determining phosphorus loss. The AM fungus can obviously reduce the runoff power of four plant grass-planting ditches, explaining the phenomenon that the runoff quantity of the phosphorus in the grass ditches is obviously reduced.

As can be seen from fig. 37, the erosion of the upward slope in the CK astragalus sinicus furrow is severe, and the entire furrow is flushed, compared with the AM treatment, the erosion degree is significantly reduced. The main erosion of the crowtoe CK treatment also occurs in the middle and upward slope, but the crowtoe treated by AM only slightly erodes in the upward slope, and the degree is far lower than that of the CK treatment. For zoysia CK, the main erosion site is on the middle and upper slope, but only slight erosion of AM treated zoysia ditches occurs. The main erosion part of CK grass groove of bermuda grass is in the middle and upward slope, the erosion groove is deeper, but the AM-treated CK grass groove erosion degree is reduced. Therefore, the main erosion and corrosion parts of the four plants are in the middle and upper slope positions, and the AM fungi effectively protect the grass ditches, reduce the sand yield of the grass ditches and further reduce the loss of phosphorus.

The invention shows that the inoculation of AM fungi can increase the overground part indexes (stem length, leaf number, tillering number, stem node number and the like) of typical water-retaining plants such as trifolium repens, bermuda grass and paspalum by 20-240 percent, and the increase range of the root system indexes (total root length, root volume, heel area and the like) of the selected typical water-retaining herbaceous plants reaches 14-480 percent; the increase in 0.05 content of WSA from white three leaves by r.intraradies and f.mosseal was 25% and 11%. The demonstration point adopts AM fungi and typical water conservation herbaceous plant measures, can realize the rapid covering of the navel orange garden table surface and the ladder wall, increase the surface roughness, reduce the phosphorus surface source pollution caused by water and soil loss of the navel orange garden, and further achieve the aim of 'source reduction'.

According to the experimental research of people, the demonstration area adopts AM fungi and ecological grass ditches, can reduce erosion sand production by 10-89%, reduce total phosphorus loss by 22-71% and granular phosphorus loss by 23-82%, and can improve the process interception efficiency by using the AM fungi.

The invention investigates the AM fungus condition of the Gannan navel orange garden and finds that the AM fungus diversity in the soil in the garden is extremely low, which is probably related to the development mode of stripping the surface soil of the local navel orange garden, the mode of excessively applying pesticide and fertilizer and the mode of clear tillage management. Therefore, human intervention is urgently needed for the restoration of the AM fungal community in the navel orange garden. According to the invention, an AM fungus propagation system based on a small hole inoculation method is explored, 10 strains of AM fungi are collected, and AM fungus germplasm resources are provided for navel orange garden inoculation.

The invention researches the possibility of applying AM fungus to enhance the grass-growing measure to the rapid protection of the table top and the ladder wall of the navel orange park, finds that the AM fungus can infect typical grass-growing plants under natural conditions, can obviously improve the stem length, the tillering number, the leaf number, the stem node number and the biomass of the overground parts of the white clover, the bermuda grass and the ditch millet, and is beneficial to the rapid coverage of the land surface of the primarily-built navel orange park; the AM fungus can obviously increase the indexes of trefoil, bermuda grass and paspalum root length, root volume, root surface area and the like, and strengthen the holding effect of grass-growing measures on soil.

The invention carries out an AM fungus-grass ditch scouring test and analyzes the contribution of AM fungus to the phosphorus retention of the drainage grass ditch in the navel orange garden. The result shows that the granular phosphorus accounts for 72-90% of the total phosphorus loss, the AM fungus can reduce the runoff power by reducing the flow rate and the runoff depth of the grass furrows, increasing the runoff width and the like, the sediment yield is averagely reduced by 62%, the total phosphorus loss is reduced by more than 50%, and the granular phosphorus loss is reduced by 61%. This shows that AM fungus plays a key role in the process of phosphorus retention in the grassland furrow, and has great application potential. Meanwhile, the use of broad-spectrum bactericides is avoided or reduced in the management of the navel orange park so as to promote the local AM fungi to exert the maximum effect.

The AM fungus inoculation-navel orange phosphorus reduction test carried out by the invention shows that along with the increase of phosphorus application level, the growth of navel oranges shows a trend of increasing firstly and then decreasing, and the optimal phosphorus application amount of the contrast, the F.mosseae inoculation and the R.intraradics inoculation is respectively 25mg/kg, 35mg/kg or 55 mg/kg. The reaction of navel orange growth on mycorrhiza is transited from parasitism to symbiosis along with the increase of phosphorus application level, and chlorophyll data shows that AM fungus improves the adaptability of navel orange to environment which is not suitable for phosphorus. However, from the perspective of growth indexes, the two AM fungi selected in the project fail to exert the effect of reducing phosphate fertilizer application, and AM fungi or artificially synthesized flora more suitable for navel oranges should be searched subsequently.

Based on the reported research and development results, a project group summarizes a phosphorus management system based on AM fungi navel orange garden, mainly comprises key points of orchard grass growing, drainage grass ditches and the like, and a project responsible person teaches a navel orange garden cleaning production management technology for local navel orange farmers in the navel orange garden of the small rural areas in the firm and thick county in Ningdu county, is widely welcomed by local fruit growers, and develops the social benefits of scientific research projects.

The foregoing is merely illustrative of the preferred embodiments of the present invention and is not to be construed as limiting the claims. The present invention is not limited to the above embodiments, and the specific structure thereof is allowed to vary. All changes which come within the scope of the invention as defined by the independent claims are intended to be embraced therein.

Claims

1. A method for reducing non-point source pollution of phosphorus in a navel orange garden based on AM fungi is characterized by comprising the following steps: the method comprises the following steps

2. The method for reducing the non-point source pollution of the phosphorus in the navel orange garden based on the AM fungus in claim 1, wherein the method comprises the following steps: the step (1) specifically comprises

(1.1) sample collection:

(1.2) extracting and detecting total DNA:

extracting total DNA of soil by using the kit, carrying out 0.8% agarose gel electrophoresis for molecular size judgment, and quantifying the DNA by using an ultraviolet spectrophotometer; whether the sample quality is qualified or not and subsequent PCRWhether or not toAmplifying effective target bands;

(1.3) DNA amplification:

after components required by PCR reaction are prepared, performing pre-denaturation on a PCR instrument at 98 ℃ for 30s to ensure that template DNA is fully denatured, and then entering an amplification cycle; in each cycle, the template is denatured by holding at 98 ℃ for 15 seconds, then the temperature is reduced to 50 ℃ and held for 30 seconds, so that the primer and the template are fully annealed; maintaining at 72 deg.C for 30s, extending the primer on the template, and synthesizing DNA to complete one cycle; repeating the cycle 25-27 times to allow the amplified DNA fragments to accumulate in large quantities; finally, keeping the temperature at 72 ℃ for 5min to ensure that the product is completely extended and stored at 4 ℃;

(1.4) building a library;

and (1.5) performing quality inspection and sequencing on the library.

3. The method for reducing the non-point source pollution of the phosphorus in the navel orange garden based on the AM fungus, according to claim 2, is characterized in that: the step (1.4) specifically comprises

a. adding 12.5 mu L A-Tailing Mix into the DNA after fragment selection;

a. adding 2.5 muL of Resuspension buffer, 2.5 muL of Ligation Mix and 2.5 muL of LDNA adapter Index into the system with A;

c. adding 5 mu of LStopLigaptionbuffer;

d. purifying the system with the joint by using BECKMAN AMPure XP beads;

4. The method for reducing the non-point source pollution of the phosphorus in the navel orange garden based on the AM fungus in claim 1, wherein the method comprises the following steps: the step (1.5) specifically comprises

(1.5.1) library quality control and quantitation

(1.5.2), sequencing

(1.5.3), sequence annotation

5. The method for reducing the non-point source pollution of the phosphorus in the navel orange garden based on the AM fungus in claim 1, wherein the method comprises the following steps: the step (2) specifically comprises the steps of uniformly paving the microbial inoculum on the table top and the surface layer of the wall soil of the navel orange garden, disinfecting seeds for 2min by using 75% alcohol, and then washing the seeds by using distilled water and sowing the seeds.

6. The method for reducing the non-point source pollution of the phosphorus in the navel orange garden based on the AM fungus in claim 1, wherein the method comprises the following steps: and the step (3) specifically comprises the steps of opening a grass ditch in the navel orange garden, planting herbaceous plants in the grass ditch, and inoculating AM fungi.