Composite combined inoculation method using arbuscular mycorrhizal fungi and plant growth-promoting rhizobacteria

Abstract

The invention relates to the technical field of agricultural micro-ecological application, and particularly discloses a composite combined inoculation method using arbuscular mycorrhizal fungi and plant growth-promoting rhizobacteria, which comprises the following steps: selecting corn plant seeds without plant diseases and insect pests; a corn planting land is randomly divided into land parcels with the area of 6 m < 2 > (2 m * 3 m), a buffer zone of 1.0 m is arranged between every two adjacent land parcels, and the cultivation mode is flat cropping; the method comprises the following steps: planting arbuscular mycorrhizal fungi inoculum comprising mycorrhiza, dry sand containing spores and ectotrophic hyphae and corn seeds in soil by using a seeder during corn sowing, and after the corn is planted for 30 days, inoculating the plant growth-promoting rhizobacteria inoculum into plants and soil by using a flood irrigation root irrigation method, when the arbuscular mycorrhizal fungi and the plant growth-promoting rhizobacteria are inoculated together, the corn rhizosphere microbial community can be changed, the richness and diversity of fungi and bacterial communities in soil are increased, and the relative abundance of plant pathogens in the soil is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural microecological applications, and particularly relates to a composite combined inoculation method using arbuscular mycorrhizal fungi and plant growth-promoting rhizobacteria. Background Art

[0002] Since the 20th century, in order to increase food production, a large amount of chemical fertilizers has been applied globally. As a major chemical fertilizer producer in China, the application amount of chemical fertilizers accounts for about 1 / 3 of the world's total, and chemical fertilizers contribute more than 40% to the increase in grain production in China. The excessive and continuous application of chemical fertilizers has long led to the weakening of the crop yield increase effect of chemical fertilizers, and the overall low utilization rate of chemical fertilizers. For example, the average utilization rates of nitrogen, phosphorus, and potassium fertilizers for the three major food crops in the current season are only 33%, 24%, and 42% respectively, and the nitrogen fertilizer productivity in many places is less than 30 kg·kg-1.

[0003] At the same time, the large amount and low efficiency of chemical fertilizer application also bring a series of environmental problems. The low utilization rate of chemical fertilizers causes a large amount of nutrients to be lost in various forms or remain in the soil, resulting in a decline in soil fertility, damage to soil structure, soil acidification, hardening, and a decrease in biodiversity. A large amount of nitrogen is lost in the form of nitrates through rainwater runoff and other means, exacerbating the emissions of ammonia and greenhouse gases, damaging the ozone layer, and continuously emerging agricultural non-point source pollution problems such as excessive nitrates in groundwater and eutrophication of water bodies.

[0004] As a substitute for traditional chemical fertilizers, microbial fertilizers have become the fertilizers with the largest annual output and application area among new fertilizers in China, and have become an important part of the green and sustainable agricultural system.

[0005] Since it is often the combined action of multiple microorganisms in nature that affects plant growth and development, the synergistic or antagonistic effects existing between different microorganisms will affect their interaction with plants, and single inoculation lacks functional diversity. Therefore, those skilled in the art have provided a method for jointly improving maize growth and soil improvement by using arbuscular mycorrhizal fungi and plant growth-promoting rhizobacteria. Summary of the Invention

[0006] The purpose of the present invention is to provide a composite combined inoculation method using arbuscular mycorrhizal fungi and plant growth-promoting rhizobacteria to solve the problems raised in the above background art.

[0007] To achieve the above purpose, the present invention provides the following technical solutions:

[0008] A composite combined inoculation method using arbuscular mycorrhizal fungi and plant growth-promoting rhizobacteria, comprising the following steps:

[0009] Step 1: Select maize plant seeds without pests and diseases;

[0010] Step 2: Randomly divide the maize planting land into plots with an area of 6 m2 (2m × 3m) plots with a 1.0-meter buffer zone between adjacent plots, and the tillage method is flat cultivation;

[0011] Step 3: When sowing corn, use a seeder to plant the arbuscular mycorrhizal fungal inoculum containing mycorrhiza, dry sand with spores, and extraradical mycelium together with corn seeds into the soil. The corn sowing method is hill-drop sowing, with a plant spacing of 40 cm and a planting density of 3,900 plants per mu. -1 When sowing, 3 corn seeds are planted in each hill.

[0012] Step 4: 30 days after planting the corn, use the method of flooding irrigation to inoculate the plant growth-promoting rhizobacteria agent into the plants and the soil;

[0013] The selected strain of the arbuscular mycorrhizal fungus with extraradical mycelium is Acaulospora laevis. It is propagated by potting at room temperature with corn as the host plant in a sterilized pure sand substrate for 90 days before inoculation and stored in a polyethylene bag at 4°C before use.

[0014] The plant growth-promoting rhizobacteria agent is Bacillus licheniformis, and the strain number is GSICC 30203.

[0015] The bacterial isolate is cultured in 50 mL of tryptic soy broth at 30°C and 120 rpm for 24 h and stored at 4°C to obtain it.

[0016] Preferably, the tryptic soy broth is composed of: tryptone: 17.0 g / L, soy peptone: 3.0 g / L, sodium chloride: 5.0 g / L, dipotassium hydrogen phosphate: 2.5 g / L, glucose: 2.5 g / L.

[0017] Preferably, the average infection rate of the arbuscular mycorrhizal fungus in the mycorrhiza is 55.3%, and each 10 g of the inoculum in the inoculated dry sand contains 65 + 17 spores.

[0018] Preferably, the physical and chemical properties of the soil are pH = 6.28, EC = 467 S·m -1 、AP = 2.63 mg·kg -1 、TC = 12.04 g·kg -1 、TN = 1.62 g·kg -1 、C / N = 7.42.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] 1. In the method used in the present invention, arbuscular mycorrhizal fungi and plant growth-promoting rhizobacteria can coexist in the rhizosphere of plants, having an obvious synergistic effect and not having any adverse effects on each other.

[0021] 2. Whether the selected arbuscular mycorrhizal fungi and plant growth-promoting rhizobacteria are inoculated alone or jointly, they can effectively promote the growth of maize. The plant yield, biomass of each part, and total biomass are all significantly higher than those of the non-inoculated control group. Among all the inoculation treatments, the combined inoculation of arbuscular mycorrhizal fungi and plant growth-promoting rhizobacteria has a significantly higher improvement effect on plants than single inoculation.

[0022] 3. When arbuscular mycorrhizal fungi and plant growth-promoting rhizobacteria are jointly inoculated, they can change the microbial community in the maize rhizosphere, increase the richness and diversity of the fungal and bacterial communities in the soil, increase the richness of the eubacterial network, reduce the relative abundance of plant pathogens in the soil, optimize the original soil and microbial ecological environment, and are beneficial to improving plant quality and yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Effect of different inoculation methods of the present invention on the total plant yield;

[0024] Figure 2 Effect of different inoculation methods of the present invention on the available phosphorus in the soil;

[0025] Figure 3 Effect of different inoculation methods of the present invention on the total organic carbon in the soil;

[0026] Figure 4 Effect of different inoculation methods of the present invention on the total nitrogen in the soil;

[0027] Figure 5 Effect of different inoculation methods of the present invention on the soil microorganisms;

[0028] Figure 6 Effect of different inoculation methods of the present invention on the plant pathogens in the soil. DETAILED DESCRIPTION OF THE INVENTION

[0029] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0030] Example 1:

[0031] 1. Select maize plant seeds that are uniform in size, healthy, plump, and free from diseases and pests. Randomly divide the maize planting area into plots with an area of 6 m2 (2 m × 3 m) for each plot, with a 1.0-meter buffer zone between adjacent plots, and the tillage method is flat cultivation.

[0032] 2. The selected arbuscular mycorrhizal fungi were propagated in a sterilized pure sand substrate at room temperature for 90 days with maize (Zea mays L.) as the host plant before inoculation. The average mycorrhizal infection rate of the final inoculum was 55.3%. Each 10 g of the inoculum in the dry sand contained 65 + 17 spores and was stored in a polyethylene bag at 4 °C before use.

[0033] 3. The selected plant growth-promoting rhizobacteria isolates were cultured in 50 mL of tryptic soy broth at 30 °C and 120 rpm for 24 h and stored at 4 °C.

[0034] 4. At the time of maize sowing, a seeder was used to plant the arbuscular mycorrhizal fungi inoculum, including mycorrhizae, dry sand containing spores, and extraradical hyphae, together with maize seeds into the soil. The maize was sown in holes with a plant spacing of 40 cm and a planting density of approximately 3900 plants·mu-1. Three maize seeds were planted in each hole during sowing.

[0035] 5. Thirty days after maize planting, the plant growth-promoting rhizobacteria were evenly inoculated into the plants and the soil using the flooding irrigation method for root drenching.

[0036] The following tests were conducted on the samples:

[0037] 1. After 120 days of maize growth, the ears of all plants in each plot were collected, manually shelled, and air-dried to determine the grain yield of the plot.

[0038] 2. After 120 days of maize growth, plants with uniform growth vigor were selected for sampling at the edge rows and inner rows in the plot. Three plants were sampled from each plot. When sampling, the maize was disassembled into five parts: roots, stems, leaves, ears, and husks. The aboveground biomass of the plants was estimated, and the biomass of the roots was measured using the corresponding roots. The aboveground biomass was disassembled into four parts: stems, leaves, husks, and ears. A soil column (40 cm deep × 35 cm long × 30 cm wide) was dug around each maize root, and then the roots were collected and carefully washed. After the samples were blanched in an oven at 105 °C for 30 minutes, they were further dried in the oven at 80 °C for at least 24 hours until constant weight and weighed using an electronic balance to measure the biomass of different parts.

[0039] 3. After 120 days of maize growth, the maize ears were air-dried, and then the length and width of the ears were measured using a vernier caliper. One hundred grains of grains were randomly selected from the grains manually shelled from the ears. When selecting, grains with significant damage or lesions were avoided, and the weight was measured using an electronic balance.

[0040] 4. The pH value of the soil was determined by potentiometry. Weigh 10.0 g of air-dried soil passing through a 2-mm sieve pore into a 50-mL beaker, add 25 mL of CO₂-free distilled water (the soil-water ratio is 2.5:1), stir with a glass rod for about 1 min to fully disperse the soil, then let it stand for 30 min. During this process, avoid the influence of CO₂, ammonia, or volatile acids in the environment, and measure the pH value of the solution with a pH meter (PHS-3C, INESA, Shanghai).

[0041] 5. The electrical conductivity of the soil was determined by a conductivity meter. Weigh 10 g of soil sample with a diameter of 2 mm, place it in a 50-ml beaker, add 50 ml of distilled water, stir well with a glass rod, and after standing and clarifying, measure the reading with a conductivity meter (DDS-307, INESA, Shanghai).

[0042] 6. Weigh 2.50 g of the sample, place it in a dry 50-ml extraction bottle, add an appropriate amount of phosphorus-free activated carbon, add 50 ml of the extractant, stopper it, place it on a constant-temperature reciprocating oscillator, and oscillate at a frequency of 200 - 220 r / min at 25 ± 1 °C for 30 ± 1 min. Immediately filter it with double-layer phosphorus-free filter paper. Discard the initial filtrate, take 10 ml from the filtrate, slowly add 0.8 ml of (1 + 1) hydrochloric acid solution, and after completely removing the bubbles, perform on-machine detection and analysis with an automatic discrete chemical analyzer.

[0043] 7. Soil organic carbon and total nitrogen were determined using a TOC / TNb analyzer. Grind the sample through a 100-mesh (diameter 0.15 mm) sieve, weigh 20 - 30 mg of the treated sample, wrap the sample with tin foil, and then put it into a TOC / TNb analyzer (vario TOC select, elementar, German) for determination.

[0044] Among them Figure 1 : The influence of different inoculation methods on the total plant yield: The abscissa shows several inoculation treatment methods including CK (control group, without inoculating the inoculant), AMF (inoculating arbuscular mycorrhizal fungi), PGPR (inoculating plant growth-promoting rhizobacteria), and AMF + PGPR (co-inoculating arbuscular mycorrhizal fungi and plant growth-promoting rhizobacteria). The yields of AMF, PGPR inoculated alone, and AMF + PGPR co-inoculated are all higher than those of the CK control group, and the yield increase effect is most significant when AMF + PGPR are co-inoculated, indicating that combined inoculation has obvious advantages in increasing the yield of maize.

[0045] Figure 2: Effects of different inoculation methods on soil available phosphorus: The abscissa is also various inoculation treatment methods, and the ordinate is the content of soil available phosphorus. It can be seen from the figure that there are differences in the content of soil available phosphorus under different inoculation methods. The treatments inoculated with microbial agents (AMF, PGPR, AMF + PGPR) have different degrees of effects on changing the availability of phosphorus in the soil compared with the control group CK.

[0046] Figure 3 : Effects of different inoculation methods on soil total organic carbon: The abscissa is the inoculation treatment, and the ordinate is the content of soil total organic carbon.

[0047] Figure 4 : Effects of different inoculation methods on soil total nitrogen: The abscissa is various inoculation treatments, and the ordinate is the content of soil total nitrogen. The changes in the content of soil total nitrogen under different inoculation treatments are shown.

[0048] Figure 5 : Effects of different inoculation methods on microbial diversity: The abscissa is various inoculation treatments, and the ordinate is the microbial Alpha diversity index.

[0049] Figure 6 : Effects of different inoculation methods on plant pathogens in the soil: The abscissa is various inoculation treatments, and the ordinate is the functional prediction of plant pathogens in the soil.

[0050] Table 1

[0051]

[0052]

[0053] It should be understood that in the development process of any actual implementation, such as in any engineering or design project, a large number of specific implementation decisions can be made. Such development efforts may be complex and time-consuming, but for those of ordinary skill in the art who benefit from this disclosure, without excessive experimentation, such development efforts will be a routine task of design, manufacturing, and production.

[0054] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A composite combined inoculation method using arbuscular mycorrhizal fungi and plant growth-promoting rhizobacteria, characterized in that, Including the following steps: Step 1: Select corn plant seeds without pests and diseases; Step 2: Randomly divide the corn planting area into plots with an area of 6 m 2 (2 m × 3 m), with a 1.0-meter buffer zone between adjacent plots, and the tillage method is flat tillage; Step 3: When sowing corn, use a seeder to plant the arbuscular mycorrhizal fungal inoculum containing mycorrhizae, dry sand with spores, and extraradical hyphae together with corn seeds in the soil. The corn sowing method is hill-drop sowing, with a plant spacing of 40 cm and a planting density of 3,900 plants per mu. -1 When sowing, plant 3 corn seeds in each hole. Step 4: 30 days after corn planting, use the flooding method of root irrigation to inoculate the plant growth-promoting rhizobacteria agent into the plants and soil; The selected strain of arbuscular mycorrhizal fungi of the extraradical mycelium is Acaulospora laevis. It is propagated in a sterilized pure sand substrate with corn as the host plant at room temperature for 90 days before inoculation and stored in a polyethylene bag at 4°C before use: The plant growth-promoting rhizobacteria agent is Bacillus licheniformis, and the strain number is GSICC 30203. The bacterial isolate is obtained by culturing in 50 mL of tryptic soy broth at 30°C and 120 rpm for 24 h and storing at 4°C.

2. The composite combined inoculation method using arbuscular mycorrhizal fungi and plant growth-promoting rhizobacteria according to claim 1, characterized in that: The tryptic soy broth is tryptone: 17.0 g / L, soy peptone: 3.0 g / L, sodium chloride: 5.0 g / L, dipotassium hydrogen phosphate: 2.5 g / L, glucose: 2.5 g / L.

3. A composite combined inoculation method using arbuscular mycorrhizal fungi and plant growth-promoting rhizobacteria according to claim 1, characterized in that: The average infection rate of the arbuscular mycorrhizal fungi of the mycorrhiza is 55.3%, and each 10 g of the inoculum in the inoculated dry sand contains 65 + 17 spores.

4. A combined inoculation method using arbuscular mycorrhizal fungi and plant growth-promoting rhizobacteria according to claim 1, characterized in that: The physical and chemical properties of the soil are pH = 6.28, EC = 467 S·m -1 , AP = 2.63 mg·kg -1 , TC = 12.04 g·kg -1 , TN = 1.62 g·kg -1 , C / N = 7.42.

Images