Stenotrophomonas rhizophila, microbial inoculant thereof and application

Abstract

This invention relates to the field of microbial technology, specifically disclosing a rhizotrophic oligotrophosome, its microbial inoculant, and its applications. The rhizotrophic oligotrophosome (Stenotrophomonas rhizophila) JB-1 has the preservation number CGMCC No. 29124. The rhizotrophic oligotrophosome JB-1 provided by this invention has a wide pH growth range and salt stress tolerance, and exhibits excellent degradation effects on various phenolic acid autotoxic substances. It also has excellent control effects on continuous cropping obstacles caused by various phenolic acids. Furthermore, rhizotrophic oligotrophosome JB-1 has good growth-promoting properties, can stably promote crop growth, and is beneficial to the healthy development of the agricultural industry and ecological environmental protection, thus possessing high practical value.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a rhizotrophic oligotrophosome, its microbial inoculum, and its applications. Background Technology

[0002] Continuous cropping obstacle refers to the phenomenon where the same crop is planted on the same land for many consecutive years, leading to abnormal crop growth, reduced yield, lower quality, and increased pests and diseases. Many factors contribute to soil continuous cropping obstacle, among which the accumulation of phenolic acid autotoxic substances released by the crop during its growth process in the soil is the main cause. Currently known phenolic acid autotoxic substances include syringic acid, sinapic acid, vanillic acid, chlorogenic acid, caffeic acid, ellagic acid, ferulic acid, lauryl acid, benzoic acid, gallic acid, salicylic acid, and p-hydroxybenzoic acid. The accumulation of these autotoxic substances in the soil leads to soil compaction, damage to soil structure, and nutrient imbalance, which in turn inhibits seed germination, affects plant root development, and ultimately hinders normal crop growth.

[0003] Currently, methods for addressing phenolic acid autotoxic substances in soil include soil replacement, chemical treatment, biochar adsorption, biochar-chemical oxidant synergistic treatment, and microbial remediation. Among these, microbial remediation is economical, efficient, and environmentally friendly, and has broad application prospects in controlling continuous cropping obstacles. However, soil acidification and salinization are widespread, and many bacterial strains exhibit poor acid and salt tolerance, hindering the full effectiveness of microbial remediation. Therefore, there is an urgent need to screen for phenolic autotoxic degrading bacteria with a wide growth pH range and tolerance to salt stress, providing excellent microbial resources for the microbial remediation of autotoxic substances in soil. Summary of the Invention

[0004] To address the issues that existing strains used for degrading phenolic acid autotoxic substances have poor acid and salt tolerance, and that their degradation efficiency for phenolic acids needs further improvement, this invention provides a rhizotrophic oligotrophosome, its microbial agent, and its applications.

[0005] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0006] In a first aspect, the present invention provides a Stenotrophomonas rhizophila JB-1, which has the accession number CGMCC No.29124.

[0007] Stenotrophomonas rhizophila JB-1 was screened from farmland around Baoding City and classified as Stenotrophomonas rhizophila. It was deposited on November 23, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 29124. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] To address the shortage of existing strains of bacteria that degrade phenolic acid autotoxic substances, especially those applicable to acidified and saline soils, this invention provides a novel rhizotrophic oligotrophic bacterium, JB-1, which exhibits broad-spectrum degradation of phenolic acid autotoxic substances, as well as a wide range of growth pH and salt stress tolerance. It can effectively prevent and control continuous cropping obstacles caused by phenolic acid autotoxic substances and has broad application prospects in agricultural production.

[0009] The biological characteristics of the rhizotrophic oligotrophoblast JB-1 provided by this invention are as follows: it is pale yellow on LB solid medium, round in shape, smooth and opaque, with a raised center, neat edges, short rod-shaped, without spores or capsules, and with a diameter of about 1 to 2 mm.

[0010] The described *Oligotrophomonas radiata* JB-1 is a Gram-negative bacterium, showing positive results for methyl red test, catalase test, citrate utilization test, organic acid test, indole test, salt tolerance test, phosphatase test, and starch hydrolysis test. It is negative for Gram staining, H2S production test, urease assay, gelatin liquefaction test, and VP test.

[0011] Secondly, the present invention also provides the application of the above-mentioned root-loving oligotrophomonas JB-1 in the prevention and control of continuous cropping obstacles in plants.

[0012] The Stenotrophomonas rhizophila JB-1 provided by this invention can tolerate NaCl concentrations of 1% to 11%, grow at pH ranges of 4 to 12, and at temperatures of 25°C to 40°C. It has a wide range of growth pH and salt stress tolerance, and also exhibits broad-spectrum degradation of phenolic autotoxic substances. It provides a new strain resource for controlling continuous cropping obstacles in plants, which is conducive to promoting the healthy development of the agricultural industry and ecological environmental protection, and has high practical value.

[0013] Thirdly, the present invention also provides the application of the above-mentioned root-loving oligotrophomonas JB-1 in the degradation of p-hydroxybenzoic acid, ferulic acid, gallic acid and / or salicylic acid.

[0014] The aforementioned root-loving oligotrophomonas JB-1 exhibits broad-spectrum tolerance to phenolic acid autotoxic substances and can grow using syringic acid, sinapic acid, vanillic acid, chlorogenic acid, caffeic acid, ellagic acid, ferulic acid, komaric acid, benzoic acid, gallic acid, salicylic acid, or p-hydroxybenzoic acid as the sole carbon source. Its degradation efficiencies for p-hydroxybenzoic acid, ferulic acid, gallic acid, and salicylic acid are 98%, 99%, 98%, and 98%, respectively.

[0015] Fourthly, the present invention also provides the application of the above-mentioned root-loving oligotrophomonas JB-1 in promoting seed germination and plant growth in saline, acidic or alkaline soils.

[0016] Fifthly, the present invention also provides the application of the above-mentioned rhizotrophic oligotrophomonas JB-1 in the preparation of indoleacetic acid.

[0017] The root-loving oligotrophoblast JB-1 provided by this invention has excellent ability to produce indoleacetic acid (IAA). After 48 hours of culture, the IAA secretion capacity can reach up to 13.56 mg / mL, which can improve seed germination rate and promote plant growth.

[0018] Furthermore, the plant is corn, wheat, cucumber, or pepper.

[0019] Furthermore, the root-loving oligotrophomonas JB-1 can significantly promote the growth of pepper plants.

[0020] In a sixth aspect, the present invention also provides the application of indoleacetic acid, a metabolite of the above-mentioned root-loving oligotrophomonas JB-1, in the preparation of plant growth promoters.

[0021] In a sixth aspect, the present invention also provides a microbial inoculant for preventing and controlling plant continuous cropping obstacles, comprising the above-mentioned root-loving oligotrophomonas JB-1.

[0022] In a seventh aspect, the present invention also provides a biocontrol agent for promoting plant growth, comprising the above-mentioned root-loving oligotrophomonas JB-1 or its metabolites.

[0023] Preferably, the metabolite contains indoleacetic acid.

[0024] This invention also provides a method for preparing the above-mentioned biocontrol agent for promoting plant growth, comprising the following steps:

[0025] The root-loving oligotrophomonas JB-1 was inoculated into LB solid medium for activation; the activated cells were inoculated into LB liquid medium and cultured at 28℃~32℃ and 110r / min~130r / min for 18h~24h to obtain the biocontrol agent that promotes plant growth.

[0026] For example, the LB solid culture medium comprises: 10g tryptone, 5g yeast extract, 10g NaCl, 20g agar, and 1000mL distilled water. The LB liquid culture medium comprises: 10g tryptone, 5g yeast extract, 10g NaCl, and 1000mL distilled water.

[0027] For example, the culture is sterilized at 121°C for 20 minutes and then placed in a clean bench for later use.

[0028] It should be noted that after liquid or solid-state fermentation of *Oligotrophomonas rhizophila* JB-1, a bacterial suspension or culture is obtained. This bacterial suspension or culture is then compounded with excipients or carriers acceptable in the art to prepare wettable powders, water-dispersible granules, suspensions, emulsions, water-in-oil emulsions, or microemulsions for controlling continuous cropping obstacles or promoting plant growth. This invention does not impose specific limitations on the process of preparing various formulations from fermentation broth or bacterial cultures; existing techniques in the art are sufficient.

[0029] Eighthly, the present invention also provides a method for preventing and controlling plant continuous cropping obstacles, wherein the aforementioned root-loving oligotrophic bacterium JB-1 is added to the soil during the early stage of plant growth.

[0030] The *Oligotrophomonas radiata* JB-1 provided by this invention has a wide pH growth range and salt stress tolerance, and exhibits excellent degradation effects on various phenolic acid autotoxic substances. It also has excellent control effects on continuous cropping obstacles caused by various phenolic acid substances. At the same time, *Oligotrophomonas radiata* JB-1 has good growth-promoting characteristics and can stably promote crop growth. It has broad application prospects in agricultural production and high promotion value. Attached Figure Description

[0031] Figure 1 This is the colony morphology of the rhizotrophic oligotrophomonas JB-1 of the present invention on LB solid medium;

[0032] Figure 2 This is a scanning electron microscope image of the rhizotrophic oligotrophomonas JB-1 of the present invention;

[0033] Figure 3 This is the phylogenetic tree constructed based on the 16S rDNA of *Oligotrophomonas rhizophila* JB-1 in this invention;

[0034] Figure 4 The figures show the effects of the rhizotrophic oligotrophozoans JB-1 of this invention on the germination of corn, wheat, cucumber, and pepper seeds. In the figures, a represents cucumber seeds cultured on day 3, b represents wheat seeds cultured on day 5, c represents corn seeds cultured on day 5, and d represents pepper seeds cultured on day 10. In each group of figures, the left figure represents seeds treated with sterile water, and the right figure represents seeds treated with rhizotrophic oligotrophozoans JB-1 bacterial solution. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0036] Solutions used in the examples:

[0037] Phenolic acid autotoxic substances were dissolved in methanol to obtain a phenolic acid autotoxic substance stock solution with a concentration of 1000 mg / L.

[0038] Culture medium used in the examples:

[0039] Inorganic salt liquid culture medium: Na2HPO4·12H2O 1.5g; CaCl2·2H2O 0.01g; KH2PO4 1.5g; MgSO4·7H2O 0.2g; (NH4)2SO4 2g; trace element solution 1mL, distilled water to a final volume of 1000mL, adjust pH to 7.0-7.2, sterilize at 121℃ for 20min, and use after cooling.

[0040] Inorganic salt solid culture medium: Na2HPO4·12H2O 1.5g; CaCl2·2H2O 0.01g; KH2PO4 1.5g; MgSO4·7H2O 0.2g; (NH4)2SO4 2g; trace element solution 1mL; agar 15g; distilled water to a final volume of 1000mL, adjust pH to 7.0-7.2, sterilize at 121℃ for 20min, and use after cooling.

[0041] Trace element solution: FeSO4·7H2O 0.004g; ZnSO4·7H2O 0.178g; MnSO4·H2O 0.008g; CuCl2·2H2O 0.05g; H3BO3 0.1g; CoCl2·6H2O 0.1832g; Na2MoO4·2H2O 0.1g; HCl 0.1M, diluted to 1000mL with distilled water.

[0042] Inorganic salt culture medium for phenolic acid autotoxic substances: Using 1000 mg / L of phenolic acid autotoxic substance as the stock solution, add the 1000 mg / L stock solution of phenolic acid autotoxic substance to the inorganic salt liquid culture medium to prepare an inorganic salt culture medium with the target final concentration of phenolic acid autotoxic substance.

[0043] LB liquid medium: 10.0g tryptone, 5.0g yeast extract, 10.0g NaCl, 1000mL distilled water, pH 7.2±0.05, sterilized at 121℃ for 20min, and placed in a clean bench for later use.

[0044] LB solid medium: 10.0g tryptone, 5.0g yeast extract, 10.0g NaCl, 20g agar, 1000mL distilled water, pH 7.2±0.05, sterilized at 121℃ for 20min, and placed in a clean bench for later use.

[0045] Example 1

[0046] 1. Screening of rhizotrophic oligotrophomonas

[0047] Microbial enrichment and isolation:

[0048] Weigh 1g of soil sample (collected from farmland around Baoding) and place it in a 250mL Erlenmeyer flask containing 100mL of sterile water and glass beads. Shake in a shaker at 30℃ for 30min to obtain a soil mixture. Then, add 10mL of the soil mixture to 90mL of inorganic salt liquid medium and add the prepared p-hydroxybenzoic acid stock solution (concentration 1000mg / L). Use inorganic salt liquid medium containing a final p-hydroxybenzoic acid concentration of 20mg / L for acclimatization culture. After 48h of culture, take 10mL of the bacterial suspension and continue acclimatization in fresh inorganic salt liquid medium containing 20mg / L p-hydroxybenzoic acid. Repeat the acclimatization process 5-6 times. Spread the bacterial suspension on LB solid medium and incubate at 30℃ until single colonies grow. Then, repeatedly streak the single colonies until uniform colonies are visible under a microscope. Transplant for at least 5 generations to ensure a pure culture strain is obtained.

[0049] Strain purification:

[0050] The isolated and screened single strains were used as test strains. The grown single colonies were then cultured sequentially on inorganic salt liquid medium containing p-hydroxybenzoic acid, ferulic acid, syringic acid, and vanillic acid at a concentration of 300 mg / L, prepared from the stock solution of phenolic acid autotoxin. The culture was carried out at 30°C until colonies grew. Strains with good growth performance on phenolic acid autotoxin medium were screened. The strains with good growth were repeatedly streaked until uniform colonies were grown under a microscope. The strains were continuously subcultured for more than 5 generations to ensure that pure culture strains were obtained.

[0051] 2. Morphological observation and physiological and biochemical identification

[0052] Strain JB-1 was inoculated onto LB solid medium and incubated in a 30°C incubator for about 48 hours. The colony morphology, color, transparency, and raised edges of the colonies on the medium were observed.

[0053] Strain JB-1, morphologically characterized on LB solid medium, is pale yellow, round, smooth, opaque, with a raised center, regular edges, no spores, and a diameter of approximately 1–2 mm. Figure 1(As shown); Cell morphology was observed using scanning electron microscopy. The cells of strain JB-1 were short rod-shaped (as shown). Figure 2 (As shown).

[0054] The obtained strain JB-1 was subjected to Gram staining, methyl red test, H2S production test, catalase test, citrate utilization test, organic acid assay, indole test, salt tolerance test, phosphatase assay, urease assay, starch hydrolysis, gelatin liquefaction, and VP assay according to the "Handbook of Systematic Identification of Common Bacteria". The results are shown in Table 1.

[0055] Table 1 Physiological and biochemical characteristics of strain JB-1

[0056]

[0057]

[0058] Molecular biological identification

[0059] Strain JB-1 was inoculated into LB liquid medium and cultured in a shaker at 30°C for 24 h. Genomic DNA of the strain was extracted using the SK8255 (bacteria) kit from Sangon Biotech (Shanghai) Co., Ltd. The extracted total DNA was then used as a template for PCR amplification using primer pairs 27F (5′-AGTTTGATCMTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′).

[0060] The PCR reaction system consisted of: 10×PCR Buffer: 2.5 μL, Mg... 2+ Ingredients: 2 mM, dNTPs: 0.5 μl (10 mmol / mL), Taq enzyme: 0.5 μL (5 U / μL), DNA template: 30–50 ng, primers: 1 μL each (10 μmol / L), ddH2O: added to 25 μL. PCR amplification program: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ primer extension for 90 s, for a total of 32 cycles, followed by a final extension at 72℃ for 10 min, and termination of the reaction at 4℃. The amplified products were subjected to 1.6% agarose gel electrophoresis to obtain the 16S rDNA band. The target band was purified and recovered, and the PCR products were sequenced by Sangon Biotech (Shanghai) Co., Ltd. The amplification results of 16S rDNA are as follows:

[0061]

[0062] The 16S rDNA sequence of *Oligotrophomonas rhizophila* JB-1 was compared with sequences in GenBank to obtain the 16S rDNA sequences of similar standard strains of the *Oligotrophomonas* genus. The evolutionary distance of this *Oligotrophomonas rhizophila* and related strains was determined, and a phylogenetic tree was constructed. Figure 3 As shown. Based on 16S rDNA sequence similarity analysis, strain JB-1 was identified as Stenotrophomonas rhizophila.

[0063] The rhizotrophic oligotrophosome JB-1 was deposited on November 23, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 29124. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0064] Example 2

[0065] Growth curve of Oligotrophomonas radiata JB-1

[0066] Activation of the strain: Root-loving oligotrophozoites JB-1, stored at -80℃, were inoculated onto LB solid medium and cultured overnight at 30℃. Single colonies were picked and inoculated into 500 mL of LB liquid medium, and cultured at 30℃ with shaking at 120 rpm for 24 h to obtain the strain enrichment culture.

[0067] The enriched culture of the above-mentioned strains was inoculated at a rate of 1% into 250 mL Erlenmeyer flasks containing 100 mL of LB liquid medium. Three parallel experiments were conducted, and the cultures were incubated at 30 °C with shaking at 120 rpm. Starting from h0, 3 mL of bacterial culture was aseptically aspirated every h, and the absorbance at 600 nm was measured. The culture was zeroed with sterile water. When the bacterial cells reached the plateau phase, the absorbance was measured every 2 h. A growth curve was plotted with time on the x-axis and absorbance on the y-axis, as shown in Table 2.

[0068] Table 2 Growth curves of *Oligotrophomonas rhizophila* JB-1

[0069]

[0070]

[0071] The results showed that the rhizotrophic oligotrophoblast JB-1 entered the logarithmic growth phase after 3 hours of culture and entered the stationary phase after 8 hours.

[0072] Example 3

[0073] Effect of NaCl concentration on the growth of *Oligotrophomonas rhizophila* JB-1

[0074] The enrichment broth of the strain from Example 2 was inoculated at a rate of 10% into 250 mL Erlenmeyer flasks containing 50 mL of LB liquid medium. The initial NaCl concentration in the LB liquid medium was set to 1%, 3%, 5%, 7%, 9%, and 11%, with each treatment repeated three times. The flasks were incubated at 30°C and 120 rpm on a shaker. After 96 h of incubation, the OD of the strain was measured. 600 The effects of different NaCl concentrations on the growth of *Oligotrophomonas rhizophila* JB-1 were compared, and the results are shown in Table 3.

[0075] Table 3. Effects of different NaCl concentrations on the growth OD of *Oligotrophomonas rhizophila* JB-1 600 Impact

[0076]

[0077] The results showed that *Oligotrophomonas rhizophila* JB-1 could grow rapidly in the range of NaCl concentrations from 1% to 11%. After 96 hours of culture, the OD of the bacterial culture was [data missing]. 600 The values ​​all reached above 1.7, and the optimal NaCl concentration was 1%.

[0078] Example 4

[0079] Effect of pH on the growth of Oligotrophomonas rhizophila JB-1

[0080] The enrichment broth of the strain from Example 2 was inoculated at a 10% inoculum into 250 mL Erlenmeyer flasks containing 50 mL of LB liquid medium. The pH of the LB liquid medium was set to 4, 5, 6, 7, 8, 9, 10, 11, and 12. Each treatment was repeated three times. The flasks were incubated at 30 °C and 120 rpm on a shaker. After 96 h of incubation, the OD of the strain was measured. 600 The effects of different pH values ​​on the growth of *Oligotrophomonas radiata* JB-1 were compared, and the results are shown in Table 4.

[0081] Table 4. Effects of different pH values ​​on the growth OD of *Oligotrophomonas rhizophila* JB-1 600 Impact

[0082]

[0083] The results showed that the rhizotrophic oligotrophosome JB-1 has a wide pH growth range, and can grow in the pH range of 4 to 12, with the optimal pH being 9.

[0084] Example 5

[0085] Effect of oxygen concentration on the growth of rhizotrophic oligotrophomonas JB-1

[0086] The enrichment broth of the strain from Example 2 was inoculated at a rate of 10% into a 250 mL serum bottle containing 50 mL of LB liquid medium. The bottle was sealed with a butyl rubber stopper and a screw cap, and a vacuum was created by purging with nitrogen. This process was repeated three times to fill the serum bottle with nitrogen. The bottle was then replaced with 100% pure oxygen at the appropriate concentrations. The headspace oxygen concentration of the serum bottle was controlled at 0%, 5%, 10%, 15%, and 21%, with each treatment repeated three times. The bottles were cultured at 30°C and 120 rpm on a shaker. After 96 hours of culture, the OD of the strain was measured. 600 The effects of different oxygen concentrations on the growth of *Oligotrophomonas radiata* JB-1 were compared, and the results are shown in Table 5.

[0087] Table 5. Effects of oxygen concentration on the growth (OD) of *Oxygen Species Oxidophilus JB-1* 600 Impact

[0088] <![CDATA[OD 600 ]]> 0.47 1.86 2.11 2.30 2.38

[0089] The results showed that the optimal oxygen concentration for the growth of *Oligotrophomonas rhizophila* JB-1 was 21%.

[0090] Example 6

[0091] Effects of different nitrogen sources on the growth of rhizotrophic oligotrophomonas JB-1

[0092] Different nitrogen sources were selected, with (NH4)2SO4, urea, beef extract, KNO3 and peptone replacing tryptone as nitrogen sources, and the concentration of each was 10 g / L. Other components of the LB liquid medium were the same as the original LB liquid medium.

[0093] The enrichment culture of the strain from Example 2 was inoculated at a rate of 10% into 250 mL Erlenmeyer flasks containing 50 mL of LB liquid medium. Each treatment was repeated in triplicate. The flasks were incubated at 30 °C and 120 rpm on a shaker. After 96 h of incubation, the OD of the strains was measured. 600 The effects of different nitrogen sources on the growth of *Oligotrophomonas rhizophila* JB-1 were compared, and the results are shown in Table 6.

[0094] Table 6. Effects of different nitrogen sources on the growth OD of *Oligotrophomonas rhizophila* JB-1 600 Impact

[0095] <![CDATA[OD 600 ]]> 1.02 1.47 1.68 2.25 2.23 2.26

[0096] The results showed that the root-loving oligotrophomonas JB-1 grew best when beef extract, peptone, and tryptone were used as nitrogen sources.

[0097] Example 7

[0098] Effects of different carbon sources on the growth of rhizotrophic oligotrophomonas JB-1

[0099] Different carbon sources were selected to replace yeast extract, namely mannitol, glucose, sucrose, soluble starch and D-galactose, with a concentration of 5 g / L. Other components of the LB liquid medium were the same as those of the original LB liquid medium.

[0100] The enrichment culture of the strain from Example 2 was inoculated at a rate of 10% into 250 mL Erlenmeyer flasks containing 50 mL of LB liquid medium. Each treatment was repeated in triplicate. The flasks were incubated at 30 °C and 120 rpm on a shaker. After 96 h of incubation, the OD of the strains was measured. 600 The effects of different carbon sources on the growth of *Oligotrophomonas rhizophila* JB-1 were compared, and the results are shown in Table 7.

[0101] Table 7. Effects of different carbon sources on the growth OD of *Oligotrophomonas rhizophila* JB-1 600 Impact

[0102] <![CDATA[OD 600 ]]> 1.53 1.76 1.56 1.71 1.44 2.23

[0103] The results showed that yeast extract was the optimal carbon source for Oligotrophic rhizogenes JB-1.

[0104] Example 8

[0105] Effects of different inorganic salts on the growth of rhizotrophic oligotrophomonas JB-1

[0106] Different inorganic salts were selected to replace NaCl in LB liquid medium, namely MnSO4·H2O, CaCO3, K2SO4, K2HPO4·3H2O, MgSO4·7H2O and CaCl2·2H2O, with a concentration of 10 g / L. Other components in LB liquid medium were the same as those in the original LB liquid medium.

[0107] The enrichment culture of the strain from Example 2 was inoculated at a rate of 10% into 250 mL Erlenmeyer flasks containing 50 mL of LB liquid medium. Each treatment was repeated in triplicate. The flasks were incubated at 30 °C and 120 rpm on a shaker. After 96 h of incubation, the OD of the strains was measured. 600 The effects of different inorganic salts on the growth of *Oligotrophomonas radiata* JB-1 were compared, and the results are shown in Table 8.

[0108] Table 8. Effects of different inorganic salts on the growth (OD) of *Oligotrophomonas radiata* JB-1. 600 Impact

[0109]

[0110] The results show that the optimal inorganic salt for *Oligotrophomonas radiata* JB-1 is CaCl2·2H2O.

[0111] Example 9

[0112] Effects of different inoculum sizes on the growth of *Oligotrophomonas rhizophila* JB-1

[0113] The enrichment broth of the strain from Example 2 was inoculated into 250 mL Erlenmeyer flasks containing 50 mL of LB liquid medium at inoculation rates of 5%, 10%, 15%, and 20%, respectively. Each treatment was repeated in triplicate. The flasks were incubated at 30 °C and 120 rpm on a shaker. After 96 h of incubation, the OD of the strains was measured. 600 The effects of different inoculum amounts on the growth of *Oligotrophomonas radiata* JB-1 were compared, and the results are shown in Table 9.

[0114] Table 9. Effects of different inoculum sizes on the growth OD of *Oligotrophomonas rhizophila* JB-1 600 Impact

[0115]

[0116] The results showed that the inoculation amount of rhizotrophic oligotrophomonas JB-1 was 5%–10%, with the optimal inoculation amount being 10%.

[0117] Example 10

[0118] Broad-spectrum tolerance of rhizotrophic oligotrophozoites JB-1 to phenolic acid autotoxic substances

[0119] The broad-spectrum activity of the bacteria against phenolic acid autotoxic substances (syringic acid, sinapic acid, vanillic acid, chlorogenic acid, caffeic acid, ellagic acid, ferulic acid, komaric acid, benzoic acid, gallic acid, salicylic acid, and p-hydroxybenzoic acid) was verified using 96-well plates.

[0120] The rhizotrophic oligotrophozoites JB-1 were activated and cultured according to the method in Example 2. The activated strain enrichment culture was inoculated into inorganic salt liquid culture medium at an inoculation rate of 10% to obtain the rhizotrophic oligotrophozoites JB-1 bacterial suspension.

[0121] The stock solution of a single phenolic acid autotoxic substance (1000 mg / L) was diluted with methanol to prepare 300 mg / L and 50 mg / L solutions of the single phenolic acid autotoxic substance.

[0122] 150 μL of the above-mentioned *Oligotrophomonas rhizophila* JB-1 bacterial suspension and 50 μL of different concentrations of single phenolic acid autotoxin dilutions were added to 96-well plates. The final concentrations of phenolic acid autotoxins in the 96-well plates were 12.5 mg / L and 75 mg / L, respectively. The single phenolic acid autotoxin was used as the sole carbon source, and the control group (CK group) was used without the addition of phenolic acid autotoxin. The plates were cultured in a shaker at 30℃ and 120 rpm, with each treatment replicated four times. The OD was measured after 96 h of culture. 600 The results are shown in Figure 10.

[0123] Table 10 Broad-spectrum tolerance of rhizotrophic oligotrophomonas JB-1 to phenolic acid autotoxic substances

[0124]

[0125]

[0126] The results showed that *Oligotrophomonas radiata* JB-1 has substrate diversity and can use syringic acid, sinapic acid, vanillic acid, chlorogenic acid, caffeic acid, ellagic acid, ferulic acid, komaric acid, benzoic acid, gallic acid, salicylic acid and p-hydroxybenzoic acid as the sole carbon source for growth and reproduction, and has good broad-spectrum tolerance to phenolic acid autotoxic substances.

[0127] Example 11

[0128] The ability of rhizotrophic oligotrophomonas JB-1 to degrade phenolic autotoxic substances

[0129] Following the method in Example 2, *Oligotrophomonas radiata* JB-1 was activated and cultured. The activated strain enrichment broth was inoculated into an inorganic salt liquid medium at a 10% inoculum. The medium used ferulic acid, p-hydroxybenzoic acid, gallic acid, and salicylic acid as the sole carbon source, respectively. The concentrations of the above-mentioned phenolic acid autotoxic substances were set at 50 mg / L and 300 mg / L, respectively. The culture was carried out in a shaker at 30°C and 120 rpm. The treatment group without *Oligotrophomonas radiata* JB-1 was used as the control group (CK group). Each treatment was repeated three times. On the third day of culture, samples were taken to determine the concentration of phenolic acid autotoxic substances. The results are shown in Table 11.

[0130] The degradation ability of *Oligotrophomonas rhizophila* JB-1 for phenolic acid autotoxic substances was determined by HPLC-MS. For each test, 5 mL of bacterial culture was centrifuged at 5000 rpm for 10 min. The supernatant was extracted twice with 5 mL of ethyl acetate. The aqueous and organic phases were then separated using a separatory funnel. The aqueous phase was discharged from the bottom, and the organic phase was poured out from the top. The mixture was dried using a nitrogen evaporator, and 5 mL of methanol was added to dissolve the solids. The dissolved solids were then drawn up with a 1 mL syringe and filtered through a 0.22 μm filter into a brown chromatographic vial for analysis.

[0131] Chromatographic conditions: Mobile phase A: 0.05% formic acid aqueous solution; Mobile phase B: methanol; Flow rate: 0.3 L / min; Column temperature: 25℃; Injection volume: 1 μL; Gradient elution program as follows:

[0132] 0 95 5 1.5 95 5 3 80 20 4 55 45 6.1 35 65 7 0 100 8 95 5 13 95 5

[0133] Mass spectrometry conditions: ESI, negative ion mode, nebulization temperature: 200℃, dry gas flow rate: 14L / min, nebulizer pressure: 35psi, sheath gas temperature: 350℃, sheath gas flow rate: 11L / min, capillary voltage: 3000V, nozzle voltage: 800V, residence time: 100, CE: 20.

[0134] Table 11 Degradation efficiency of phenolic autotoxic substances by rhizotrophic oligotrophomonas JB-1

[0135]

[0136] The results showed that at a concentration of phenolic acid autotoxic substances of 50 mg / L, the degradation efficiencies of *Oligotrophomonas rhizophila* JB-1 for p-hydroxybenzoic acid, ferulic acid, gallic acid, and salicylic acid were 95%, 99%, 96%, and 97%, respectively. At a concentration of 300 mg / L, the degradation efficiencies of *Oligotrophomonas rhizophila* JB-1 for p-hydroxybenzoic acid, ferulic acid, gallic acid, and salicylic acid were 98%, 99%, 98%, and 98%, respectively.

[0137] Example 12

[0138] The ability of rhizotrophic oligotrophomonas JB-1 to secrete indoleethylene (IAA)

[0139] Following the method in Example 2, *Oligotrophomonas radiata* JB-1 was activated and cultured. The activated strain's enriched culture was inoculated at a 1% inoculum into LB liquid medium containing 500 mg / L L-tryptophan (analytical grade). The medium was cultured on a shaker at 30°C and 120 rpm for 48 h. The bacterial culture was then centrifuged at 4°C and 10,000 rpm for 10 min. 3 mL of the supernatant was added to an equal volume of Salkowski reagent (150 mL concentrated sulfuric acid dissolved in 250 mL deionized water, with 7.5 mL of 0.5 mol / L FeCl3·6H2O solution (81.1 g FeCl3 dissolved in 1000 mL 0.1 mol / L dilute hydrochloric acid)). The mixture was incubated at room temperature in the dark for 30 min. The OD value was then measured. 530 The absorbance was measured using a UV spectrophotometer, and the obtained absorbance was substituted into the IAA standard curve to obtain the IAA yield of this strain. It was found that the concentration of IAA secreted by *Oligotrophomonas rhizophila* JB-1 reached 13.56 mg / L.

[0140] Example 13

[0141] Effects of root-loving oligotrophomonas JB-1 on seed germination rates of maize, wheat, cucumber, and pepper.

[0142] Following the method in Example 2, *Oligotrophomonas radiata* JB-1 was activated and cultured to obtain an activated *Oligotrophomonas radiata* JB-1 enriched culture medium with an effective viable count ≥3 billion / mL.

[0143] Seeds of corn, wheat, cucumber, and pepper were soaked in 95% alcohol for 5 seconds, followed by soaking in 75% alcohol for 5 minutes to sterilize the seed surface. The sterilized seeds were then thoroughly rinsed five times in sterile water. Afterward, the seeds were soaked in sterile water and a JB-1 enrichment culture of *Oligotrophomonas radiata* for 6 hours, with each treatment repeated three times, and 30 seeds per treatment. After soaking, the seeds were drained and placed in petri dishes containing two layers of sterile filter paper moistened with sterile water. The petri dishes were covered to prevent moisture evaporation from affecting seed germination. Each petri dish was labeled, and then placed in an intelligent light incubator to observe seed germination.

[0144] On day 3 of cultivation, the germination rate of cucumber seeds treated with sterile water and JB-1 bacterial solution was 100%. On day 5, the germination rate of corn seeds treated with sterile water and JB-1 bacterial solution was 94.44%, while the germination rates of wheat seeds treated with sterile water and JB-1 bacterial solution were 75.56% and 74.44%, respectively. On day 10, the germination rates of pepper seeds treated with sterile water and JB-1 bacterial solution were 57.78% and 51.52%, respectively. Furthermore, it was found that the sprouts of corn, wheat, cucumber, and pepper seeds treated with JB-1 bacterial solution grew significantly better than those treated with sterile water. Significant mold growth was observed in the corn and wheat seeds treated with sterile water, while the seeds in the JB-1 bacterial solution group remained healthy. Figure 4 As shown, this demonstrates that *Oligotrophomonas radiata* JB-1 promotes the growth of seed buds in maize, wheat, cucumber, and pepper.

[0145] Example 14

[0146] Effects of root-loving oligotrophomonas JB-1 on pepper growth

[0147] The chili pepper variety used was *Chili tamarind*, and the soil sample was ordinary farmland soil. After the soil was brought back to the laboratory, large stones and plant roots were removed, and the soil was sieved through a 5mm sieve and air-dried for later use. One chili pepper seedling of uniform growth was planted in each pot (150mm top diameter × 100mm bottom diameter × 150mm height) containing 1.5kg of soil that had passed through a 5mm sieve. Simultaneously, 300mL of *Oligotrophomonas radiata* JB-1 bacterial suspension (effective viable count ≥3 billion / mL) and 300mL of sterile water were added to the pots, with each treatment replicated three times. The pots were placed in a location avoiding direct sunlight, and the soil was kept moist during the growing period. The plant height of the chili pepper seedlings was recorded every 5 days to observe the growth of the chili peppers. On day 75 of cultivation, the chili pepper plant height in the sterile water treatment was 40cm, and the plant height in the bacterial suspension treatment was 44cm. This demonstrates that *Oligotrophomonas radiata* JB-1 significantly promoted the growth of chili pepper plant height.

[0148] In summary, the rhizotrophic oligotrophomonas JB-1 with accession number CGMCC NO.29124 provided by this invention has excellent salt and acid tolerance, can prevent and control continuous cropping obstacles caused by various phenolic acid autotoxic substances, and can also improve the seed germination rate of corn, wheat, cucumber and pepper, and promote the growth of corn, wheat, cucumber and pepper. It is a multifunctional strain with good ability to degrade phenolic acid autotoxic substances and promote plant growth, which is of great significance for ensuring the healthy and sustainable development of agriculture and has broad application prospects.

[0149] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A root-loving oligotrophomonad ( Stenotrophomonas rhizophila JB-1, characterized in that, Its accession number is CGMCC No.29124.

2. The application of the root-loving oligotrophomonas JB-1 as described in claim 1 in controlling plant continuous cropping obstacles, characterized in that, The continuous cropping obstacle is caused by p-hydroxybenzoic acid, ferulic acid, gallic acid and / or salicylic acid.

3. The use of the rhizotrophic oligotrophomonas JB-1 as described in claim 1 in the degradation of p-hydroxybenzoic acid, ferulic acid, gallic acid and / or salicylic acid.

4. The use of the rhizotrophic oligotrophoblast JB-1 as described in claim 1 in the preparation of indoleacetic acid.

5. A microbial inoculant for preventing and controlling continuous cropping obstacles in plants, characterized in that, The bacterium JB-1, which contains the root-loving oligotrophomonas according to claim 1, wherein the continuous cropping obstacle is caused by p-hydroxybenzoic acid, ferulic acid, gallic acid and / or salicylic acid.

6. A method for preventing and controlling continuous cropping obstacles in plants, characterized in that, In the early stages of plant growth, the root-loving oligotrophic monocytogenes JB-1 as described in claim 1 is added to the soil, wherein the continuous cropping obstacle is caused by p-hydroxybenzoic acid, ferulic acid, gallic acid and / or salicylic acid.

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