Simulated root exudates and application thereof in promoting growth of phosphate solubilizing bacteria and rhizosphere colonization

Abstract

The invention provides simulated root exudates and application of the simulated root exudates in promoting growth of phosphate solubilizing bacteria and rhizosphere colonization. The simulated root exudates comprise glucose, fructose, myristic acid and luteolin. The simulated root exudates can promote the plants to utilize phosphorus and grow, and can improve the colonization, growth and phosphorus dissolving and growth promoting effects of the Domibacillus indicus D99 (Domibacillus indicus) D99.

Description

Technical Field

[0001] The present invention belongs to the technical fields of agricultural microbiology and environmental protection. Specifically, the present application provides a simulated root exudate and its application in promoting the growth and rhizosphere colonization of phosphate-solubilizing bacteria. Background Art

[0002] Phosphorus is one of the macronutrients essential for crop growth and development. At present, agricultural production in China generally relies on a large amount of phosphate fertilizer input to ensure crop yields. However, the utilization rate of phosphate fertilizer applied to farmland is low, and most of it accumulates in the soil in different forms such as available phosphorus, insoluble inorganic phosphorus, and organic phosphorus. Specifically, 30%-65% of the total phosphorus in the soil exists in the form of organic phosphorus, and in soils with high organic matter, this proportion can even be as high as 90%. Therefore, improving the utilization efficiency of soil organic phosphorus by crops is crucial for the sustainable development of modern agriculture.

[0003] Soil microorganisms are the main driving force for organic phosphorus mineralization, and most of the enzymes that can convert organic phosphorus into inorganic phosphorus are also produced by microorganisms. Such phosphate-solubilizing microorganisms are abundant in the rhizosphere of plants. After the application of bacterial agents made from rhizosphere phosphate-solubilizing bacteria, it can effectively promote the dissolution and mineralization of soil organic phosphorus. The stable survival of phosphate-solubilizing bacteria in the soil and their colonization in the rhizosphere of crops and becoming the dominant bacterial community can effectively improve fertilizer efficiency and stability. Root exudates are the key substances mediating the colonization and growth of phosphate-solubilizing bacteria in rhizosphere soil and can promote the colonization of phosphate-solubilizing bacteria. Many reports have found that single root exudates have the effect of promoting the colonization of phosphate-solubilizing bacteria. For example, citric acid and fumaric acid secreted by tomato roots promote the colonization of Pseudomonas fluorescens; D-galactose in cucumber root exudates can significantly promote the biofilm formation of Bacillus velezensis; malic acid secreted by Arabidopsis thaliana roots can promote Bacillus subtilis to form a biofilm on the root surface. However, root exudates are a complex mixture of various substances, and not only one substance plays a role. For example, the carbohydrates and fatty acids contained in maize root exudates can serve as carbon sources for phosphate-solubilizing bacteria, and substances such as flavonoids can serve as signal molecules. Therefore, simulating the complex combination of multiple exudates may be an important way to improve the colonization of phosphate-solubilizing bacteria.

[0004] Glucose, myristic acid, and luteolin have been found to be key components in root exudates. Currently, there are no reports on using the compound of glucose, myristic acid, and luteolin as a simulated root exudate and applying it to improve the dissolution of soil organic phosphorus and promote plant growth by Bacillus megaterium var. phosphaticum. Summary of the Invention

[0005] The applicant isolated Domibacillus indicus D99 from the rhizosphere soil of corn used in greenhouse pot experiments in the College of Resources and Environment, China Agricultural University and deposited it. This strain has the ability to dissolve soil organic phosphorus, can effectively promote plant growth, and also has a growth-promoting effect on other phosphorus-solubilizing bacteria. How to better promote the colonization and function of this bacterium is a prerequisite for its full utilization, but there is currently a lack of relevant research on this bacterium.

[0006] On the one hand, the present application provides a kind of simulated root exudates, and the simulated root exudates include: 18 - 36 parts of carbon source, 23 - 46 parts of fatty acid, 28 - 36 parts of flavonoid substances.

[0007] Further, the carbon source is glucose and / or fructose; the fatty acid is myristic acid; the flavonoid substance is luteolin.

[0008] Further, the simulated root exudates are composed of 18 parts by weight of glucose, 23 parts by weight of myristic acid and 28 parts by weight of luteolin; or the simulated root exudates are composed of 18 parts by weight of glucose, 18 parts by weight of fructose, 23 parts by weight of myristic acid and 28 parts by weight of luteolin.

[0009] On the other hand, the present application provides a liquid simulated root exudates, and the liquid simulated root exudates are made from the above-mentioned simulated root exudates, surfactant, co-surfactant and oil phase.

[0010] Further, the liquid simulated root exudates are made from 1 part by weight of the above-mentioned simulated root exudates, 1 volume part of Tween 80, 0.5 ml volume part of isopropyl myristate and 1 volume part of 1,2 - propanediol.

[0011] On the other hand, the present application provides the use of the above-mentioned simulated root exudates or liquid simulated root exudates in promoting plant phosphorus uptake.

[0012] On the other hand, the present application provides the use of the above-mentioned simulated root exudates or liquid simulated root exudates in improving the growth-promoting effect of Domibacillus indicus D99 on plants. The Domibacillus indicus D99 is deposited in the Guangdong Provincial Microbial Culture Collection Center, and the deposit number is GDMCC No.65675.

[0013] The plant is preferably corn.

[0014] On the other hand, the present application provides the use of the above-mentioned simulated root exudates or liquid simulated root exudates in promoting the growth of Domibacillus indicus D99, and the Domibacillus indicus D99 is preserved in the Guangdong Provincial Microbial Culture Collection Center with the preservation number of GDMCC No. 65675.

[0015] On the other hand, the present application provides the use of the above-mentioned simulated root exudates or liquid simulated root exudates in promoting the colonization of Domibacillus indicus D99, and the Domibacillus indicus D99 is preserved in the Guangdong Provincial Microbial Culture Collection Center with the preservation number of GDMCC No. 65675.

[0016] Furthermore, in the above application, the dosage of the simulated root exudates is 0.3 - 0.7 g / kg of soil.

[0017] The present invention discloses that specific simulated root exudates are obtained by compounding carbohydrates and fatty acid substances as carbon sources and flavonoids as signal substances, and optimizing the microemulsion system to improve the solubility of flavonoid substances, thus forming a corresponding preparation method. At the same time, it is found that the simulated root exudates can increase the growth rate and maximum biomass of the phosphate-solubilizing bacterium Domibacillus indicus in the culture medium, and improve the preparation efficiency of the microbial inoculum.

[0018] In addition, the application of such simulated root exudates in promoting organic phosphorus mineralization, improving the colonization ability of Domibacillus indicus and promoting plant growth is also disclosed. When the simulated root exudates are applied to the soil, they can not only significantly promote the activity of soil microorganisms, promote soil organic phosphorus mineralization and plant growth by themselves, but also be applied in combination with Domibacillus indicus D99 to improve the soil colonization ability of Domibacillus indicus D99, thereby significantly improving the ability of Domibacillus indicus D99 to dissolve soil organic phosphorus and promote plant growth. Description of the Drawings

[0019] Figure 1 It is the effect diagram of solubilizing the simulated root exudates by using the emulsion system.

[0020] Figure 2 It is the comparison diagram of the growth promotion effect of the simulated root exudates on corn.

[0021] Figure 3 It shows the effects of the simulated root exudates on the above-ground biomass of corn, plant phosphorus uptake and phosphatase activity.

[0022] Figure 4To simulate the effect of root exudates on the colonization ability enhancement of Domibacillus indicus D99 in farmland soil.

[0023] Figure 5 To simulate the growth-promoting effect of root exudates on Domibacillus indicus D99 during the fermentation process.

[0024] Figure 6 To simulate the comparison chart of the role of root exudates in improving the ability of Domibacillus indicus D99 to promote soil organic phosphorus dissolution and plant growth.

[0025] Figure 7 To simulate the effect diagram of root exudates improving the colonization ability of Domibacillus indicus D99 in the maize rhizosphere. Specific implementation mode

[0026] The following examples facilitate a better understanding of the present invention, but are not limited thereto. These examples are for illustrative purposes only and do not limit the protection scope of the present invention.

[0027] Example 1 Preparation method of liquid simulated root exudates

[0028] In a 500 mL beaker, 1 mL of surfactant (Tween 80), 0.5 mL of oil phase (isopropyl myristate), and 1 mL of co-surfactant (1,2-propanediol) were added according to a volume ratio of 1:0.5:1, and then stirred and mixed evenly with a magnetic stirrer. Then, powdery root exudates not exceeding the total mass of the surfactant and the auxiliary agent were added to form a mixture. The final ratio was surfactant:oil phase:auxiliary agent:root exudates = 1:0.5:1:1, (v:v:v:m). Magnetic stirring was carried out until a homogeneous milky white (or yellow or colorless) emulsion was formed, and then distilled water was slowly added while stirring. First, 5 mL was added at a time, and then another 5 mL was added after mixing evenly (finally adding up to 20 - 30 mL). Then, it was diluted to 400 mL with distilled water to form a clear and transparent microemulsion. After standing for a period of time, it did not separate into layers and there was no precipitation. The identification method of the microemulsion was the centrifugation method, 3000r·min -1 After centrifuging for 10 min, observe whether it separates into layers and remains clear. If it does not separate into layers and remains clear, it is a microemulsion system.

[0029] Example 2 Pot experiment on the addition of simulated root exudates to promote phosphorus uptake by maize

[0030] (1) Test method

[0031] Four kinds of simulated root exudates were prepared with different substances and different proportions: Simulated root exudate 1 was composed of 18 parts of glucose and 23 parts of myristic acid; Simulated root exudate 2 was composed of 23 parts of myristic acid and 28 parts of luteolin; Simulated root exudate 3 was composed of 18 parts of glucose, 23 parts of myristic acid and 28 parts of luteolin; Simulated root exudate 4 was composed of 18 parts of glucose, 18 parts of fructose, 23 parts of myristic acid and 28 parts of luteolin.

[0032] The test plant was maize, and the variety was Zhengdan 958. The black soil from Dali, Yunnan, the fluvo-aquic soil from Shangzhuang, Beijing, and the brown soil from Tai'an, Shandong were sieved through a 2-mm sieve and filled into pots at 2 kg per pot. The control group was set without adding any substances. There were 4 treatment groups in total, with 4 replicates for each treatment. And about 0.5 g / kg of 4 kinds of simulated root exudates were directly mixed into 3 kinds of soils. The selected maize seeds were soaked in 2.5% sodium hypochlorite solution for 10 min, then surface-sterilized with 75% alcohol for 1 min, and rinsed 6 - 7 times with sterile water. After that, the seeds were first placed on a culture dish and cultured for 24 h. After germination, the seeds were ready to be sown into flower pots, and 5 seeds were sown in each flower pot and covered with soil on the surface. After growing for 40 days, the plant height, dry weight, aboveground phosphorus content of maize, and rhizosphere soil phosphatase activity were measured, and the mineralization effect of simulated root exudates on soil organic phosphorus and the promoting effect on phosphorus uptake by maize were analyzed.

[0033] (2) Test results

[0034] The results of the pot experiment are shown in Table 1. The results show that different simulated root exudates have different effects on the growth and promotion of maize. The promoting effect of simulated root exudates on the aboveground biomass of maize in three kinds of soils will be affected by the soil.

[0035] After data analysis and effect evaluation, the results show that the promoting effect of simulated root exudate 3 on the growth of maize is stable in three kinds of soils. Compared with the control, the increase in aboveground biomass of maize in this treatment ranges from 0.5% to 79.03%, and the highest increase in aboveground phosphorus uptake of maize is 217.07%. The rhizosphere soil phosphatase activity reflects the ability of organic phosphorus mineralization. Compared with the control group, simulated root exudate 3 significantly increases the acid phosphatase activity of maize roots, with an increase of 9.2% - 102.17%; the alkaline phosphatase activity increases by 27.63% - 134.16%. The effect of simulated root exudate 4 is also significant. Compared with the control, the increase in aboveground biomass of maize in this treatment ranges from 2.6% to 50.3%, and the highest increase in aboveground phosphorus uptake of maize is 191.22%. The rhizosphere soil phosphatase activity reflects the ability of organic phosphorus mineralization. Compared with the control group, simulated root exudate 4 significantly increases the acid phosphatase activity of maize roots, with an increase of 10.9% - 140.46%; the change in alkaline phosphatase activity is -16.65% - 150.6%.

[0036] Table 1 Effects of Different Simulated Root Exudates in Three Kinds of Soils on Maize Growth

[0037]

[0038]

[0039] Note: * indicates that there is a significant difference in this index between the control group and the treatment group

[0040] Example 3 Effects of Simulated Root Exudate Addition on the Growth of Domibacillus indicus D99 in Clean Room

[0041] Domibacillus indicus D99 was isolated from the rhizosphere soil of maize used in greenhouse pot experiments in the College of Resources and Environment, China Agricultural University. After being identified by colony characteristics, metabolic characteristics and molecular biology, it was classified into Domibacillus indicus and called Domibacillus indicus according to the Chinese name of CCTCC. This strain is preserved in the Guangdong Provincial Microbial Culture Collection Center; the preservation number is GDMCC No.65675; the preservation time is December 25, 2024; the address is the 5th Floor, Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences. This strain has the ability to dissolve soil organic phosphorus, with an acid phosphatase activity of 17.32 and an alkaline phosphatase activity of 1.3; it can promote plant growth, increase the aboveground biomass by 50 - 115%, significantly reduce the available organic phosphorus in black soil by 14.75 mg / kg, and has a growth-promoting effect on indigenous phosphorus-solubilizing bacteria in the soil.

[0042] (1) Test Method

[0043] Inoculate Domibacillus indicus D99 into M9 medium containing 1% glucose and shake-culture (30 °C, 200 rpm) for 8 hours to obtain a seed solution. Centrifuge the fermentation broth of D99 at 5000 rpm for 5 min, discard the supernatant, and re-suspend and adjust the OD value to 0.5 with sterile water to obtain a seed solution.

[0044] Four treatment groups were set up in the experiment: D99_G was M9 medium with 1% glucose, D99_M was M9 medium supplemented with 500 μM myristic acid; D99_GM was M9 medium with 1% glucose supplemented with 500 μM myristic acid; D99MLG was M9 medium with 1% glucose supplemented with 500 μM myristic acid and luteolin. In each treatment group, the seed solution of Bacillus amyloliquefaciens D99 was inoculated at an inoculation amount of 1%, and there were 5 replicates in each treatment group. The culture device used a 100-well honeycomb plate, and the culture instrument and detection device used a Bioscreen C microbial automatic growth analyzer. The detection wavelength was 600 nm, the detection interval was 30 min, and the cumulative culture time was 48 h.

[0045] (2) Experimental results

[0046] Bacillus amyloliquefaciens D99 did not grow in the pure myristic acid treatment group (D99_M), indicating that this strain could not utilize fatty acids alone. It grew well in the M9 medium with 1% glucose treatment group (99_G) and reached the maximum growth value at 16 h, with an OD 600 value of 0.72. In the fermentation broth of M9 medium with 1% glucose supplemented with 500 μM myristic acid, D99 showed a faster growth rate and better growth amount. The maximum growth value at 16 h had an OD 600 value of 0.92. When 500 μM myristic acid and luteolin solution were added to the M9 medium with 1% glucose, the growth value of D99 could be greatly increased to 1.045, and at 48 h, the OD 600 value could still be maintained at 0.935. This indicates that the simulated root exudates have a significant promoting effect on the growth of Bacillus amyloliquefaciens D99 during the fermentation process.

[0047] Example 4 Effect of adding simulated root exudates on the colonization of Bacillus amyloliquefaciens D99 in farmland soil

[0048] Detecting the colonization ability of Bacillus amyloliquefaciens D99 in field soil. The premise for the bacterial agent to play a role is to stably survive in the soil, and the survival quantity and relative abundance represent the action potential of this bacterium in farmland soil.

[0049] (1) Preparation of the bacterial agent

[0050] Bacillus amyloliquefaciens D99 was inoculated into LB liquid medium and cultured with shaking in a shaker (30 °C, 200 rpm) for 48 hours to obtain a liquid bacterial agent. The bacterial agent was centrifuged at 5000 rpm for 5 min, the supernatant was removed, and the bacteria in the centrifuge tube were resuspended with sterile water and then centrifuged again. After repeating this 2 times, it was adjusted to OD 600 = 10, and the concentration was about 2×10 7CFU / mL to form a bacterial agent.

[0051] (2) Experimental materials and methods

[0052] Fresh soil was taken from the field of Quzhou Experimental Station in Handan City, Hebei Province and stored at 4°C. In the experimental group, 10 mL of liquid bacterial agent was added to 200 g of fresh soil, and 3 mL of simulated root exudates made of glucose, myristic acid, and luteolin with a concentration of 0.345 g / kg of soil was mixed in, and then filled into a special soil incubation device; while in the control group, only 10 mL of liquid bacterial agent was added, and 4 replicates were set for each treatment group. The soil incubation device was buried in the corn field of Quzhou Experimental Station in Handan City, Hebei Province, harvested after 3 months of cultivation, and the colonization of the strain in the farmland soil was detected by 16S rRNA gene sequencing.

[0053] (3) Test results

[0054] The results of the bacterial colonization test in farmland soil showed that Bacillus amyloliquefaciens D99 could survive stably in farmland soil with a relative abundance of 0.14%. However, after adding the simulated root exudates, the relative abundance of Bacillus amyloliquefaciens D99 increased significantly to 1.43%, which was 9.21 times higher than that of the control group. This indicates that the addition of simulated root exudates has a significant promoting effect on the soil colonization of Bacillus amyloliquefaciens.

[0055] Example 5 Verification of the promoting effect of simulated root exudates on the growth of plants by Bacillus amyloliquefaciens D99

[0056] After the Bacillus amyloliquefaciens D99 bacterial agent was added to the soil, it would face the competition of the original bacterial resources in the soil. In order to detect whether the simulated root exudates could improve the promoting effect of Bacillus amyloliquefaciens D99 on plant growth, a pot experiment was designed.

[0057] (1) Test method

[0058] Beijing Shangzhuang fluvo-aquic soil was used in the experiment. After sieving through a 2-mm sieve, it was sterilized by γ-ray and reserved for use. Each pot was filled with 2 kg of soil. Nine indigenous bacterial strains were obtained from the soil in the early stage: Bacillus sp. C70, Domibacillus sp. C94, Bacillus sp. C67, Bacillus sp. A113, Bacillus sp. A7, Fictibacillus sp. A103, Streptomyces sp. C92, Streptomyces sp. D61, and Streptomyces sp. D1. The 9 strains of bacteria were inoculated in LB medium respectively and cultured at 30°C and 200 rpm for 24 h. The supernatant was removed by centrifugation, and the cells were resuspended with deionized water to adjust the OD 600= 0.5. The bacterial suspensions of each strain were mixed in equal volumes to form a synthetic bacterial community suspension, and 180 mL / kg of the suspension was added to restore the soil bacterial community. Bacillus amyloliquefaciens D99 was also made into a bacterial agent in the above manner. A control group was designed in the experiment without adding the Bacillus amyloliquefaciens D99 bacterial agent; Treatment Group 1 was only added with the Bacillus amyloliquefaciens D99 bacterial agent at an addition amount of 20 mL / kg; Treatment Group 2 was added with 20 mL / kg of the Bacillus amyloliquefaciens D99 bacterial agent and 0.69 g of simulated secretion 3 was incorporated. The selected corn seeds were soaked in 2.5% sodium hypochlorite solution for 10 min, then surface-sterilized with 75% alcohol for 1 min, rinsed 6 - 7 times with sterile water, and then the seeds were placed on a culture dish and cultured for 24 h. After germination, the seeds were ready to be sown into flower pots. 5 seeds were sown in each flower pot and covered with soil on the surface. Thinning was carried out after 7 days to ensure 1 corn plant per pot. After growing for 40 days, the growth of the corn was measured to analyze the effect of simulated root exudates on enhancing the promoting effect of Bacillus amyloliquefaciens D99 on plant growth.

[0059] (2) Experimental results

[0060] The results of the pot experiment are shown in Table 2. Compared with the control group, the treatment with the Bacillus amyloliquefaciens D99 bacterial agent significantly increased the above-ground biomass, with an increase amplitude of 27.8%; while in the treatment group with both bacteria inoculation and addition of simulated root exudates, the above-ground biomass was significantly higher, 97.47% higher than the control treatment. The activity of rhizosphere soil phosphatase reflects the ability of organic phosphorus mineralization. Compared with the control group, inoculation with the bacterial agent significantly increased the activity of soil acid phosphatase by 26.83%; the increase amplitude of the treatment group with both bacteria inoculation and addition of simulated root exudates was greater, which was 32.53%. In addition, compared with the control group, inoculation with the bacterial agent significantly increased the activity of soil alkaline phosphatase by 55.4%; the increase amplitude of the treatment group with both bacteria inoculation and addition of simulated root exudates was greater, which was 129.7%.

[0061] The sequencing results of the rhizosphere soil showed that when only the bacterial agent was added, the relative abundance of Bacillus amyloliquefaciens D99 in the rhizosphere was 0.013%, and after adding the simulated root exudates, the relative abundance increased to 0.38%. The treatment with the synergist could increase the rhizosphere colonization of Bacillus amyloliquefaciens D99 by 30 times.

[0062] Table 2 Effects of Bacillus amyloliquefaciens D99 on the mineralization of organic phosphorus in black soil

[0063]

Claims

1. A simulated root exudate, characterized in that: The simulated root exudates include: 18-36 parts of carbon sources, 23-46 parts of fatty acids, and 28-36 parts of flavonoids.

2. The simulated root exudate according to claim 1, wherein the carbon source is glucose and / or fructose; the fatty acid is myristic acid; and the flavonoid is luteolin.

3. The simulated root exudate according to claim 2, wherein the simulated root exudate consists of 18 parts by weight of glucose, 23 parts by weight of myristic acid and 28 parts by weight of luteolin; or the simulated root exudate consists of 18 parts by weight of glucose, 18 parts by weight of fructose, 23 parts by weight of myristic acid and 28 parts of luteolin.

4. A liquid simulated root exudate, characterized in that: The liquid simulated root exudate is made of the simulated root exudate according to any one of claims 1 to 3, a surfactant, a co-surfactant and an oil phase.

5. The liquid simulated root exudate according to claim 4, wherein the liquid simulated root exudate is prepared from 1 part by weight of the simulated root exudate according to any one of claims 1 to 3, 1 part by volume of Tween 80, 0.5 ml part by volume of isopropyl myristate, and 1 part by volume of 1,2-propylene glycol.

6. Use of the simulated root exudate according to any one of claims 1 to 3 or the liquid simulated root exudate according to claim 4 or 5 in promoting phosphorus absorption in plants.

7. Use of the simulated root exudate according to any one of claims 1 to 3 or the liquid simulated root exudate according to claim 4 or 5 in improving the promoting effect of Indian cleanroom Bacillus (Domibacillus indicus) D99 on plant growth, wherein the Indian cleanroom Bacillus D99 is deposited in Guangdong Microbial Culture Collection Center with a deposit number of GDMCC No.65675.

8. Use of the simulated root exudate according to any one of claims 1 to 3 or the liquid simulated root exudate according to claim 4 or 5 in promoting the growth of Domibacillus indicus D99, wherein the Indian cleanroom Bacillus D99 is deposited in Guangdong Microbial Culture Collection Center with a deposit number of GDMCC No. 65675.

9. Use of the simulated root exudate according to any one of claims 1 to 3 or the liquid simulated root exudate according to claim 4 or 5 in promoting the colonization of Domibacillus indicus D99, wherein the Indian cleanroom Bacillus D99 is deposited in Guangdong Microbial Culture Collection Center with a deposit number of GDMCC No. 65675.

10. The use according to any one of claims 6 to 9, wherein the amount of the simulated root exudates used is 0.3-0.7 g / kg soil.

Images