A method for promoting growth of elymus plants and soil improvement by using composite strains

Abstract

The application discloses a method for promoting growth of plants of the genus Puccinellia and soil improvement by using composite bacteria, and the method mainly inoculates composite bacterial manure composed of arbuscular mycorrhizal fungi, rhizobium and bacillus, so that the total biomass, plant height and stem diameter of Puccinellia distans are significantly improved, and soil quality and structure are improved. The composite bacterial manure can not only reduce overuse of traditional chemical fertilizer, improve the use efficiency of the chemical fertilizer, reduce the agricultural production cost, but also relieve ecological environment pollution, maintain the balance of soil rhizosphere microorganisms, increase the nitrogen content of the soil, promote soil nutrient circulation, improve the agricultural ecological environment, and has great significance for sustainable development of green agriculture and animal husbandry in China and maintenance of the health of the agricultural ecological system.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of ecological restoration, in particular, the field of degraded grassland restoration, and specifically provides a method for promoting the growth of plants of the genus Puccinellia and soil improvement by using a complex microbial strain. BACKGROUND

[0002] In recent years, due to intensified human interference activities and global climate change, the terrestrial ecosystems in China are in a state of degradation, and the ecological service functions are declining. The natural ecological environment of the alpine meadow on the Qinghai-Tibet Plateau is fragile, overgrazing and frequent occurrence of rodent and insect pests, which have made the degradation trend of the alpine meadow increasingly intensified. The plants of the genus Puccinellia, as the main perennial herbaceous forage grass, are mainly distributed in the meadow, grassland and river beach areas of the Qinghai-Tibet Plateau, and are not only the excellent forage grass resources in the alpine region, but also the excellent grass species for artificial grassland construction and vegetation restoration, which play an important role in the ecological construction and livestock production in the alpine region. Elymus

[0003] At present, the main measure for soil nutrient improvement of artificial grassland is fertilization, and the components are mainly nitrogen fertilizer and phosphorus fertilizer. Large-scale application has led to soil fertility decline, soil and water pollution, and decline of rhizosphere microbial flora. The use of beneficial microorganisms to regulate plant growth and development has become the focus of research in agriculture, ecology and environment, and the development of new fertilizers to replace chemical fertilizers has become an urgent need for the development of agriculture and animal husbandry. In recent years, microbial fertilizers have attracted widespread attention due to their environmental friendliness and promotion of plant growth. At present, microbial fertilizers are developing from single microbial fertilizer to complex microbial fertilizer. The development and production of complex microbial fertilizer by using multiple microorganisms will have great significance for the development of green agriculture and animal husbandry in China.

[0004] Because multiple microorganisms often coexist in nature and affect plant growth and development, the synergistic or antagonistic effects between different microorganisms will affect the interaction between them and plants. Single inoculation lacks functional diversity, and double inoculation often cannot reflect the comprehensive effect of multiple microorganisms on plants. Therefore, the present application provides a method for promoting plant growth and soil improvement by using complex microbial fertilizer to solve the above problems. SUMMARY

[0005] To solve the above problems, the applicant has developed a method for promoting the growth of plants of the genus Puccinellia and improving the soil by using arbuscular mycorrhizal fungi, Bacillus and rhizobium.

[0006] In one aspect, the present application provides a method for promoting the growth of plants of the genus Puccinellia and soil improvement by using a complex microbial strain, which comprises using a microbial agent comprising one or more selected from AMF, Bacillus and rhizobium during the planting of the plants of the genus Puccinellia.

[0007] ​Further, the method comprises adding a composite microbial agent composed of AMF fungus soil, bacillus liquid and rhizobium liquid into the soil in which the Puccinellia plant is planted.

[0008] Further, the Puccinellia plant is Puccinellia distans.

[0009] Further, the AMF fungus is Glomus mosseae. Funneliformis mosseae ).

[0010] Further, the bacillus is Bacillus megaterium. Bacillus megaterium ).

[0011] Further, the rhizobium is Mesorhizobium intermedium. Mesorhizobium ).

[0012] Further, in the composite microbial agent composed of AMF fungus soil, bacillus liquid and rhizobium liquid, the ratio of AMF fungus soil, bacillus liquid and rhizobium liquid is 3:2:2 g:mL:mL.

[0013] Further, the AMF fungus soil contains 129 spores of Glomus mosseae per gram of AMF fungus soil, the bacillus liquid contains 1×10 8 CFU / mL of Bacillus megaterium, and the rhizobium liquid contains 4.30×10 8 CFU / mL of Mesorhizobium intermedium.

[0014] Further, the method comprises:

[0015] Step one: selecting Puccinellia plant seeds that are uniform in size, healthy and full, and free of diseases and pests;

[0016] Step two: sterilizing and disinfecting the selected seeds;

[0017] Step three: sterilizing and disinfecting the original soil and river sand as the substrate material for seed germination;

[0018] Step four: placing the Puccinellia plant seeds treated in step two evenly in the sterilized seedling box for seed germination, each hole containing sterilized soil, to obtain Puccinellia plant seedlings. During the seed germination process, water is supplemented once every 1-3 days.

[0019] Step 5: After the seedlings have grown for 15 days, transplant them into sterilized flowerpots: Remove each seedling from the seedling tray and add compound microbial agent to 4 / 5 of the soil in the flowerpot. Then cover the surface of the microbial agent with 200g of mixed soil. Finally, transplant them into flowerpots containing 3kg of mixed substrate. Place all flowerpots in a smart greenhouse for growth and cultivation, and randomly change the position of the flowerpots every 3 weeks. During the seedling growth process, water them every 2-3 days according to their specific conditions. At the same time, regularly check the growth status of the seedlings within one week of transplanting. If any seedlings die, replace them with healthy Leymus chinensis seedlings in time.

[0020] The sterilization and disinfection process involves soaking the seeds of *Leymus* in an 8% hydrogen peroxide (H2O2) solution for 10-20 minutes and rinsing them repeatedly with deionized water 3-5 times. The sterilization and disinfection of the native soil and river sand involves passing the native soil through a 2mm sieve and the river sand through a 3mm sieve. Both are then sterilized in a high-temperature, high-pressure steam sterilizer at 121℃ for 2 hours. After cooling, the two are mixed evenly and spread in flowerpots with a diameter of 15cm.

[0021] In step five, select seedlings of the genus Leymus that are growing uniformly, in good health, and in equal numbers, and transplant them into flowerpots. A total of 45 seedlings are selected and transplanted into each flowerpot.

[0022] In step five, the filtered soil and river sand are mixed evenly at a volume ratio of 2:1 and then spread in the flowerpot.

[0023] On the other hand, this application provides an application of a compound microbial agent in the remediation or improvement of degraded grassland soil. In this application, after adding the compound microbial agent to the degraded grassland soil, plants of the genus Leymus are planted. The compound microbial agent includes AMF bacteria, Bacillus, and Rhizobium.

[0024] Furthermore, the compound microbial agent is composed of *Tetranychus mosierifolius* (…). Funneliformis mosseae ), Bacillus megaterium ( Bacillus megaterium ) bacterial solution and intermediate rhizobia ( Mesorhizobium The bacterial solution composition; the alkali grass plant is *Elymus chinensis*.

[0025] Furthermore, the compound microbial agent contains *Morchella mosierifolia* (… Funneliformis mosseae ), Bacillus megaterium ( Bacillus megaterium ) bacterial solution and intermediate rhizobia ( Mesorhizobium The ratio of bacterial solution is 3:2:2 g:mL:mL.

[0026] The bacterial strains, sources, and amplification methods used in the examples are described below. Subsequent verification showed that similar results could be achieved by using other strains of the same bacterial species from other sources.

[0027] Arbuscular mycorrhizal fungi strains were obtained by the following method: *Morchella mosierifolia* ( Funneliformis mosseae The strain originated from the Beijing Academy of Agricultural and Forestry Sciences and was propagated by the Guizhou Academy of Agricultural Sciences. Each gram of inoculum contains approximately 129 spores / g. The strain number is BGCYN05, and the National Natural Science and Technology Resource Platform number is 1511C0001BGCAM0013.

[0028] Bacillus inoculum was obtained by the following method: *Bacillus megaterium* was propagated using beef extract peptone medium, and its growth curve was determined. *Bacillus megaterium* (… Bacillus megaterium The strain Bary was provided by the China Agricultural Microbial Culture Collection Center, strain number ACCC10011. The formula for beef extract peptone medium is as follows: 10.0 g peptone, 3.0 g beef extract, 5.0 g NaCl, 1000 mL distilled water, pH 7.0, and 18.0-20.0 g agar added to the solid medium. The medium was then autoclaved at 121℃ for 30 min. Two loops of inoculum were scraped from the slant culture medium and transferred to 30 mL of liquid beef extract peptone medium. The culture was incubated on a shaker at 28°C and 220 rpm for 24 h to obtain a seed culture. 0.5 mL of this seed culture was transferred to a 50 mL Erlenmeyer flask containing 30 mL of liquid beef extract peptone medium and incubated on a shaker at 28°C and 220 rpm. Samples were taken periodically, using uninoculated liquid medium as a control. Each sample was measured in triplicate (with distilled water absorbance as 0). The absorbance of the bacterial culture was measured at 600 nm using a spectrophotometer to plot the growth curve of *Bacillus megaterium*. The culture in the logarithmic growth phase was then inoculated to produce a final product of 1×10⁻⁶ cells / mL. 8 CFU / mL bacterial suspension.

[0029] Rhizobium inoculum was obtained by the following method: Rhizobium was propagated using YMA medium, and its growth curve was measured. Intermediate rhizobium ( Mesorhizobium) was provided by China Agricultural University Culture Collection Center, and the strain number was CCBAU13029. The YMA medium formula was as follows: mannitol 10.0 g, K2HPO4 0.25 g, KH2PO4 0.25 g, MgSO4·7H2O 0.20 g, NaCl 0.10 g, yeast powder 3.0 g, distilled water 1000 mL, pH 6.8-7.0, and 15.0 g of agar was added to the solid medium, and high-pressure steam sterilization was carried out at 121 DEG C for 30 min. Two single colonies were scraped from the plate medium with an inoculation ring into 30 mL of liquid YMA medium, and seed liquid was obtained by culturing at 28 DEG C and 220 rmp for 44 h. 0.5 mL of the seed liquid was taken into a 50 mL conical flask containing 30 mL of liquid YMA medium, and the culture was carried out at 28 DEG C and 220 rmp, and the sample was taken every 4 h, and each time the sample was measured 3 times, and the uninfected liquid medium was used as a control (the absorbance of distilled water was 0), and the absorbance of the bacterial liquid was measured at a wavelength of 600 nm by using a spectrophotometer, and the growth curve of rhizobium was drawn, and the bacterial liquid in the logarithmic growth phase was inoculated, and finally 4.30 x 10 8 CFU / mL bacterial suspension.

[0030] The beneficial effects of the present application are that the AMF, rhizobium and bacillus (plant rhizosphere growth promoting bacteria) in the method used by the present application can exist in the plant rhizosphere at the same time, have obvious synergies, and do not have adverse effects on each other. The plant hormones such as indole acetic acid and abscisic acid secreted by the microorganisms are beneficial to plant growth. In addition to symbiotic nitrogen fixation with leguminous plants to form root nodules, the rhizobium can also coexist with non-leguminous plants and form rhizobium-like nodules with them, improve the absorption efficiency of nitrogen elements of plants, and promote the growth of non-leguminous plants. When the AMF, rhizobium and bacillus are inoculated together, the rhizosphere microbial community is changed, the richness and diversity of the soil fungal community are increased, the relative abundance of the symbiotic nutrition type is increased, the relative abundance of the pathogenic nutrition type is reduced, the original microbial ecological environment is optimized, and the plant quality and yield are improved. At the same time, the AMF uses the widely distributed extraradical hyphae to obtain nutrients from the soil, and the carbon produced by plant photosynthesis is also transferred to the soil microenvironment through this pathway, and these hyphal networks provide a suitable survival interface for the heterotrophic bacteria in the soil to benefit from the hyphal networks, further promoting the sustainable development of the environment. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Effects of inoculation of different strains on aboveground biomass and underground biomass;

[0032] Figure 2 Effects of inoculation of different strains on root crown ratio and total biomass;

[0033] Figure 3 Effect of different inoculated strains on stem diameter and plant height;

[0034] Figure 4 Effect of different inoculated strains on soil pH and electrical conductivity;

[0035] Figure 5 Effect of different inoculated strains on soil available nitrogen;

[0036] Figure 6 Effect of different inoculation ratios on soil electrical conductivity and pH;

[0037] Figure 7 Effect of different inoculation ratios on soil total nitrogen and total carbon;

[0038] Figure 8 Effect of different inoculation ratios on total biomass of plants;

[0039] Figure 9 Effect of different inoculation ratios on aboveground and underground biomass of plants;

[0040] Figure 10 Effect of different inoculation ratios on total nitrogen and total carbon of plants. DETAILED DESCRIPTION

[0041] The following examples facilitate a better understanding of the present application, but are not limited thereto, and are only for illustrative purposes, and in no way limit the protection scope of the present application.

[0042] Example 1 Basic operation steps

[0043] Step one: select uniform size, healthy and full, no disease and insect pests of Elymus nutans seeds;

[0044] Step two: sterilization and disinfection treatment of selected seeds;

[0045] Step three: sterilization and disinfection of the original soil taken from Haibei Tibetan Autonomous Prefecture of Qinghai Province and river sand as seed germination matrix material;

[0046] Step four: place the treated Elymus nutans seeds in step two evenly in the sterilized seedling box for seed germination, each hole is filled with sterilized soil, and Elymus nutans seedlings are obtained. During the seed germination process, water is supplemented once every 1-3 days;

[0047] Step five: After the seedlings grow for about 15 days, the seedlings are transplanted into sterilized plastic flowerpots. Specifically, a single seedling is taken out of the seedling box, and a bacterial strain is added to 4 / 5 of the soil in the flowerpot, and then 200 g of mixed soil is covered on the surface of the bacterial agent. All the flowerpots are placed in an intelligent greenhouse for growth culture (12g / 12h (L / D), 25±1℃, w: 25%), and the flowerpot positions are randomly changed every 3 weeks. During the growth of the seedlings, water is poured every 2-3 days according to the specific situation, and the growth status of the seedlings is regularly detected during the first week after transplantation. Once the seedlings die, they are replaced with healthy Elymus nutans seedlings in time. The samples are subjected to the following tests:

[0048] (1) After the plants grow for 70 days, the samples are dried in an oven at 65℃ for 72 h to constant weight, and the dry weights of the aboveground and underground parts are measured by an electronic balance.

[0049] (2) After the plants grow for 70 days, 3 complete root systems are randomly taken and stored in a 4℃ refrigerator for determination of root morphology. The root system is completely unfolded with tweezers, scanned with a root system scanner (EPSON PERFECTION V700 PHOTO), and analyzed with analysis software WinRHIZO Pro to determine the root-shoot ratio.

[0050] (3) After the plants grow for 70 days, the thickest part of the root stem is measured with a vernier caliper to determine the stem thickness, and the height from the root neck to the highest point of plant growth is measured with a tape measure to determine the plant height.

[0051] (4) After the plants grow for 70 days, the soil is dried, impurities are removed, and then 10 g of soil is weighed, 25 mL of deionized water is added, and after shaking, the sample is shaken at room temperature (HY-4A digital multi-purpose speed oscillator) for 30 min, and after standing for 1 h, the supernatant of the sample solution is measured with a pH conductivity meter to determine the pH and conductivity.

[0052] (5) After the plants grow for 70 days, 10 g (accurate to 0.01) of fresh soil sample passing through a 2 mm sieve is weighed in a 250 mL conical flask, 100 mL of 1 mol / L potassium chloride is added, the flask is tightly plugged, and the shaking speed is set to 140 rpm on a shaking table (HY-4A digital multi-purpose speed oscillator) at room temperature for 1 h. The filtrate is filtered with qualitative filter paper, and the soil ammonium nitrogen and nitrate nitrogen are determined within 24 h using an AA3 type continuous flow analyzer.

[0053] (6) After the plants grow for 70 days, the soil sample is crushed with a ball mill (Retsch MM400), and the total nitrogen and total carbon of the soil are determined with an elemental analyzer (Germany, Elementar Vario MICRO CUBE).

[0054] Experimental results and analysis of Example 2

[0055] The inoculation of the bacteria includes:

[0056] No inoculation control group (CK);

[0057] AMF inoculation treatment group (AMF): 30g AMF bacteria;

[0058] Rhizobium inoculation treatment group (R): 20mL rhizobium bacteria solution;

[0059] Bacillus inoculation treatment group (B): 20ml bacillus bacteria solution;

[0060] AMF and bacillus inoculation treatment group (A+B): 30g AMF bacteria + 20ml bacillus bacteria solution;

[0061] AMF and rhizobium inoculation treatment group (A+R): 30g AMF bacteria + 20mL rhizobium bacteria solution;

[0062] Bacillus and rhizobium inoculation treatment group (B+R): 20mL rhizobium bacteria solution + 20ml bacillus bacteria solution;

[0063] AMF, rhizobium and bacillus inoculation treatment group (A+B+R): 30g AMF bacteria + 20ml bacillus bacteria solution + 20mL rhizobium bacteria solution.

[0064] Figure 1 Effects of the above 8 methods on aboveground biomass and underground biomass; Figure 2 Effects of the above 8 methods on root-shoot ratio and total biomass; Figure 3 Effects of the above 8 methods on stem diameter and plant height; Figure 4 Effects of the above 8 methods on soil pH and conductivity; Figure 5 Effects of the above 8 methods on soil available nitrogen.

[0065] By Figures 1-5It can be seen that the co-inoculation of A+B+R significantly increased the aboveground biomass and underground biomass of Elymus nutans and the root-shoot ratio, and effectively increased the plant height and stem diameter. The co-inoculation of A+B+R significantly reduced the soil electrical conductivity and increased the soil pH, increased the soil available nitrogen content, and effectively improved the soil quality for plant growth. The above results show that the interaction between different strains can promote plant growth and development and improve soil quality. Soil microbial activity participates in the nitrogen cycle process through nitrification and ammonification, improves the effectiveness of soil nitrogen utilization, and improves soil structure by producing glomalin to bind soil particles and form soil aggregates, thereby having a positive effect on soil structural properties and fertility. At the same time, the synergistic effect of microorganisms not only affects the soil microbial community structure and function, but also controls plant pathogens in the soil, and improves plant nutrition by producing growth regulators and / or by supplying and promoting soil nutrient absorption, thereby improving soil production efficiency and promoting plant growth.

[0066] Inoculation methods of different strain ratios:

[0067] Inoculation of AMF, rhizobium and bacillus in a ratio of 3:2:2 (CK): 30g AMF strain + 20ml bacillus bacterial solution + 20mL rhizobium bacterial solution;

[0068] Inoculation of AMF, rhizobium and bacillus in a ratio of 1:1:1 (A): 20g AMF strain + 20ml bacillus bacterial solution + 20mL rhizobium bacterial solution;

[0069] Inoculation of AMF, rhizobium and bacillus in a ratio of 3:4:4 (B): 30g AMF strain + 40ml bacillus bacterial solution + 40mL rhizobium bacterial solution;

[0070] Inoculation of AMF, rhizobium and bacillus in a ratio of 3:1:1 (C): 30g AMF strain + 10ml bacillus bacterial solution + 10mL rhizobium bacterial solution.

[0071] Figure 6 Effects of the above four methods on soil electrical conductivity and pH; Figure 7 Effects of the above four methods on soil total nitrogen and total carbon; Figure 8 Effects of the above four methods on plant total biomass; Figure 9 Effects of the above four methods on plant aboveground and underground biomass; Figure 10 Effects of the above four methods on plant total nitrogen and total carbon.

[0072] From the above results, it can be seen that the co-inoculation of A+B+R significantly increased the aboveground biomass and underground biomass of Elymus nutans and the root-shoot ratio, and effectively increased the plant height and stem diameter. The co-inoculation of A+B+R significantly reduced the soil electrical conductivity and increased the soil pH, increased the soil available nitrogen content, and effectively improved the soil quality for plant growth. The above results show that the interaction between different strains can promote plant growth and development and improve soil quality. Soil microbial activity participates in the nitrogen cycle process through nitrification and ammonification, improves the effectiveness of soil nitrogen utilization, and improves soil structure by producing glomalin to bind soil particles and form soil aggregates, thereby having a positive effect on soil structural properties and fertility. At the same time, the synergistic effect of microorganisms not only affects the soil microbial community structure and function, but also controls plant pathogens in the soil, and improves plant nutrition by producing growth regulators and / or by supplying and promoting soil nutrient absorption, thereby improving soil production efficiency and promoting plant growth. Figures 6-10The results showed that the inoculation of AMF, rhizobium and bacillus in the ratio of 3:2:2 significantly increased the soil pH and reduced the conductivity, promoted the accumulation of aboveground and underground biomass and total biomass, and increased the total carbon and nitrogen content in soil and plants. The above results showed that the effects of different ratios of bacteria were different. Due to the different types, characteristics and functions of microorganisms, and the limitation of host plants, climate, soil type and indigenous microorganisms, the best ratio of fungal symbiont and root bacteria strains was used to achieve the purpose of plant actively absorbing the surrounding and adjacent environmental nutrient elements. Therefore, the combined inoculation of AMF, rhizobium and bacillus in the best ratio of 3:2:2 could form a combination to promote plant growth, which could maximize the relief of the plight of artificial grassland soil nutrient deficiency and promote the sustainable and green production of Elymus nutans.

Claims

1. A method for promoting growth of Elymus plants and soil improvement by using a complex microbial inoculant, characterized in that, The method comprises adding a composite microbial agent consisting of AMF fungus soil, bacillus liquid and rhizobium liquid into soil in which a plant of the Puccinellia genus is planted; the AMF fungus is Glomus mosseae, Funneliformis mosseae the bacillus is Bacillus megaterium, Bacillus megaterium the rhizobium is Mesorhizobium intermedium, Mesorhizobium the ratio of the AMF fungus soil, the bacillus liquid and the rhizobium liquid in the composite microbial agent is 3:2:2 g:mL:mL; the AMF fungus soil contains 129 spores / g, the bacillus liquid is a 1×10 8 CFU / mL bacterial suspension, and the rhizobium liquid is a 4.30×10 8 CFU / mL bacterial suspension; and the plant of the Puccinellia genus is Puccinellia distans.

2. Use of a complex microbial agent for the restoration or improvement of degraded grassland soils, characterized in that, The application plants the Puccinellia plant in the degenerated grassland soil after adding the compound microbial agent in the application, the compound microbial agent is a compound microbial agent composed of AMF fungus soil, bacillus bacterial liquid and rhizobium bacterial liquid; the AMF fungus is Glomus mosseae ( Funneliformis mosseae ), the bacillus is Bacillus megaterium ( Bacillus megaterium ), and the rhizobium is Mesorhizobium intermedium ( Mesorhizobium ); the ratio of the AMF fungus soil, the bacillus bacterial liquid and the rhizobium bacterial liquid in the compound microbial agent is 3:2:2 g:mL:mL; the AMF fungus soil contains 129 spores / g, the bacillus bacterial liquid is a bacterial suspension of 1×10 8 CFU / mL, and the rhizobium bacterial liquid is a bacterial suspension of 4.30×10 8 CFU / mL; and the Puccinellia plant is Puccinellia distans.

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