Rhodococcus thermotolerans strain for degrading phenolic acid and application of the strain in preparing microbial inoculants for crop growth

Abstract

The application discloses a strain of thermophilic Rhodococcus RA1 capable of efficiently degrading phenolic acid and application of the strain in preparation of a crop biocontrol microbial agent. The technical solution of the application is characterized by the following: the strain of thermophilic Rhodococcus RA1 capable of efficiently degrading phenolic acid is preserved in the China General Microbiological Culture Collection Center on December 26, 2022, and has a preservation number of CGMCC No. 26299; the phenolic acid is single phenolic acid or mixed phenolic acid; the application further specifically discloses a separation and purification method of the thermophilic Rhodococcus and application of the thermophilic Rhodococcus in the crop biocontrol microbial agent. The thermophilic Rhodococcus RA1 can efficiently degrade single phenolic acid or mixed phenolic acid with a concentration of up to 1200 mg / L, thereby relieving direct stress of the phenolic acid on plant growth, and can inhibit growth of plant pathogenic fungi, thereby preventing the plant pathogenic fungi from invading the plants, and provides a new strain resource for development of the crop biocontrol microbial agent.

Description

Technical Field

[0001] This invention belongs to the field of microbial agents for alleviating plant autotoxicity and inhibiting the growth of plant pathogenic fungi, specifically involving a highly efficient thermophilic Rhodococcus thermophilus strain that degrades phenolic acids and its application in the preparation of crop biocontrol microbial agents. Background Technology

[0002] Food is the foundation of life, and agricultural production has always been a key factor in the development of human society. However, with the increase in global population and the advancement of production technology, the types of crops cultivated on the same land have become more specialized, and the planting density is higher. This leads to an increased accumulation of plant secretions in the soil. Phenolic acids are benzene ring compounds containing several phenolic hydroxyl groups, mainly produced by plant root secretions and the decomposition of plant residues or litter. These phenolic acids can have autotoxic effects, directly inhibiting plant growth or stimulating the growth of pathogenic fungi, leading to frequent plant diseases and pests, and seriously threatening the healthy growth of plants.

[0003] Some microorganisms can utilize phenolic acids as a carbon source to transform phenolic acids in the environment, thereby mitigating their direct or indirect harm to plant growth. Therefore, developing beneficial microorganisms that degrade phenolic acids is of great significance for sustainable agricultural production. However, currently, the development and application of microbial strains capable of efficiently degrading high concentrations of phenolic acids are still very limited. The innovation of this invention lies in isolating and obtaining a thermophilic Rhodococcus thermophilus strain RA1 capable of efficiently degrading high concentrations of phenolic acids, and effectively applying it to alleviate the inhibitory effect of phenolic acid stress on plant growth and inhibit plant pathogenic fungi. Summary of the Invention

[0004] The technical problem solved by this invention is to provide a highly efficient thermophilic Rhodococcus thermophilus RA1 strain that degrades phenolic acids, its isolation and purification method, and its application in the preparation of crop biocontrol microbial agents.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: a highly efficient thermophilic Rhodococcus thermophilus RA1 strain, characterized in that: the thermophilic Rhodococcus thermophilus was deposited at the China General Microbiological Culture Collection Center on December 26, 2022, with the accession number: CGMCC No. 26299, and the phenolic acid is a single phenolic acid or a mixture of phenolic acids.

[0006] Further specifying, the single phenolic acid is ferulic acid, vanillic acid, or p-hydroxybenzoic acid, and the mixed phenolic acid is at least two of ferulic acid, vanillic acid, or p-hydroxybenzoic acid.

[0007] Further specified, the mixed phenolic acid is composed of ferulic acid, vanillic acid and p-hydroxybenzoic acid in a mass ratio of 1:1:1.

[0008] Further specified, the concentration of the phenolic acid is as high as 1200 mg / L.

[0009] The method for isolating and purifying Rhodococcus thermophilus RA1 described in this invention is characterized by the following specific steps: Rhizosphere soil from yam is collected using the root-shaking method. 10g of the rhizosphere soil is added to a conical flask containing 100mL PBS, shaken at 180rpm for 1 hour, and allowed to stand for 30 minutes. Then, 10mL of the supernatant is added to an inorganic salt culture medium containing a mixed phenolic acid concentration of 10mg / L. This mixed phenolic acid consists of ferulic acid, vanillic acid, and p-hydroxybenzoic acid in a mass ratio of 1:1:1. The medium is shaken and cultured for 10-20 days. Another 10mL is transferred to a culture medium containing a mixed phenolic acid concentration of 30mg / L and cultured for 10-20 days. The concentration of the mixed phenolic acid is gradually increased to enrich the microorganisms that degrade phenolic acid. The turbidity of the culture medium in the conical flask is observed. When the mixed phenolic acid concentration reaches 60mg / L, the inorganic salt culture medium shows... When the turbidity of the culture medium no longer increased, it was selected as the termination concentration for enrichment culture. Finally, 100 μL of the culture solution at the termination concentration was plated onto an inorganic salt solid medium with a mixed phenolic acid concentration of 60 mg / L and incubated at 37℃ for 72 h. The components of the inorganic salt solid medium, in g / L, were: ammonium sulfate 2 g / L, potassium dihydrogen phosphate 2 g / L, sodium chloride 5 g / L, disodium hydrogen phosphate 3 g / L, agar 20 g / L, and the remaining component was water. The pH was natural. When the colonies on the solid plate grew well, a single colony was picked and streaked onto the solid inorganic salt medium for isolation and purification to obtain the bacterial strain, namely Rhodococcus thermophilus.

[0010] The application of Rhodococcus thermophilus RA1 described in this invention in the preparation of crop biocontrol microbial agents.

[0011] The application of Rhodococcus thermophilus RA1 described in this invention in the preparation of microbial agents that inhibit the growth of plant pathogenic fungi.

[0012] The application of Rhodococcus thermophilus RA1 described in this invention in alleviating the growth-inhibiting effect of phenolic acid stress on plants.

[0013] The application of Rhodococcus thermophilus RA1 described in this invention in the preparation of a growth inhibitory agent for phenolic acid-dependent plant pathogenic fungi.

[0014] The present invention has the following advantages and beneficial effects: The present invention uses a shake-flask enrichment and isolation method to obtain a bacterial strain from the rhizosphere soil of yam. It was identified as Rhodococcus thermophilus by 16S rDNA sequencing and has the preservation number CGMCC No. 26299. This strain can efficiently degrade single or mixed phenolic acids at concentrations up to 1200 mg / L, thereby alleviating the direct stress of phenolic acids on plant growth. Moreover, it can inhibit the growth of plant pathogenic fungi, thereby controlling the invasion of pathogenic fungi on plants. This provides a new strain resource for the development of crop biocontrol microbial agents. Attached Figure Description

[0015] Figure 1 It refers to the degradation capacity of Rhodococcus thermophilus RA1 for single and mixed phenolic acids. A: 60 mg / L single or mixed phenolic acids; B: 60-1500 mg / L mixed phenolic acids.

[0016] Figure 2 It is the ability of HPLC-MS to detect the degradation of 1200 mg / L of single or mixed phenolic acids by Rhodococcus thermophilus RA1.

[0017] Figure 3 The inhibitory effect of phenolic acids on plant seed germination was relieved by Rhodococcus thermophilus RA1. A: Blank control; B: Phenolic acid degradation group; C: Phenolic acid control group.

[0018] Figure 4 This describes the inhibitory effect of Rhodococcus thermophilus RA1 on common plant pathogenic fungi (partial). T represents the treatment group; CK represents the control group. A: Fusarium oxysporum B: Fusarium proliferatum C: Fusarium fujikuroi .

[0019] Figure 5 It is the effect of Rhodococcus thermophilus RA1 in inhibiting phenolic acid-induced spore germination and hyphal growth of pathogenic fungi. Detailed Implementation

[0020] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0021] Rhizosphere soil from yam was collected using the root-shaking method. 10g of rhizosphere soil was added to a conical flask containing 100mL of PBS, shaken at 180rpm for 1 hour, and allowed to stand for 30 minutes. Then, 10mL of the supernatant was added to an inorganic salt medium containing a 10mg / L concentration of mixed phenolic acids (ferulic acid:vanillic acid:p-hydroxybenzoic acid = 1:1:1, hereinafter the same). The medium was shaken and cultured for 10-20 days. Another 10mL was transferred to a medium containing 30mg / L of mixed phenolic acids and cultured for 10-20 days. The concentration of mixed phenolic acids was gradually increased to enrich phenolic acid-degrading microorganisms. The turbidity of the medium in the conical flask was observed. If the microbial density in the enrichment medium significantly decreased at a certain mixed phenolic acid concentration, that concentration was selected as the termination concentration. In this experiment, the turbidity in the inorganic salt medium was observed to be no greater when the mixed phenolic acid concentration was 60mg / L; therefore, this concentration was selected as the termination concentration for the enrichment culture. Finally, 100 μL of the final concentration of culture solution was plated onto an inorganic salt solid medium with a mixed phenolic acid concentration of 60 mg / L and incubated at 37°C for 72 h. The inorganic salt solid medium consisted of the following components (g / L): ammonium sulfate 2 g / L, potassium dihydrogen phosphate 2 g / L, sodium chloride 5 g / L, disodium hydrogen phosphate 3 g / L, agar 20 g / L, and water as the remaining component, with a natural pH. When the colonies on the solid plates were growing well, single colonies were picked and streaked onto the inorganic salt solid medium for isolation and purification, yielding the bacterial strain Rhodococcus thermophilus RA1. The genetic characteristics of this bacterial strain are as follows: on the inorganic salt solid medium, the colonies are orange-yellow, round, and have a smooth, moist surface within 72 h; as the incubation time increases, the colonies gradually turn orange-red. 16S rDNA sequencing and BLAST alignment results showed that this strain had 92% similarity to Rhodococcus thermophilus. Its sequencing sequence is shown in SEQ ID NO:1. The purified strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26299 and deposit date of December 26, 2022.

[0022] Rhodococcus thermophilus has the ability to degrade high concentrations of phenolic acids: Example 1

[0023] Determination of the degradation ability of Rhodococcus thermophilus RA1 by ultraviolet spectrophotometer:

[0024] A single colony of *Rhodococcus thermophilus* RA1 was picked up using a sterile pipette tip and placed in an Erlenmeyer flask containing 100 mL of LB liquid medium. The flask was then incubated at 37°C and 180 rpm for 40 h using a shaker. 50 mL of the fermentation broth was taken, centrifuged at 5000 rpm for 2 min, the supernatant was removed, and the bacterial cells were resuspended in sterile water to achieve a concentration of 1 × 10⁻⁶. 8 CFU·mL -1 (OD)600 =1) Add 2 mL of the resuspended solution to 98 mL of inorganic salt culture medium (2 mL of sterile water was added for the control). The culture medium used three single phenolic acids or mixtures thereof as the sole carbon source, with total phenolic acid concentrations of 60 mg / L, 300 mg / L, 600 mg / L, 1000 mg / L, 1100 mg / L, 1200 mg / L, 1300 mg / L, 1400 mg / L, and 1500 mg / L, respectively, with three replicates for each treatment. The mixture was incubated at 37℃ in a shaker, and samples were taken every 24 hours. After centrifugation at 8000 rpm for 10 min, the supernatant was analyzed using UV-Vis spectrophotometry to determine the absorbance at the maximum absorption wavelength of the corresponding phenolic acid substance, and the degradation rate was calculated. The maximum absorption wavelengths for each substance were: ferulic acid 310 nm, vanillic acid 251 nm, p-hydroxybenzoic acid 246 nm, and the three mixed phenolic acids 248 nm. Degradation rate (DR) = (control absorbance - absorbance of treated sample) / control absorbance × 100%.

[0025] from Figure 1 As shown in Figure A, at a phenolic acid concentration of 60 mg / L, *Rhodococcus thermophilus* RA1 exhibited high degradation rates for both the three single phenolic acids and the mixed phenolic acids after 24 hours of cultivation. Furthermore, there was no significant difference in the final degradation rates among the four treatments, all exceeding 95%. Further, as... Figure 1 As shown, when the concentration of mixed phenolic acids was below 1200 mg / L, the final degradation rate of mixed phenolic acids by Rhodococcus thermophilus RA1 reached over 95% after 96 hours of shake-flask culture. At a phenolic acid concentration of 1300 mg / L, the degradation rate of phenolic acids by Rhodococcus thermophilus RA1 still reached over 95% after 192 hours. However, as the phenolic acid concentration continued to increase, the degradation ability of this Rhodococcus thermophilus rapidly decreased. Therefore, considering both degradation time and degradation rate, Rhodococcus thermophilus RA1 exhibits optimal degradation ability at a maximum phenolic acid concentration of 1200 mg / L. Example 2

[0026] HPLC-MS was used to verify the degradation ability of Rhodococcus thermophilus RA1:

[0027] After activation, Rhodococcus thermophilus RA1 was inoculated into 100 mL of LB medium and placed in a constant temperature shaking incubator. The culture was incubated at 37°C and 180 rpm for 40 h. Then, 20 mL of the fermentation broth was transferred to a centrifuge tube and centrifuged at 5000 rpm for 2 min. The supernatant was discarded, and the cells were resuspended in sterile water to achieve a concentration of 1 × 10⁻⁶. 8 CFU·mL -1 (OD) 600 =1) Take 2mL OD 600=1 bacterial culture was added to 98 mL of 1200 mg / L inorganic salt culture medium (2 mL of sterile water was added to the control). Each treatment was repeated in triplicate. The culture was carried out at 37 °C on a shaker, and samples were taken every 24 h. The fermentation broth was filtered through a 0.22 μm filter membrane and stored in brown bottles for later use.

[0028] Preparation of mixed phenolic acid standards: Weigh 0.004 g each of ferulic acid, vanillic acid, and p-hydroxybenzoic acid standards, dissolve them in 10 mL of inorganic salt culture medium to prepare a 1200 mg / L stock solution. Dilute the stock solution with inorganic salt culture medium to prepare a series of standard solutions of different mass concentrations (30 mg / L). -1 60 mg·L -1 120 mg·L -1 300mg·L -1 600mg·L -1 1200mg·L -1 Filtered through a 0.22μm filter membrane and stored in a brown bottle for later use.

[0029] Chromatographic conditions: Mobile phase: 0.1% (volume fraction, same below) formic acid (A) and methanol (B); Gradient elution program: 0–1 min, 5% B, 1–2 min, 30% B, 2–3 min, 95% B, 3–5.5 min, 95% B, 5.5–6 min, 5% B; Flow rate: 0.3 mL / min -1 Column temperature: 30℃; Injection volume: 10μL.

[0030] Mass spectrometry conditions: Electrospray ionization (ESI) source: negative ion scanning, electrospray voltage 3500V, ion source temperature 350℃, nebulization temperature 350℃, sheath gas pressure 40Arb, auxiliary gas pressure 10Arb, make-up gas pressure 0Arb. Plot the peak area (mAu) on the ordinate (Y) and the concentration of the mixed standard solution (mg·L⁻¹) on the analyte. -1 Plot a standard curve with X as the x-axis, and calculate the linear regression equation and correlation coefficient of the three mixed phenolic acid standards.

[0031] like Figure 2 As shown, at a phenolic acid concentration of 1200 mg / L, the degradation rate of the three single phenolic acids or their mixtures reached over 95% after 72 hours of inoculation, with a slight increase thereafter. This indicates that *Rhodococcus thermophilus* RA1 can utilize high concentrations of single or mixed phenolic acids as its energy source, making it a strain with significant potential application value in alleviating plant autotoxicity. Example 3

[0032] The effect of Rhodococcus thermophilus RA1 on relieving the stress of phenolic acids on plant growth:

[0033] Chinese ginseng seeds were selected as the experimental subject. The seeds were soaked in 75% ethanol for 2 minutes, then rinsed three times with sterile water for disinfection. A single colony of *Rhodococcus thermophilus* RA1 was picked up using a sterile pipette tip and placed in an Erlenmeyer flask containing 100 mL of LB liquid medium. The flask was then incubated at 37℃ and 180 rpm for 40 hours. 50 mL of the fermentation broth was centrifuged at 5000 rpm for 2 minutes, the supernatant was removed, and the bacterial cells were resuspended in sterile water to achieve a concentration of 1 × 10⁻⁶. 8 CFU·mL -1 (OD) 600 =1) Add 2 mL of the resuspended bacterial solution to 98 mL of 1 / 2 MS medium (add 2 mL of sterile water to the control). The culture medium uses three mixed phenolic acids as the sole carbon source, with a phenolic acid concentration of 1200 mg / L. Three parallel experiments are set up for each treatment. The mixture is incubated at 37℃ in a shaker for 72 h, centrifuged at 5000 r / min for 2 min, and the supernatant is retained for later use. Three experimental groups are designed: A is the blank control (sterile 1 / 2 MS medium), B is the supernatant after degradation, and C is 1 / 2 MS medium with a phenolic acid concentration of 1200 mg / L. Sterilized *Clerodendrum inerme* seeds are placed in sterile petri dishes, with 20-50 seeds per treatment, and three replicates are set up. The mixture is incubated at 25℃ for 60 h, and the germination of *Clerodendrum inerme* seeds is observed, and root length is recorded.

[0034] The results are as follows Figure 3 Compared with the phenolic acid control group (C), the fermentation broth treated with Rhodococcus thermophilus RA1 significantly promoted the germination of Chinese green seeds, and the root length could almost recover to the level of the blank control, indicating that Rhodococcus thermophilus RA1 can relieve plant growth inhibition caused by phenolic acid substances. Example 4

[0035] The inhibitory effect of Rhodococcus thermophilus RA1 on plant pathogenic fungi:

[0036] Select pathogenic fungal hyphae and inoculate them onto potato dextrose agar (PDA) medium. Incubate at 28°C for five days. Select areas with uniform mycelial morphology and good growth, and inoculate each area with a 7.5 mm diameter mycelial disc onto the PDA. At a distance of 2 cm from the mycelial disc, streak in 2 μL of *Rhodococcus thermophilus* RA1 culture in the logarithmic growth phase. Use PDA inoculated only with the pathogen as a control. Incubate the petri dishes at 28°C for 3–5 days. Observe the inhibitory effect of *Rhodococcus thermophilus* RA1 on the pathogenic fungus. Figure 4 Rhodococcus thermophilus RA1 Fusarium proliferatum , Fusarium fujikuroi and Fusarium oxysporum It has a certain degree of direct inhibitory effect. Compared with CK, the main manifestation is that the mycelial layer of pathogenic fungi is thin near RA1 and cannot grow normally. Example 5

[0037] The effects of phenolic acids on the spore germination and mycelial growth of *Rhodococcus thermophilus* RA1 were confirmed using a liquid shake-flask experiment. The specific experimental procedure was as follows: A single colony of *Rhodococcus thermophilus* RA1 was picked up using a sterile pipette tip and placed in an Erlenmeyer flask containing 100 mL of LB liquid medium. The flask was then incubated at 37°C and 180 rpm for 40 h using a shaker. 50 mL of the fermentation broth was taken, centrifuged at 5000 rpm for 2 min, the supernatant was removed, and the bacterial cells were resuspended in sterile water to achieve a concentration of 1 × 10⁻⁶. 8 CFU·mL -1 (OD) 600 =1). 2 mL of the resuspended bacterial solution was added to an inorganic salt culture medium containing three mixed phenolic acids as the sole carbon source, serving as the treatment group. The inorganic salt culture medium and the inorganic salt culture medium containing the mixed phenolic acids served as controls. The total phenolic acid concentrations for the treatment group and the control group (inorganic salt culture medium containing the mixed phenolic acids) were set at 30 mg / L, 60 mg / L, 120 mg / L, 300 mg / L, 600 mg / L, and 1200 mg / L, with three replicates for each treatment. The mixture was incubated at 37℃ in a shaker for 72 h, centrifuged, and the supernatant from each control and treatment group was collected and stored for later use. 5 mL of the pathogenic fungal spore suspension was added to 30 mL of different supernatants and incubated with shaking at 28℃. Samples were taken every 2 h for microscopic examination. During microscopic examination, five fields of view were randomly selected from each sample, with at least 30 spores in each field of view. Spore germination rate and hyphal length were recorded. The criteria for spore germination were: germ tube length exceeding half the spore diameter; for spores that were not perfectly round, the shorter diameter was used; and germ tubes exceeding twice the diameter of the mycelium were considered to have developed into mycelium. The spore germination and mycelial growth after 48 hours of treatment are shown below. Figure 5 After 48 hours of treatment, the spore germination rates of the blank control group, phenolic acid control group, and bacterial suspension treatment group were 28.67%, 100%, and 34.35%, respectively. The lengths of the germinating hyphae differed significantly. The hyphae in the blank control group and bacterial suspension treatment group were approximately 10 μm long, while the hyphae in the phenolic acid control group were significantly longer than the other two groups, ranging from 10 to 60 μm. Therefore, *Rhodococcus thermophilus* RA1 can indirectly inhibit the germination of pathogenic microorganism spores and the growth of hyphae by degrading phenolic acids, thereby inhibiting the growth of pathogenic microorganisms.

[0038] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles of the present invention, and all such changes and modifications fall within the protection scope of the present invention.

Claims

1. A thermophilic Rhodococcus strain that efficiently degrades phenolic acids Rhodococcus aetherivorans RA1, characterized in that: The thermophilic Rhodococcus was deposited on December 26, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26299. The phenolic acid is a single phenolic acid or a mixture of phenolic acids. The single phenolic acid is ferulic acid, vanillic acid, or p-hydroxybenzoic acid. The mixture of phenolic acids is at least two of ferulic acid, vanillic acid, or p-hydroxybenzoic acid.

2. The thermophilic Rhodococcus thermophilus that efficiently degrades phenolic acids according to claim 1. Rhodococcus aetherivorans RA1, characterized in that: The mixed phenolic acid is composed of ferulic acid, vanillic acid and p-hydroxybenzoic acid in a mass ratio of 1:1:

1.

3. The thermophilic Rhodococcus thermophilus that efficiently degrades phenolic acids according to claim 1. Rhodococcus aetherivorans RA1, characterized in that: The concentration of the phenolic acid is as high as 1200 mg / L.

4. The thermophilic rhodococcus according to any one of claims 1 to 3 Rhodococcus aetherivorans Application of RA1 in the preparation of crop biocontrol microbial agents.

5. The thermophilic rhodococcus as described in any one of claims 1 to 3 Rhodococcus aetherivorans Application of RA1 in the preparation of microbial agents that inhibit the growth of plant pathogenic fungi.

6. The thermophilic Rhodococcus according to any one of claims 1 to 3 Rhodococcus aetherivorans Application of RA1 in the preparation of phenolic acid-dependent growth inhibitors for plant pathogenic fungi.

7. The thermophilic rhodococcus according to any one of claims 1 to 3 Rhodococcus aetherivorans Application of RA1 in the preparation of microbial agents that alleviate the inhibitory effect of phenolic acid stress on plant growth.

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