CN112662589B - Development and application of plant rhizosphere biofilm co-colonization type multifunctional complex microbial inoculum - Google Patents
Development and application of plant rhizosphere biofilm co-colonization type multifunctional complex microbial inoculum Download PDFInfo
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Abstract
The invention provides development and application of a co-colonization type composite microbial inoculum based on a plant rhizosphere biological membrane. The plant probiotic compound microbial inoculum consists of bacillus amyloliquefaciens SQR9 and pseudomonas stutzeri RFPse-2. The strain RFPse-2 is separated from the rhizosphere of cucumber inoculated with a single strain SQR9, can be metabolized and intercropped with the strain SQR9 to form a biological membrane together, and the biomass of the biological membrane formed together is obviously higher than that of a single bacterium. The fluorescence labeling strain is inoculated to the plant rhizosphere, and the strain SQR9 (green) and RFPse-2 (purple) can be co-colonized and generate synergistic probiotic effect on the plant. The plant probiotic composite microbial inoculum can not only remarkably promote the growth of cucumbers, but also help plants to resist salt in soil with salt content of 0.3%.
Description
Technical Field
The invention belongs to the field of agricultural microorganisms, and relates to development and application of a plant growth-promoting composite microbial inoculum based on rhizosphere biofilm co-colonization.
Background
The compound microbial agent is a microbial agent product which is formed by two or more than two microbes. The nature of the compound microbial agent is artificially designed and synthesized microbial ecology, and the compound microbial agent is mainly used for reasonably designing a microbial combination or an ecosystem with novel functions and behaviors aiming at a specific purpose. Compared with single microorganism and natural microorganism group, the synthetic microorganism community has the advantages of low complexity, high controllability, good stability and the like. The application research of the synthetic microbial community, namely the composite microbial inoculum, in agriculture is still in the initial stage at present, how to reasonably design and construct a stable and efficient synthetic microbial community by utilizing the existing ecological theory, and the exertion of the stable related biological functions is the main research direction in the future.
After the microbial fertilizer is applied to soil, on one hand, the microbial fertilizer needs to face the problem of living environment pressure caused by abiotic factors such as soil physicochemical properties and the like, and on the other hand, the microbial fertilizer also faces the problems of competition for living space and the like with indigenous microorganisms, so that the colonization ability of growth-promoting bacteria at the rhizosphere is restricted, the field effect stability of the microbial fertilizer is influenced, and the development of the microbial fertilizer is limited. Rhizosphere biofilms are the primary form of microbial stabilization occurring in the rhizosphere of plants. Effective rhizosphere colonization is a prerequisite for the growth-promoting and biocontrol effects of rhizosphere growth-promoting bacteria.
The bacillus and the pseudomonas are two types of microorganisms which are most widely researched by plant growth-promoting rhizobacteria, wherein the bacillus is a gram-positive bacterium, and the pseudomonas is a gram-negative bacterium. More than 70 percent of production strains in the registered products of 6400 microbial fertilizers in China are various rhizosphere probiotic bacillus. Spores formed by the bacillus have strong environmental adaptability, can keep biological activity in most environments, and enter a growth state again under the condition of proper conditions, so that a bacillus agent product can be better stored in the preparation process of the microbial agent. Although the pseudomonas microbial fertilizer has short shelf life, the pseudomonas microbial inoculum capable of keeping vitality can obviously promote the growth of plants and protect the plants against various biotic and abiotic stresses. How to scientifically combine bacillus and pseudomonas, the functional characteristics of two microorganisms are fully exerted, and the formation of a microbial inoculum product with various functions is a scientific problem with great application prospect.
Disclosure of Invention
The invention aims to enhance the application effect of the microbial fertilizer, develop and develop a compound microbial agent product which can jointly form a biological membrane at the rhizosphere of a plant, and provide theoretical and technical support for scientifically combining the compound microbial agent product.
Still another object of the present invention is to provide the use of the multifunctional plant probiotic complex agent.
The purpose of the invention can be realized by the following technical scheme:
a multifunctional composite microbial inoculum based on plant rhizosphere biofilm co-colonization is characterized by consisting of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SQR9 and Pseudomonas stutzeri (Pseudomonas stutzeri) RFPse-2; the bacillus amyloliquefaciens SQR9 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation date is 2012, 2 and 27 days, and the preservation number is CGMCC NO. 5808; the Pseudomonas stutzeri RFPse-2 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation date is 12 months and 7 days in 2020, and the preservation number is CGMCC NO. 21329.
The plant growth promoting compound microbial inoculum consists of the following components after being screened according to the following principle:
(1): the selected strains were all from the plant rhizosphere. Wherein the Pseudomonas stutzeri RFPse-2 is screened from the rhizosphere of cucumber inoculated with the strain SQR9, and the number of Pseudomonas stutzeri is increased remarkably after the strain SQR9 is inoculated.
(2): the selected strains are capable of metabolic cross-feeding in a culture medium.
(3): the biomass of the biomembrane formed by mixed bacteria is obviously higher than that of a single-bacterium biomembrane when the selected strains are co-cultured.
(4): after the selected strain is inoculated to the plant rhizosphere, the selected strain can co-colonize on the surface of the plant root and form a biofilm structure, so that a stable cooperative relationship is formed with the plant.
The preparation method of the plant probiotic composite bacterial agent comprises the step of respectively inoculating the activated strains SQR9 and RFPse-2 into a TSB liquid culture medium at 37 ℃ for 170r min -1 Culturing for 24 h; centrifuging and cleaning to obtain wet thalli, and resuspending the wet thalli by using equivalent sterile ultrapure water; adjusting bacterial liquid OD 598 And 2, mixing the two kinds of bacteria liquid in equal amount to obtain the plant probiotic composite bacterial agent.
The plant probiotic composite inoculant is inoculated in soil with normal salt concentration and soil with high salt concentration (0.3%) to plant cucumbers.
Advantageous effects
The invention screens the symbiotic bacterium Pseudomonas stutzeri RFPse-2 of the strain SQR9 from the rhizosphere of cucumber inoculated with the strain SQR9, and finds that the strain RFPse-2 and the strain SQR9 can jointly form a biofilm structure with higher biomass and can be co-colonized in the rhizosphere of plants. The mixed fungicide of bacillus and pseudomonas stutzeri is applied to the rhizosphere of the cucumber planted in the conventional soil, so that the plant height and the dry weight of the cucumber can be obviously improved by 103.6 percent and 125 percent respectively (compared with a control CK). Compared with the application effect of a single inoculation microbial agent (Bacillus amyloliquefaciens SQR9), the height and the dry weight of the cucumber plant by the mixed inoculation microbial agent are respectively increased by 17% and 35%; compared with Pseudomonas stutzeri RFPse-2, the improvement is 12.17 percent and 28.5 percent respectively. Cucumber is planted in soil with salt content of 0.3%, and the inoculation of the mixed microbial inoculum of bacillus and pseudomonas also promotes the plant height and dry weight of cucumber to be respectively increased by 81.2% and 102% (compared with control CK). Compared with the application effect of a single inoculation microbial inoculum (Pseudomonas stutzeri RFPse-2), the cucumber plant height and dry weight of the mixed inoculation microbial inoculum are respectively increased by 16.6 percent and 40.6 percent compared with the single inoculation Pseudomonas stutzeri. Treatment with Bacillus amyloliquefaciens SQR9 also significantly helped plants tolerate salt stress and promote cucumber growth.
Drawings
FIG. 1 phylogenetic Tree of Bacillus and Pseudomonas stutzeri
FIG. 2 Metabolic cross-feeding pattern of Bacillus and Pseudomonas stutzeri in culture medium. Acetyl levorotatory dextro-alkali, acetyl carnitine, valeric acid, tropine, cinnamic acid and the like secreted by the bacillus amyloliquefaciens can be utilized and metabolized by pseudomonas stutzeri RFPse-2; and the L-citrulline, biological purine, guanine, 7-methylguanosine and 2-O-methylguanosine secreted by the Pseudomonas stutzeri RFPse-2 can be metabolized and utilized by the Bacillus amyloliquefaciens SQR 9.
FIG. 3A mixture of inoculated colonies and co-cultured biofilm formation phenotype of Bacillus and Pseudomonas stutzeri, where green represents strain SQR9 and purple represents strain RFPse-2.
FIG. 4A representation of the rhizospheric biofilm co-colonizing phenotype of Bacillus sp and Pseudomonas stutzeri after mixed inoculation of the rhizosphere, wherein green represents strain SQR9 and purple represents strain RFPse-2.
FIG. 5 plant growth promotion test of plant probiotic complex
CK: no inoculation, S: SQR9, P: RFPse-2, PS: the two bacteria are inoculated together.
FIG. 6 potted plant test with plant probiotic to help plants tolerate salt stress
CK: no inoculation, S: SQR9, P: RFPse-2, PS: the two bacteria are inoculated together.
Biological material preservation information
Bacillus amyloliquefaciens SQR9 has been disclosed in ZL 201710797445.0.
Pseudomonas stutzeri (Pseudomonas stutzeri) RFPse-2 is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, the preservation date is 12 months and 7 days in 2020, the preservation address is the institute of microbiology of China academy of sciences No. 3, West Lu No. 1 Hopkins, Chaoyang district, Beijing, and the preservation number is CGMCC NO. 21329.
Detailed Description
1. Isolation and identification of strains
The cucumber rhizosphere growth promoting bacillus SQR9 has a strain preservation number of CGMCC N0.5808 and is obtained by laboratory prophase separation. Pseudomonas stutzeri RFPse-2 was screened in the rhizosphere of cucumber inoculated with strain SQR 9: taking out the root system of the cucumber from the soil, and collecting the soil adhered to the surface of the root. Adding 0.1g of rhizosphere soil into a 1.5mL centrifuge tube filled with 1mL of 0.9% sodium chloride and glass beads, shaking for 20 minutes at maximum speed to form soil suspension, and respectively sucking 10 -6 ,10 -7 ,10 -8 0.1mL of diluted soil suspension is coated on a TSB plate, each concentration is repeated at 3 times, colonies with different forms are selected on the plate for purification after 2-7 days of culture at 30 ℃, and the colonies are inoculated into a TSB liquid culture medium for 18-24h and stored in a 15% glycerol tube at-80 ℃. The obtained bacterium is named as RFPse-2, and the colony on the TSB plate is small and is light yellowHas a dark yellow halo and certain viscosity. Gram staining is negative, and the thallus is uniformly colored. Strain RFPse-2 was identified as Pseudomonas stutzeri based on the genome-wide sequence of the strain (NCBI accession number ASM974018v 1). It is stored in CGMCC with the preservation number of CGMCC NO. 21329.
The preparation method of the TSB culture plate comprises the following steps of preparing 1L of culture medium: 30g of TSB and 20g of agar, the volume is adjusted to 1000mL, the pH is natural, and the sterilization is carried out for 30min at 115 ℃. .
2. Co-culture of composite microbial inoculum and biological membrane
SQR9 and RFPse-2 strains preserved in-80 ℃ glycerol tubes were activated by streaking on TSB solid medium plates and cultured in an incubator at 30 ℃ for 2 days. Picking single colony in 3mL liquid TSB test tube at 30 deg.C for 170r min -1 Shake culturing overnight, and adjusting OD 600 1, the same amount of the suspension was inoculated into a 24-well plate at an inoculum size of 1% (v/v), and cultured at 30 ℃ for 24 hours. And (3) observing the cell distribution of the biomembrane by using a laser confocal microscope. The result is shown in figure 3, and the result in figure 3 shows that Bacillus amyloliquefaciens SQR9 and Pseudomonas stutzeri RFPse-2 can well coexist in the biomembrane at the liquid-gas boundary of the solid culture medium colony and the liquid culture medium, and a close relation is established to share the special ecological niche built by the biomembrane extracellular matrix.
3. Co-colonization of composite microbial inoculum rhizosphere (laser confocal)
Rhizosphere colonization of the complex inoculant was performed on the model plant Arabidopsis thaliana. The arabidopsis seeds are subjected to surface disinfection by adopting a 2% sodium hypochlorite solution for 10min, then washed for 5 times by using sterile distilled water, then placed on an MS plate, vernalized and pregerminated for 3 days at 4 ℃, and then transferred to a constant temperature box at 28 ℃ for culture for 7 days to root. 16h light, 8h dark. Seedlings with root system of 8-12mm are selected and transferred to a 6-well culture plate, and 2.7mL of MSNg culture solution is added.
SQR9 (green fluorescence labeling) and RFPse-2 (purple fluorescence labeling) strains preserved in-80 ℃ glycerol tubes are streaked and activated on TSB solid medium plates, and are cultured in an incubator at 30 ℃ for 2 days. Picking single colony in 3mL liquid TSB test tube respectively, 30 deg.C, 220r min -1 Shake culturing overnight, centrifuging at 4 deg.C, collecting thallus, washing with MSNg culture solution for 3 times, and adjusting OD 600 0.2, at 10% (v)V) (0.3mL) were inoculated in 6-well plates. The plate was incubated at 28 ℃ and 90rpm in a rotary shaker. And taking out the arabidopsis seedlings after 24h, washing the arabidopsis seedlings for 3 times by using sterile water, removing non-adhesive bacteria, transferring the arabidopsis seedlings onto a glass slide, dropwise adding 20 mu L of sterile water, covering a cover glass, and observing the colonization of the bacteria on the root surface by using a laser confocal microscope. The results are shown in FIG. 4, and it can be seen from FIG. 4 that Bacillus amyloliquefaciens SQR9 and Pseudomonas stutzeri RFPse-2 can co-colonize on the surface of the plant root system to form a biofilm structure, and the green fluorescent SQR9 strain and the purple fluorescent RFPse-2 strain have deep overlap in a partial colonization region, which indicates that the two rhizosphere bacteria can well coexist in the plant root system. The quantitative data of rhizosphere colonization show that the strains SQR9 and RFPse-2 in mixed bacteria colonization are in the same order of magnitude as single bacteria rhizosphere colonization, and the total colonization amount is higher than that of single bacteria.
4. Plant growth promotion test of plant probiotic composite agent
The cucumber potting test is carried out in a greenhouse of a high-tech solid organic waste resource research key laboratory (Yixing) in Jiangsu province from 7 months, 2 days to 9 months, 5 days in 2020, and the tested cucumber variety is Jinchun No. 4 purchased from agricultural academy in Jiangsu province. The surface of cucumber seeds is disinfected by 2% sodium hypochlorite solution for 10min, then the cucumber seeds are treated in warm water at 55 ℃ for 10min, then the cucumber seeds are soaked in the warm water at 30 ℃ for 8h, then the cucumber seeds are put in a constant temperature box at 30 ℃ for accelerating germination, the humidity is controlled to be about 80%, and the dark environment is maintained until the cucumber seeds germinate.
And when the seeds germinate, selecting the plump and uniform-sprouting seeds, and sowing the seeds in a hole tray filled with a seedling raising matrix, wherein one seed is put in each hole. After the seeding is finished, the plug tray is placed in a greenhouse with good illumination, and water is maintained irregularly according to the state of the matrix, so that the growth of the cucumber is guaranteed. After 2 true leaves grow out, seedlings with consistent growth vigor are selected for pot experiment. The experiment set up four treatments: 1) CK, no bacterium treatment;
2) adding a single-bacterium bacillus amyloliquefaciens SQR9 microbial agent for treatment; 3) adding pseudomonas stutzeri RFPse-2 microbial inoculum for treatment; 4) adding mixed bacteria (Bacillus SQR9+ Pseudomonas RFPse-2) for treatment. Each 10 pots are treated, each pot is filled with 2kg of soil, and the bacterial agent filling amount is 2.5 multiplied by 10 7 CFU/g soil (dry basis). Conventional management, after 58d respectivelyThe plant height and the dry weight of the aerial parts of the plants were determined. The results are shown in FIG. 5, from which it can be seen that the treatment with co-inoculation of two strains is significantly higher in both cucumber plant height and dry weight than the single-strain inoculated cucumber plants (strains SQR9 and RFPse-2) and the control cucumber without the inoculated strain, indicating that the double-strain inoculation has a significant synergistic effect in promoting cucumber plant growth.
5. Potted plant effect of plant probiotic composite inoculant for helping plants to resist salt stress
The cucumber seed treatment was as above. The soil is artificially simulated to be subjected to salt stress treatment, and NaCl solution is added to cultivate the soil, so that the final soil salt content is 0.3%. The experiment set up four treatments: 1) CK, no bacterium treatment; 2) adding a single-bacterium bacillus amyloliquefaciens SQR9 microbial agent for treatment; 3) adding pseudomonas stutzeri RFPse-2 microbial inoculum for treatment; 4) adding mixed bacteria (Bacillus SQR9+ Pseudomonas RFPse-2) for treatment. Each 10 treatment pots are filled with 2kg of soil, and the bacteria filling amount of the microbial inoculum is 2.5 multiplied by 10 7 CFU/g soil (dry basis). And (5) performing conventional management, and measuring the plant height and the dry weight of the overground part of the plant after 58 d. The results are shown in FIG. 6, and it can be seen from FIG. 6 that under salt stress, the treatment of co-inoculation of two strains is significantly higher in both plant height and dry weight of cucumber plants than the treatment of single-strain inoculated cucumber plants (strain RFPse-2) and the control cucumber without strain inoculation, and the single-strain inoculated SQR9 treatment also has better growth promoting effect.
Claims (6)
1. A multifunctional composite microbial inoculum based on plant rhizosphere biofilm co-colonization is characterized by consisting of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SQR9 and Pseudomonas stutzeri (Pseudomonas stutzeri) RFPse-2; the bacillus amyloliquefaciens SQR9 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation date is 2012, 2 and 27 days, and the preservation number is CGMCC NO. 5808; the Pseudomonas stutzeri RFPse-2 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation date is 12 months and 7 days in 2020, and the preservation number is CGMCC NO. 21329.
2. The multifunctional complex microbial inoculum of claim 1, which is prepared byPrepared by inoculating the activated strains SQR9 and RFPse-2 to TSB liquid medium at 30 deg.C for 170r min -1 Culturing for 24 hours; centrifuging and cleaning to obtain thalli, and resuspending the thalli by using equal amount of sterile ultrapure water; adjusting bacterial liquid OD 598 And 2, mixing the two bacterial liquids in equal quantity to obtain the multifunctional composite bacterial agent.
3. The multifunctional complex microbial inoculant according to claim 1, wherein the selected strains are capable of nutrient metabolic cross-feeding in a culture medium.
4. The method for preparing the multifunctional complex microbial inoculum of claim 1, which is characterized by respectively inoculating the activated strains SQR9 and RFPse-2 to a TSB liquid culture medium at 30 ℃ for 170r min -1 Culturing for 24 hours; centrifuging and cleaning to obtain thalli, and resuspending the thalli by using equal amount of sterile ultrapure water; adjusting bacterial liquid OD 598 And 2, mixing the two bacterial liquids in equal quantity to obtain the multifunctional composite bacterial agent.
5. The use of the multifunctional complex microbial inoculum of claim 1 in promoting the growth of cucumbers.
6. The use of the multifunctional complex microbial inoculum of claim 1 for assisting cucumbers in resisting soil salt stress.
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| CN114015629B (en) * | 2021-12-20 | 2022-04-29 | 南京林业大学 | Complex microbial inoculum for promoting growth of soybeans and improving abundance of probiotic microbial populations |
| CN114574401A (en) * | 2022-04-07 | 2022-06-03 | 南京工业大学 | Application of acetoin in improving rhizosphere growth-promoting bacteria in plant rhizosphere colonization |
| CN116333937B (en) * | 2023-04-03 | 2024-03-29 | 南京农业大学 | Plant rhizosphere probiotic strain with greenhouse gas nitrous oxide emission reduction function and application thereof |
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