Pseudomonas for promoting plant growth and increasing content of paris saponins and application thereof

Abstract

The application discloses a pseudomonas for promoting plant growth and increasing content of paris saponin and application thereof, and belongs to the technical field of microbial application. Pseudomonas palleroniana The pseudomonas is classified and named as P6, has a preservation number of CCTCC M 20232513, and is derived from rhizosphere soil of two-year paris polyphylla Smith var. yunnanensis (fisch.) Hand.-Mazz. The application has the following beneficial effects: (1) the prepared fermentation bacterial liquid has the effects of dissolving phosphorus and decomposing potassium, is green, safe and environmentally-friendly compared with a large amount of chemical fertilizer, and can be used as a starting strain of functional microbial fertilizer; (2) the prepared bacterial liquid can produce IAA and has a good growth promoting effect on ryegrass, can be applied to forage production, increases yield and improves economic benefits; and (3) the prepared bacterial liquid promotes the growth of paris polyphylla Smith var. yunnanensis (fisch.) Hand.-Mazz and the content of paris saponin, is beneficial to paris polyphylla Smith var. yunnanensis (fisch.) Hand.-Mazz planting and resource development of paris saponin.

Description

Technical Field

[0001] This invention belongs to the field of bacterial application research technology, specifically relating to a Pseudomonas bacterium that promotes plant growth and increases the content of Paris polyphylla saponins, and its applications. Background Technology

[0002] Plant growth-promoting bacteria are a class of bacteria beneficial to plant growth and development, influencing plant health through various pathways. In recent years, increasing research has confirmed the importance of plant growth-promoting bacteria for agricultural production and ecosystems. Previous reports have shown that most of these microorganisms possess functions such as phosphorus solubilization, potassium solubilization, or nitrogen fixation, and can produce auxins, siderophores, and other substances that promote plant growth. Phosphorus is a key element for plant DNA, RNA, and energy metabolism (ADP and ATP), and is essential for plants. Potassium plays a crucial role in plant growth and development, stress resistance, yield, and quality. Typically, soil microorganisms gradually decrease in the rhizosphere and accumulate in the roots over time; therefore, soil nutrition is crucial for plant growth and the accumulation of effective components. Long-term, excessive application of chemical fertilizers to the soil can lead to soil compaction, fertilizer waste, reduced fertilizer efficiency, and environmental pollution. Microbial fertilizers, on the other hand, are products that utilize the life activities of microorganisms to induce specific fertilizer effects in plants. They are a type of fertilizer used in agricultural production and have become a good alternative to chemical fertilizers. The development of strains that can efficiently dissolve phosphorus and potassium and promote plant growth can improve crop nutrition conditions, create a favorable soil microecological environment, increase the content of available phosphorus and potassium in the soil, and promote crop growth.

[0003] Paris polyphylla, a traditional Chinese medicine, is widely used in prepared Chinese medicines and prescriptions. It is a major ingredient in over 80 prepared Chinese medicines, including Yunnan Baiyao, Gongxue Ning, Paris polyphylla detoxifying tincture, Loulian capsules, Sanqi Xueshang Ning capsules, pediatric antipyretic granules, nasopharyngeal detoxifying granules, Jinfukang oral liquid, Reduqing, and Jishengde snake medicine tablets, indicating significant market demand. As mentioned earlier, my country is the center of Paris polyphylla diversity and has the richest resources. Among them, Yunnan Paris polyphylla is one of the most representative and influential authentic medicinal plants in Yunnan, dominating industrial utilization, with wild resources being its main source. According to incomplete statistics, the annual demand for Paris polyphylla is around 1,500 tons, far exceeding the total wild Paris polyphylla reserves. Reports indicate that since the 1980s, the market price of Paris polyphylla has increased more than 400 times, from 2.7 yuan / kg to 1,100 yuan / kg in 2017. In recent years, the market price of Paris polyphylla has remained high. Driven by huge profits, long-term predatory harvesting has led to even greater scarcity of wild Paris polyphylla resources. Furthermore, long-term over-harvesting has damaged the growing environment of Paris polyphylla, and coupled with its low reproduction rate and long regeneration cycle (average 8-12 years), some varieties are on the verge of extinction. Therefore, in recent years, artificial cultivation of Paris polyphylla has gradually increased. However, the long cultivation cycle, high requirements for the growing environment and artificial cultivation techniques, and high investment risk of Paris polyphylla have not fundamentally changed, resulting in a still low actual yield. Therefore, the invention of a microbial fertilizer that can promote the growth and saponin synthesis of Paris polyphylla is of great significance for the utilization of Paris polyphylla resources. Summary of the Invention

[0004] The main objective of this invention is to provide a Pseudomonas bacterium that promotes plant growth and increases the saponin content of Paris polyphylla, and its application, which has great potential for use in the preparation of functional microbial fertilizers.

[0005] To achieve the above objectives, the present invention mainly provides the following technical solutions:

[0006] This invention provides a Pseudomonas bacterium that promotes plant growth and increases the content of Paris polyphylla saponins. The bacterium is Pseudomonas palleroniana P6, with accession number CCTCC M 20232513; the deposit location is China Center for Type Culture Collection; and the deposit date is December 8, 2023.

[0007] The present invention also provides applications of the aforementioned Pseudomonas, including for phosphorus solubilization, potassium solubilization, IAA production; and for promoting plant growth; particularly for promoting ryegrass growth.

[0008] Specifically, it is used to promote an increase in the height, root length, and number of roots of ryegrass.

[0009] The present invention also provides another application of the aforementioned Pseudomonas aeruginosa for promoting the growth of Paris polyphylla.

[0010] This includes methods to promote Paris polyphylla biomass and methods to increase the content of Paris saponin VII and Paris saponin I in Paris polyphylla.

[0011] The present invention also provides a Pseudomonas inoculum agent, which is prepared by fermentation culture of the aforementioned Pseudomonas.

[0012] The preparation method of the microbial agent includes the following steps:

[0013] Pseudomonas palleroniana P6 was inoculated into PDB liquid medium and placed in a shaker at 180-200 r / min. The culture temperature was 28-30℃, and the culture was carried out for 1-4 days to complete the preparation of the fermentation broth.

[0014] This invention also provides the application of the aforementioned Pseudomonas inoculant. When applied to planting soil, the Pseudomonas microbial inoculant promotes the content of available phosphorus and potassium in the soil, further promoting plant growth and, more importantly, increasing the content of Paris saponins in Paris polyphylla. In particular, it significantly increases the content of Paris saponins VII and I.

[0015] The selection culture medium for the bacteria is:

[0016] (1) Inorganic phosphorus culture medium: glucose 10.0g, sodium chloride 0.3g, magnesium sulfate heptahydrate 0.3g, ferrous sulfate heptahydrate 0.03g, manganese sulfate 0.02g, potassium chloride 0.3g, ammonium sulfate 0.5g, calcium phosphate 5.0g, agar 20.0g / L, distilled water 1.0L, pH=7.2±0.2;

[0017] (2) Potassium feldspar culture medium: glucose 10.0g, magnesium sulfate heptahydrate 0.5g, calcium chloride 0.1g, ferric chloride 0.005g, potassium feldspar powder 1.0g, distilled water 1.0L, pH=7.2±0.2.

[0018] This invention uses King's medium with / without L-tryptophan to test whether Pseudomonas produces IAA. The medium ratio is as follows: 0.5g soluble starch, 0.3g sodium pyruvate, 0.5g glucose, 0.5g casein amino acids, 0.5g tryptone, 0.5g yeast extract, 0.3g potassium dihydrogen phosphate, 0.05g magnesium sulfate heptahydrate, 0.0g / 0.5g L-tryptophan, 1.0L distilled water, pH 7.2±0.2.

[0019] The Pseudomonas microbial agent of the present invention, when applied to the soil in which Paris polyphylla is planted, can promote the content of available phosphorus and available potassium in the soil; can promote the biomass of Paris polyphylla; and can increase the content of Paris polyphylla saponins in Paris polyphylla.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0021] Compared with the application of large amounts of chemical fertilizers, the fermentation liquid prepared by this invention is green, safe and environmentally friendly, and can be used as the starting strain for functional microbial fertilizers. It has a good growth-promoting effect on ryegrass and can be applied to forage production in the future to increase yield and improve economic benefits. It also improves the growth of Paris polyphylla and the content of Paris polyphylla saponins, which is beneficial to the planting of Paris polyphylla and the resource development of Paris polyphylla saponins. Attached Figure Description

[0022] Figure 1 The growth morphology of strain Pseudomonas palleroniana P6 on both sides of the culture medium.

[0023] Figure 2 The effect of applying Pseudomonas palleroniana P6 fermentation broth on ryegrass growth.

[0024] Figure 3 The weight of Paris polyphylla roots in the experimental and control groups (***indicates p<0.05), (a) fresh weight; (b) dry weight.

[0025] Figure 4 Information on high performance liquid chromatography-mass spectrometry (HPLC-MS) for standards and representative samples. (a) HPLC chromatogram of standards; (b) HPLC chromatogram of FP sample; (c) HPLC chromatogram of FC sample; (d) Retention time and ion fragmentation information for each saponin. Detailed Implementation

[0026] The present invention is further illustrated by the following embodiments, but is not limited to these embodiments.

[0027] Example 1: Isolation and Identification of Phosphate-Solubilizing and Potassium-Solubilizing Microorganisms

[0028] In the Jiudingshan area of ​​Dali City, Yunnan Province (25°20′24″N, 100°29′18″E), rhizosphere soil samples from two-year-old Paris polyphylla plants were selected for isolation and screening to identify strains with phosphorus-solubilizing and potassium-solubilizing functions. 1g of rhizosphere soil samples were placed in 9mL of sterile water and rapidly shaken to prepare a soil-bacterial suspension. After settling, the supernatant was collected for 10... -4 10 -5 10 -6Serial dilutions were performed, with 100 μL of each serially diluted soil suspension spread onto inorganic phosphorus and potassium feldspar solid media. After inoculation, all media were sealed with sterile film and incubated at 30°C. Observations were made every 8 hours, and new colonies were picked and inoculated onto their respective solid media, repeating this process for 7 consecutive days. Colonies on the newly selected media were then streaked 3-4 times until a single colony was confirmed to grow. The screening medium formulations are as follows: Inorganic phosphorus medium: glucose 10.0g, sodium chloride 0.3g, magnesium sulfate heptahydrate 0.3g, ferrous sulfate heptahydrate 0.03g, manganese sulfate 0.02g, potassium chloride 0.3g, ammonium sulfate 0.5g, calcium phosphate 5.0g, distilled water 1.0L, pH=7.2±0.2; Potassium feldspar medium: glucose 10.0g, magnesium sulfate heptahydrate 0.5g, calcium chloride 0.1g, ferric chloride 0.005g, potassium feldspar powder 1.0g, distilled water 1.0L, pH=7.2±0.2. Liquid medium is prepared according to the above formulations, and the solid medium formulation is prepared by adding 20g / L agar. The prepared medium is sterilized at 121℃ before use.

[0029] A strain capable of dissolving both inorganic phosphorus and potassium feldspar was obtained through isolation and screening using two selective culture media. DNA was extracted from the strain using a bacterial genome extraction kit. Genome integrity was assessed using 1.5% agarose gel electrophoresis, and the quality and concentration of the genomic DNA were determined using a NanoDrop ND-1000 UV spectrophotometer. PCR amplification was performed using the universal 16S primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-AGAGTTTGATCCTGGCTCAG-3'). The PCR products were sequenced by Sangon Biotech (Shanghai) Co., Ltd., and the data was assembled using Bioedit software and uploaded to the NCBI database (https: / / www.ncbi.nlm.nih.gov / ). BLAST sequence alignment and homology analysis were performed, and a phylogenetic tree was constructed using MEGA X software using the neighbor-joining method. Pseudomonas palleroniana P6 showed high homology with the genus Pseudomonas. The growth of Pseudomonas palleroniana P6 on the culture medium is as follows: Figure 1 As shown.

[0030] Example 2: Verification of the multifunctionality of the strain

[0031] Nitrogen is a fundamental element in the formation of important organic compounds such as proteins, amino acids, nucleic acids, and chlorophyll in plants, and it has a significant impact on plant growth, development, disease resistance, and environmental adaptability. Four culture media were set up to validate the multifunctionality of the strains: potato dextrose medium, inorganic phosphorus medium, potassium feldspar medium, and Assumption nitrogen-fixing medium. PDA medium consisted of 200g potato, 20g glucose, 20.0g agar, and 1.0L distilled water, with a natural pH. Assumption nitrogen-fixing medium consisted of 10g mannitol, 5.0g calcium carbonate, 0.2g sodium chloride, 0.2g magnesium sulfate heptahydrate, 0.2g potassium dihydrogen phosphate, 0.1g calcium sulfate, 20.0g agar, and 1.0L distilled water, with a pH of 7.2 ± 0.2. A small amount of the purified *Pseudomonas palleroniana* P6 from Example 1 was transferred to a sterile centrifuge tube using a sterile toothpick. A bacterial suspension was prepared by adding PBS buffer. 2 μL of the suspension was inoculated into the center of each culture medium, with three replicates for each medium. The tubes were incubated at 30°C for 7 days, and their growth was observed. The colony diameter on each medium was measured daily as an indicator of growth rate. Growth status was categorized into four levels based on the average colony diameter on day 7: "-, +, ++, and +++". "-" indicates no growth on that medium, "+" indicates growth rate, and more "+" signs indicate a larger average colony diameter.

[0032] The multiplate growth experiments of Pseudomonas palleroniana P6 are shown in Table 1. Compared with PDA medium, it grew fastest on inorganic phosphorus medium and potassium feldspar medium, but hardly grew on Assumption nitrogen-fixing medium. Pseudomonas palleroniana P6 has phosphorus-solubilizing and potassium-solubilizing effects, but does not have nitrogen-fixing function.

[0033] Table 1 Growth rate of strains on different culture media

[0034]

[0035] A "-" indicates growth failure on this medium, while more "+"s indicate faster growth and the formation of larger lysis zones on this medium.

[0036] Example 3: Application of Functional Strains in Pots

[0037] 20 kg of soil from the plants grown in Example 1 was collected and brought back to the laboratory. Plant debris and large stones were removed, large clumps of soil were broken up and passed through a 2 mm sieve. The sieved soil was thoroughly mixed and then placed into 12 disposable small basins, each 8 cm in diameter and 10 cm high. The *Pseudomonas palleroniana* P6 strain was inoculated into PDB liquid medium and placed in a shaker at 180-200 rpm and 28-30°C for 2-4 days to obtain the fermentation broth. The fermentation broth was centrifuged to collect the bacterial cells, resuspended in PBS, and centrifuged again. The collected bacterial cells were diluted with sterile water and then applied to the small basins containing soil, with approximately 1 × 10⁻⁶ cells per basin. 9 Six pots were used as biological replicates, with one CFU inoculated with the inoculant, and the remaining six pots were used as a control group without inoculation but with an equal amount of sterile water. To ensure the full phosphorus and potassium solubilizing effects of the inoculant, the pots were placed in a light incubator, and the soil was kept moist by watering daily at 25°C for one month. Five ryegrass seeds were then planted in each pot, and the pots were placed in a light incubator at 25°C, with a light intensity of 1000 lx and a light-dark ratio of 10 h:14 h. After planting, the soil was kept moist by watering daily, and the growth of the ryegrass plants was observed. On the 15th day after germination, the ryegrass was harvested, washed, and the plant height, root length, and average number of roots per plant were measured.

[0038] Ryegrass germinated on the 7th day after planting. After 15 days of germination, there was a significant difference in the growth of ryegrass between the control group and the experimental group. Figure 2 The average plant height of ryegrass in the control group was 12.32 cm, while that in the experimental group reached 14.36 cm, representing a growth rate of 14.70%. The average root length in the control group was 4.72 cm, while that in the experimental group was 6.75 cm, representing a growth rate of 43.01%. The average number of roots per plant in the control group was 1.12, while that in the experimental group was 1.69, representing a growth rate of 50.89% (Table 2). The application of this treatment had a significant positive impact on the plant height, root length, and average number of roots per plant of ryegrass. Among these, strain Pseudomonas palleroniana P6 had a significant effect on the root growth of ryegrass, significantly promoting root formation and, to some extent, increasing the yield. Therefore, strain Pseudomonas palleroniana P6 has good application potential in promoting growth.

[0039] Table 2. Statistics on the growth of ryegrass in the experimental and control groups.

[0040]

[0041] Example 4: Validation of IAA production

[0042] The strain *Pseudomonas palleroniana* P6 from Example 1 was inoculated into 50 mL of PDB liquid medium and cultured on a shaker at 180-200 rpm and 28-30°C for 1 day. 100 μL of the bacterial suspension was inoculated into test tubes containing 2 mL of King's medium without L-tryptophan and King's medium containing L-tryptophan. Controls consisted of equal volumes of King's medium without L-tryptophan, King's medium containing L-tryptophan, and sterile water. Three biological replicates were established. The culture was then incubated on a shaker at 180-200 rpm and 28-30°C for 5 days. After centrifugation at maximum speed for 5 min, 1 mL of the bacterial suspension was taken, and 200 μL of the supernatant was added to a centrifuge tube containing an equal volume of S2 colorimetric solution. The mixture was thoroughly mixed, incubated in the dark for 30 min, and the color change was observed. Regardless of the addition of L-tryptophan, the culture medium containing the strain *Pseudomonas palleroniana* P6 turned red after the colorimetric reaction, while the culture medium without the strain remained unchanged. The absorbance was measured at 530 nm using a microplate reader. Compared to the culture medium without the strain, the absorbance of the culture medium containing *Pseudomonas palleroniana* P6 was significantly increased at 530 nm, with the increase being even greater in the medium containing L-tryptophan. Specific absorbance test results are shown in Table 3.

[0043] The King's medium with / without L-tryptophan is prepared as follows: 0.5g soluble starch, 0.3g sodium pyruvate, 0.5g glucose, 0.5g casein amino acids, 0.5g tryptone, 0.5g yeast extract, 0.3g potassium dihydrogen phosphate, 0.05g magnesium sulfate heptahydrate, 0.0g / 0.5g L-tryptophan, 1.0L distilled water, pH 7.2±0.2.

[0044] Preparation of S2 colorimetric solution: Dissolve 4.5g FeCl3 in 300mL distilled water, then slowly add 587.4mL of 98% concentrated sulfuric acid. After the solution cools, bring the volume to 1L.

[0045] Table 3 Absorbance after colorimetric reaction in culture medium

[0046]

[0047] Example 5: Field test verification of functional strains

[0048] The experimental field was located at the Paris polyphylla planting base in Nantangtian Village, Fengyi Town, Dali City, Yunnan Province (100°20′34″E, 25°31′8″N). The region has a humid and warm climate with an average temperature of 15.1℃, an average annual precipitation of 1078.9 mm, and an average annual sunshine duration of 2228.1 h. All Paris polyphylla plants were grown in greenhouses with semi-shaded, moist humus soil. The greenhouses implemented uniform water and fertilizer management (basal fertilizer was applied, but no top dressing was applied before the inoculant was administered). Seeds from closely related mother plants of the same ancestor were sown in the Paris polyphylla seedbed, and the plants showed uniform and good growth. Twelve quadrats (1m × 1m) were selected as experimental sites for inoculant application.

[0049] Pseudomonas palleroniana P6 was inoculated into 1L shake flasks containing 600mL of PDB liquid medium and incubated on a constant-temperature shaker at 28-30℃ and 180-200rpm for 12 hours to obtain the seed culture. This seed culture was then inoculated into ten 1000mL shake flasks containing 600mL of medium each and incubated at 28-30℃ and 180-200rpm for 1-4 days to ensure a bacterial concentration greater than 1×10⁻⁶. 9 The bacterial cells were collected at 10,000 rpm for 5 min at 4 °C. After removing the supernatant, the cells were resuspended and washed with PBS buffer, and then centrifuged again at 10,000 rpm for 20 min. The collected bacterial precipitate was resuspended in PBS buffer. The PBS suspension was added to a special cryovial and frozen overnight at -80 °C or below. The frozen bacterial solution was placed in a vacuum freeze dryer. Under vacuum, the bacterial sample in the cryovial was gradually heated at a low temperature (-50 °C) to sublimate the water and dry for 8 h. The freeze-dried bacterial cells were collected and stored at -20 °C.

[0050] The seedlings of Paris polyphylla typically emerge from the soil around mid-March each year. The inoculum agent is added approximately one month before this time. Six quadrats are randomly selected as the experimental group (OP), and the remaining six quadrats serve as the control group (OC). The freeze-dried bacterial strain is diluted and dissolved in 6L of water, divided evenly into six portions, and each portion is diluted to 20L before being applied to each experimental quadrat. The inoculum solution is sprayed evenly onto the soil surface of the quadrats twice a day, once in the morning and once in the afternoon, with a 6-hour interval. The control quadrats are sprayed with the same amount of water in the same manner as a control.

[0051] Before applying the inoculant, 2 kg of soil samples from a depth of 3-15 cm were taken from each quadrat using a 5-point sampling method and thoroughly mixed. These samples were used as the original soil samples, designated as OC1-OC6 for the control group and OP1-OP6 for the experimental group. Nine months after applying the inoculant, 2 kg of soil samples from a depth of 3-15 cm were taken from each quadrat again using the 5-point sampling method and thoroughly mixed. These samples were designated as FC1-FC6 for the control group and FP1-FP6 for the experimental group. Simultaneously, the plants from the soil samples were collected and brought back to the laboratory to determine their plant growth indicators and saponin content.

[0052] The collected soil was air-dried, ground, and passed through a 2mm sieve. The physicochemical properties of the soil were analyzed according to published national standard testing methods. Soil pH determination: 10.00g of soil sample was weighed and placed in a 100mL Erlenmeyer flask. 25mL of ultrapure water (water:soil = 2.5:1) that had been ultrasonically purified to remove oxygen and carbon dioxide was added. The soil suspension was vigorously stirred with a glass rod for 2 minutes, then allowed to stand for 30 minutes. The pH value of the soil suspension was then measured using an electronic pH meter (PHSJ-4A, Leici Shanghai, China). Total organic carbon (TOC) and total nitrogen (TN) in the soil were determined using a carbon and nitrogen analyzer (LECO, St. Joesph, MI, USA). Soil hydrolyzable nitrogen (WSN) was determined by mixing 3g of soil with 30ml of water (water:soil = 10:1), shaking at 170rpm for 30min in a shaker, centrifuging at 8000rpm for 20min, and filtering the collected supernatant through a 0.45μm filter membrane. Soil total phosphorus (TP) and total potassium (TK) were determined by the NaOH fusion method. Soil available phosphorus (AP) and available potassium (AK) were determined by the sulfuric acid-hydrochloric acid extraction method and the ammonium acetate extraction method, respectively.

[0053] Determination of growth indicators of Paris polyphylla: Five Paris polyphylla plants were randomly selected from each quadrat. After removing the stems and leaves, the tuberous roots were washed, the fibrous roots were trimmed, and the roots were dried with absorbent paper. The weight of each tuberous root was recorded as fresh weight (FW). After measuring the fresh weight, the roots were placed in a constant temperature oven at 60℃ and dried to constant weight. The weight of each root was then recorded as dry weight (DW).

[0054] Analysis of Paris polyphylla saponin content: Paris polyphylla roots dried to constant weight in a 60℃ oven were ground into powder less than 0.2 mm and extracted using a Soxhlet extractor based on the extraction method in the Chinese Pharmacopoeia (2015). 0.100 g of dried Paris polyphylla powder sample was accurately weighed, and 25 mL of methanol solution was added. The mixture was weighed at room temperature, then refluxed at 80℃ for 30 min. After cooling to room temperature, the weight was replenished with methanol to the level before reflux. The solution was diluted a certain factor and the saponin content was determined by high-performance liquid chromatography (HPLC). Standards for the corresponding Paris polyphylla saponins I, II, VI, VII, and diosgenin were mixed at specific concentrations, dissolved in methanol, and serially diluted to obtain standard samples. These samples were injected together with the test sample to determine the content of each saponin. Measure 10 μL of standard solution sequentially from low to high concentration. Plot a standard curve with injection volume (mg / mL) as the x-axis and peak area as the y-axis. Calculate the correlation coefficient and the corresponding linear range to ensure good linearity of the standards. Inject the same sample at least three times and record the peak area to ensure instrument precision and good repeatability of the experimental method. A Shimadzu LC-20AD series high-performance liquid chromatography system equipped with an SPD-20A UV-Vis detector was used. An ACQUITY UPLC™ BEH C18 column (217 mm × 2.1 mm, 1.7 μm) was employed at a column temperature of 25 °C. The mobile phases were acetonitrile (A) and water (B), with a gradient elution program as follows: 0–5 min, 45–45% A; 5–7 min, 45–30% A; 7–9 min, 30–2% A; 9–15 min, 2–2% A; 15–16 min, 2–45% A. The injection volume was 10 μL, and the flow rate was 1.0 mL / min.

[0055] Effects of strain P6 on the physicochemical properties of the rhizosphere soil of Paris polyphylla. The physicochemical properties of the soil before and after application of Pseudomonas palleroniana P6 are shown in Table 4. The results showed that soil pH changed significantly between the two sampling periods. In the experimental group without inoculant application, the soil pH decreased from 6.10 (OC) before inoculant application to 5.83 (FC). The pH decrease was even greater in the strain-added group FP, decreasing from 6.14 in the OP group to 5.50 in the FP group, a decrease of 0.33 pH units compared to the control group. The contents of total organic matter (TOC), total nitrogen (TN), and dissolved nitrogen (WSN) in the soil decreased significantly before and after planting: TOC decreased from 73.90 g / kg and 74.71 g / kg in the OC and OP groups to 69.46 g / kg and 69.74 g / kg, respectively; TN decreased from 2.79 g / kg and 2.72 g / kg in the OC and OP groups to 2.40 g / kg and 2.39 g / kg in the FC and FP groups, respectively; and WSN decreased from 0.28 g / kg in the OC and OP groups to 0.21 g / kg in the FC and FP groups. The contents of total phosphorus in the soil decreased from 0.75 g / kg and 0.74 g / kg in the OC and OP groups to 0.71 g / kg and 0.70 g / kg in the FC and FP groups, respectively, a decrease of 0.4 g / kg; while total potassium decreased by 1.16 g / kg and 1.12 g / kg, respectively. In contrast, the available phosphorus content in the FP group did not decrease significantly, while in the FC group it decreased from 0.17 g / kg in the OC group to 0.14 g / kg. The available potassium content in the FP group even increased significantly, from 0.24 g / kg in the OP group to 0.26 g / kg, while the available potassium content in the FC group decreased significantly by 0.04 g / kg from 0.24 g / kg in the OC group. Overall, with the growth of *Paris polyphylla*, soil pH, total organic carbon, total nitrogen, dissolved nitrogen, total phosphorus, total potassium, available phosphorus, and available potassium all decreased significantly. However, the available phosphorus and available potassium contents in the FP group with added microbial agents did not show a decreasing trend; in fact, the available potassium content increased significantly. Compared to FC, the FP group with added *Pseudomonas palleroniana* P6 significantly reduced soil pH and significantly increased available phosphorus and available potassium contents.

[0056] Table 4. Physicochemical properties of soil from sample plots

[0057]

[0058]

[0059] The effect of Pseudomonas palleroniana P6 on the biomass of Paris polyphylla tubers was investigated. The dry and fresh weights of 60 Paris polyphylla tubers from the experimental (FP) and control (FC) groups were measured. Figure 3 The results showed that the addition of Pseudomonaspalleroniana P6 significantly increased the fresh and dry weight of Paris polyphylla tubers. The average fresh weight of Paris polyphylla tubers in the FC group was 2.06 g, while the average fresh weight of Paris polyphylla tubers in the FP group reached 2.54 g, an increase of 0.48 g (23.30%) per tuber; while the average dry weight of Paris polyphylla tubers in the FP group increased from 0.76 g in the FC group to 0.93 g, a significant increase of 22.37%.

[0060] The content of saponins in Paris polyphylla roots was determined using high performance liquid chromatography-mass spectrometry (HPLC-MS). Figure 4 (Table 5) The results showed that the highest content of saponins in the roots of the experimental group (FP) was Paris saponin II, followed by Paris saponin VII and Paris saponin I. The average content of Paris saponin II in the experimental group (FP) reached 30.38 mg / g, while the average content of Paris saponin II in the control group (FC) was 26.63 mg / g. The application of Pseudomonas palleroniana P6 significantly increased the content of Paris saponin II by 14.08%. After the application of Pseudomonas palleroniana P6, the average contents of Paris saponins VII and I were 4.89 mg / g and 4.46 mg / g, respectively, which were significantly increased by 66.33% and 30.79% compared with 2.94 mg / g and 3.41 mg / g in the control group. There was no significant difference in the contents of Paris saponins VI and diosgenin between the experimental and control groups.

[0061] Table 5. Content of various saponins in the roots of Paris polyphylla in the control and experimental groups.

[0062]

[0063] VII, VI, II, I, and N represent Paris saponins VII, VI, II, and I, and diosgenin, respectively. Different letters indicate significant differences determined by the Turkey test.

Claims

1. A Pseudomonas bacterium that promotes plant growth and increases the content of Paris polyphylla saponins, characterized in that, The aforementioned Pseudomonas is *Pseudomonas pallensii* (…). Pseudomonas palleroniana P6, accession number CCTCC M 20232513.

2. The application of the *Pseudomonas* strain according to claim 1, characterized in that, Used for dissolving phosphorus, releasing potassium, and producing IAA.

3. The application of the *Pseudomonas* strain according to claim 1, characterized in that, Used to promote the growth of ryegrass.

4. The application according to claim 3, characterized in that, It is used to promote an increase in plant height, root length, and root number of ryegrass.

5. The application of the *Pseudomonas* strain according to claim 1, characterized in that, It is used to promote the growth of Paris polyphylla.

6. The application according to claim 5, characterized in that, Used to promote Paris polyphylla biomass.

7. The application of the *Pseudomonas* strain according to claim 1, characterized in that, It is used to increase the content of Paris saponin VII and Paris saponin I in Paris polyphylla.

8. A Pseudomonas aeruginosa inoculum, characterized in that, It is prepared by fermentation culture of Pseudomonas as described in claim 1.

9. The microbial agent according to claim 8, characterized in that, The preparation method of the bacterial agent includes the following steps: preparing *Pseudomonas pallens* (…). Pseudomonas palleroniana P6 is inoculated into PDB liquid medium and placed in a shaker at 180-200 r / min. The culture temperature is 28-30℃, and the culture is carried out for 1-4 days to complete the preparation of fermentation broth inoculum.

10. The application of the Pseudomonas agent according to claim 8 or 9, characterized in that, Applying Pseudomonas microbial agents to the planting soil can increase the content of available phosphorus and potassium in the soil and promote the growth of ryegrass.

11. The application of the Pseudomonas agent according to claim 8 or 9, characterized in that, It is used to increase the content of Paris saponins in Paris polyphylla.

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