Pseudomonas putida and application thereof in agaricus bisporus planting

Abstract

A strain of *Pseudomonas putida* and its application in the cultivation of *Agaricus bisporus*, wherein the *Pseudomonas putida* is *Pseudomonas putida* ( Pseudomonas putida AT130, has been deposited at the China Center for Type Culture Collection (CCTCC) on September 5, 2023, with the strain accession number CCTCC NO: M20231607. This invention relates to *Pseudomonas putida* (*Pseudomonas putida*). Pseudomonas putida AT130 is a stable and highly efficient associative fungus for the growth of button mushrooms. Biological agents made from it can effectively improve the nutrient environment of the casing soil, accelerate fruiting, and improve the quality of button mushroom fruiting bodies. This fungus is a plant rhizosphere probiotic with advantages such as easy cultivation, good preservation, environmental friendliness, and safety for humans and animals, and it shows promise for development and application as a novel microbial resource.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a strain of *Pseudomonas putida* and its application in the cultivation of *Agaricus bisporus*. Background Technology

[0002] Button mushrooms (Agaricus bisporus) are one of the most widely cultivated edible fungi in the world. The casing soil used for button mushrooms directly affects their yield, quality, and growth cycle. Different casing substrates have a significant impact on mycelial growth and fruiting body formation. Although there is considerable research on casing substrates, the interaction mechanism between casing substrates and button mushrooms remains unclear.

[0003] Button mushrooms are saprophytic fungi that rely on absorbing organic nutrients from the substrate for growth. They convert the organic matter in the fermented plant material into carbohydrates, proteins, water, carbon dioxide, and minerals. During their growth and development, button mushrooms exhibit strong dependence on and varying requirements for environmental nutrients, humidity, and ventilation at different growth stages. Furthermore, their growth is significantly influenced by microorganisms. These microorganisms are primarily found in the casing soil, and the casing soil microorganisms and its physiological environment can, to a certain extent, determine the growth of button mushrooms. Studies have shown that bacteria, rather than molds, actinomycetes, or yeasts, play a major role in the growth of button mushrooms in the casing soil. The bacteria that promote fruiting in button mushrooms have been identified as primarily *Pseudomonas putida*. This bacterium can remove the self-inhibiting volatile substances produced during the vegetative growth stage of button mushrooms, thereby relieving inhibition, promoting primordia formation, and ultimately promoting button mushroom growth. Controlling the interaction between this edible fungus and key microorganisms can further regulate the cultivation process, save raw materials, reduce costs, and improve product quality.

[0004] Microbial inoculants are live bacterial preparations made by industrially propagating target microorganisms (effective bacteria) and then using porous materials as adsorbents to adsorb the fermentation broth of the bacteria. When applied to the soil, these inoculants can directly or indirectly improve soil quality, restore fertility, prevent soil-borne diseases, maintain the balance of the rhizosphere microbiome, and degrade toxic substances. Proper use of agricultural microbial inoculants can increase crop yields, improve crop quality, reduce fertilizer use, lower costs, improve soil, and protect the ecological environment. Therefore, the utilization and development of pollution-free microbial inoculants demonstrates their application advantages and promising development prospects. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of *Pseudomonas putida* and its application in the cultivation of *Agaricus bisporus*.

[0006] To achieve the above and other related objectives, the technical solution provided by this invention is: a strain of *Pseudomonas putida*, wherein *Pseudomonas putida* AT130 has been deposited at the China Center for Type Culture Collection (CCTCC) on September 5, 2023, with the strain accession number CCTCC NO: M20231607.

[0007] To achieve the above and other related objectives, the technical solution provided by this invention is as follows: Pseudomonas putida AT130 is prepared as a biological agent and sprayed onto the casing soil for Agaricus bisporus cultivation; the Pseudomonas putida AT130 is deposited at the China Center for Type Culture Collection (CCTCC) on September 5, 2023, with the strain accession number CCTCC NO: M20231607.

[0008] The preferred technical solution is that the bacterial content of *Pseudomonas putida* AT130 in the biological agent is 10. 8 -10 9 cfu / ml.

[0009] Due to the application of the above technical solution, the advantages of this invention compared with the prior art are:

[0010] The *Pseudomonas putida* AT130 of this invention is a stable and highly efficient as a by-product of *Agaricus bisporus* growth. Biological agents made from this fungus can effectively improve the nutrient environment of casing soil, accelerate fruiting, and improve the quality of *Agaricus bisporus* fruiting bodies. This fungus is a beneficial rhizosphere bacterium with advantages such as easy cultivation, good preservation, environmental friendliness, and safety for humans and animals, and shows promise for development and application as a novel microbial resource. Attached Figure Description

[0011] Figure 1 This is a photograph of the colony morphology of strain AT130 on a plate.

[0012] Figure 2 Gram staining micrograph of strain AT130.

[0013] Figure 3 This is a scanning electron microscope image of strain AT130.

[0014] Figure 4 Phylogenetic tree of strain AT130.

[0015] Figure 5 This refers to the protein content of button mushrooms.

[0016] Figure 6 This refers to the fat content in button mushrooms.

[0017] Figure 7 This refers to the moisture content of button mushrooms.

[0018] Figure 8 This refers to the ash content in button mushrooms.

[0019] Figure 9 This refers to the soluble sugar content in button mushrooms.

[0020] Figure 10 This represents the available phosphorus content in the cover soil.

[0021] Figure 11 This represents the content of available potassium in the cover soil.

[0022] Figure 12 The images show the planting of the experimental and control groups of button mushrooms; the top row represents the experimental group, and the bottom row represents the control group. Detailed Implementation

[0023] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in these embodiments.

[0024] Please see Figure 1-12 It should be noted that the structures, proportions, sizes, etc., illustrated in the accompanying drawings are merely for illustrative purposes and to aid those skilled in the art in understanding and reading the invention. They are not intended to limit the scope of the invention and therefore have no substantial technical significance. Any modifications to the structure, changes in proportions, or adjustments to the size are not permitted. The following embodiments are provided to better understand the invention, but are not intended to limit it. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0025] Preservation of biological materials:

[0026] Pseudomonas putida AT130 has been deposited at the China Center for Type Culture Collection (CCTCC) on September 5, 2023, with accession number CCTCC NO: M20231607. The address of CCTCC is 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0027] Unless otherwise specified, all reagents or materials described in the following examples are commercially available.

[0028] Example 1: A strain of *Pseudomonas putida* and its application in *Agaricus bisporus* cultivation

[0029] I. Isolation, screening and purification of Pseudomonas putida AT130

[0030] 1. Raw material preparation

[0031] (1) Soil sample preparation

[0032] Soil samples were collected from the mushroom cultivation workshop of Funan Lianmei Agricultural Products Co., Ltd., Funan County, Fuyang City, Anhui Province. Soil samples were randomly collected from the top, middle, and bottom layers of the mushroom casing soil using a 5-point sampling method. After thorough mixing, the soil samples were placed in an ice bucket and brought back to the laboratory for storage at 4°C for later use.

[0033] (2) Culture medium formulation

[0034] Pseudomonas isolation broth: 10g peptone, 10g casein peptone, 1.4g magnesium chloride, 10g potassium sulfate, 0.025g irgasan, pH 7.0±0.2, 20ml glycerol, heated and dissolved in 980ml distilled water, autoclaved at 121℃ for 15min, ready for use.

[0035] Pseudomonas CFC selective medium: 16g gelatin peptone, 10g acid-hydrolyzed casein, 10g potassium sulfate, 1.4g magnesium chloride, 12g agar, pH 7.1±0.2, 10g glycerol. Dissolve in 1000ml distilled water by heating. Autoclave at 121℃ for 15 minutes. When cooled to about 50℃, add one vial of Pseudomonas CFC selective medium additive to every 200ml, mix well, and set aside.

[0036] Additives for selective culture medium for Pseudomonas CFC: cetanetrimethylammonium bromide 10 mg / L, sodium clostridium 10 mg / L, cefotaxime 50 mg / L.

[0037] 2. Strain isolation and purification

[0038] 1.0g of casing soil sample was weighed in the clean bench and transferred to an Erlenmeyer flask containing 99.0mL of Pseudomonas spp. Ten small glass beads were placed in the Erlenmeyer flask, the flask was sealed with a sealing film and shaken thoroughly. A casing soil solution diluted 100 times was prepared using this sample solution and incubated in a bacterial incubator at 30℃ and 150r / min for 24h before being stored for later use.

[0039] The above-mentioned soil-covered culture solution was serially diluted, with dilution gradients of 10... -4 10 -5 10 -6 and 10 -7100 μL of the solution was evenly spread onto Pseudomonas CFC selective medium and incubated at 30°C for 24 hours. Single colonies with good growth were picked and streaked onto Pseudomonas CFC selective medium for further incubation. After incubation, the culture was stored at 4°C. The selected Pseudomonas strain was named *Pseudomonas putida* AT130.

[0040] II. Strain Identification

[0041] 1. Morphological identification

[0042] After activating the above-mentioned *Pseudomonas putida* AT130 with *Pseudomonas cfc* selective medium, the results were as follows: Figure 1 As shown in the figure. Observation of its colony morphology characteristics revealed that the strain forms pale yellow, round colonies with a raised surface and neat edges on the culture medium. The colony surface is moist, smooth, and glossy. After Gram staining, the bacterial morphology was observed under a microscope, and the results are as follows. Figure 2 As shown. Gram-negative, rod-shaped bacteria, no spores observed.

[0043] The above-mentioned bacterial strains were subjected to scanning electron microscopy, and the results are as follows: Figure 3 As shown. Scanning electron microscopy (SEM) results show that the bacteria have smooth ends and are short rod-shaped.

[0044] 2. Physiological and biochemical identification

[0045] The Vitek 2 Compact GN identification card identified the bacterium as *Pseudomonas putida*. The biochemical reaction results are shown in Table 1 below.

[0046] Table 1: Results of Physiological and Biochemical Reactions

[0047]

[0048]

[0049] Note: "+" indicates a positive reaction, and "-" indicates a negative reaction.

[0050] 3. Molecular biological identification

[0051] The purified *Pseudomonas putida* AT130 strain was identified using 16S rDNA amplification and sequence analysis. Specifically, genomic DNA was extracted from the purified strain using an Ezup column-based bacterial genomic DNA extraction kit. The 16S rDNA of this strain was amplified using the following universal primers: 27F (upstream primer, sequence [omitted]).

[0052] The amplification system and conditions for the 5'-AGTTTGATCMTGGCTCAG-3' primer and the 1492R primer (sequence 5'-GGTTACCTTGTTACGACTT-3') are detailed in Tables 2 and 3 below. The amplified products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0053] Table 2: 16S rDNA amplification system

[0054]

[0055] Table 3: 16S rDNA amplification conditions

[0056]

[0057] The PCR products amplified using the above method were sequenced and BLAST-aligned on the NCBI website. A phylogenetic tree was then constructed using MEGA11 software with a neighbor-joining method. Figure 4 As shown, the results indicate that *Pseudomonas putida* AT130 shares 99.86% sequence similarity with *Pseudomonas putida* PP112420. Furthermore, it shares over 99% similarity with multiple *Pseudomonas* strains. Combined with the culture, morphological, and physiological / biochemical characteristics of *Pseudomonas putida* AT130, this strain can be identified as *Pseudomonas putida*. A phylogenetic tree of *Pseudomonas putida* AT130 was constructed based on the new sequence data from NCBI, and the results are shown below. Figure 4 As shown.

[0058] Example 2: A strain of *Pseudomonas putida* and its application in *Agaricus bisporus* cultivation

[0059] Application of Pseudomonas putida AT130 in the cultivation of button mushrooms

[0060] I. Preparation of biological inoculants and cultivation of button mushrooms

[0061] The purified *Pseudomonas putida* AT130 was cultured at 30°C to the logarithmic growth phase, then transferred to physiological saline and adjusted to a viable count of 102. 8 -10 9 cfu / ml. 50ml of bacterial solution was sprayed per kg of soil covering, while a control group was sprayed with 50ml of physiological saline per kg of soil covering.

[0062] The soil covering weight for each basket was 4 kg, with 3 replicates per group, and a total of 6 baskets of button mushrooms were cultivated in the experiment. Laboratory conditions were simulated for the mushroom house: initially, the temperature was maintained at 21–22℃ and the humidity at 80%–85%. Once the mycelium had covered more than 80% of the mushroom bed area, the temperature was controlled at 17–18℃ and the humidity at 90%, with occasional light spraying of a fruiting-promoting solution to encourage fruiting. The cultivation cycle was 21 days.

[0063] A batch of mushrooms was harvested and promptly preserved and transferred to the laboratory for physicochemical analysis.

[0064] II. Determination of Physicochemical Indicators of Agaricus bisporus

[0065] Mature button mushrooms were harvested and quickly transferred to a laboratory refrigerator for storage and sample preparation for subsequent experiments. The harvested control group button mushrooms were cut into cap parts (CKU) and stem parts (CKS), and similarly, the experimental group button mushrooms were also divided into cap parts (APU) and cap parts (APS).

[0066] 1. Determination of protein content

[0067] Protein content was determined using the method specified in GB / T15673-2009. The results are as follows: Figure 5 As shown, the protein content of *Agaricus bisporus* cultured in casing soil enhanced with strain AT130 was significantly higher in both the cap (41.41±0.20%) and stem (29.1±0.23%) than that in the control group (34.94±0.28%) and stem (26.06±0.78%). This result indicates that the addition of strain AT130 to the casing soil may have interacted with genes related to protein synthesis in *Agaricus bisporus*, promoting protein synthesis.

[0068] 2. Determination of fat content

[0069] The fat content was determined using the method specified in GB 5009.6-2016. The results are as follows: Figure 6 As shown. Fat is an important macronutrient, essential for maintaining normal bodily functions. Adequate fat intake is crucial for maintaining cell membrane health, vitamin absorption, the transport of fat-soluble vitamins, and other physiological functions. When strain AT130 is added to the casing soil, the enzymes or organic matter it produces may directly or indirectly promote the synthesis of fat within the plant. Figure 6It was found that the fat content in the cap of the enhanced button mushroom was significantly higher than that in the control group, while there was no significant change in the stem. This is likely because during the growth of button mushrooms, nutrients and water are transported to the cap, while the stem, with its higher cellulose content, primarily provides support. The APU's fat content (7.36±0.31%) still classifies it as a low-fat food, and the increased fat content contributes to its texture and flavor.

[0070] 3. Determination of moisture content

[0071] The moisture content was determined according to the method in GB 5009.3-2016. Results Figure 7 As shown, the difference in moisture content between the experimental group and the control group was not significant. The moisture content of the cap was slightly higher than that of the stem, presumably because the cap of the button mushroom receives more moisture from the air during its growth in the mushroom house, and a small amount of water is lightly sprayed in the early stage of fruiting to promote fruiting, hence the slightly higher moisture content of the cap.

[0072] 4. Ash content determination

[0073] Moisture content was determined according to the method in GB 5009.4-2016. The results are as follows: Figure 8 As shown in the figure. The ash content in button mushrooms mainly consists of minerals, mineral salts, and other inorganic substances. The ash content in button mushrooms was determined, and the results are shown below. Figure 8 As shown, the ash content of button mushrooms modified by strain AT130 decreased in both the cap and stem parts compared to the control group.

[0074] 5. Determination of soluble sugar content

[0075] The anthrone-sulfuric acid method was used, with slight modifications, to determine the soluble sugar content. The results are as follows: Figure 9 As shown in the figure, soluble sugars serve as energy and carbohydrate reserves within the button mushroom. This helps support the growth and development of the button mushroom. The figure above shows that the soluble sugar content significantly increased in button mushrooms after the addition of AT130 strain. Particularly in the stem, the APS content in the stem reached as high as 223.66±9.21 mg / g, indicating that strain enhancement improved the stress resistance of button mushrooms and provided stronger protection for the fruiting body. Furthermore, we found that the soluble sugar content in the stem was higher than that in the cap, which is consistent with the growth characteristics of button mushrooms.

[0076] 6. Determination of Vitamin Content

[0077] The vitamin content was determined using LC-MS, and the results are shown in the table below:

[0078] Table 4: Content of the three vitamins

[0079]

[0080] Note: The means of different superscript letters differed significantly (one-way ANOVA; P<0.05).

[0081] As shown in the table above, for *Agaricus bisporus*, the vitamin C content is higher in the stem than in the cap, and the vitamin C content decreases in *Agaricus bisporus* cultured after improvement with this patented strain. For vitamins B1 and B2, their distribution is greater in the cap than in the stem. After improvement, the vitamin B1 content did not change significantly, but the vitamin B2 content decreased. This indicates that the addition of strain AT130 inhibits the vitamin synthesis mechanism in *Agaricus bisporus*.

[0082] 7. Determination of organic acids

[0083] The organic acid content was determined using LC-MS, and the results are shown in the table below:

[0084]

[0085] Note: The means of different superscript letters differed significantly (one-way ANOVA; P<0.05).

[0086] As shown in the table above, regarding the organic acids in *Button Mushroom*, after strain enhancement, the changes in tartaric acid and quinic acid were minimal, but the contents of malic acid and fumaric acid increased significantly. It is worth noting that the contents of various organic acids were significantly higher in the cap of *Button Mushroom* than in the stem. This is consistent with the structural characteristics of different parts of the fruiting body of edible fungi.

[0087] Example 3

[0088] Improvement of casing soil fertility for button mushrooms by *Pseudomonas putida* AT130

[0089] Regularly sample the soil covering the mushroom cultivation area. The samples were then quickly transferred to a -20°C freezer in the laboratory for subsequent experiments.

[0090] 1. Determination of available phosphorus in soil

[0091] The available phosphorus content in the soil was determined by extraction with sodium bicarbonate solution and then by the molybdenum-antimony colorimetric method. The results are as follows: Figure 10As shown in the figure, the casing soil of *Buttonella spp.* was sampled at different stages to record the changes in available phosphorus throughout the entire process from the start of casing to the harvest of the first flush of mushrooms. The data in the figure show that the changes in available phosphorus between the experimental and control groups were not significant during the 0-4 day period. However, after 4 days, the increase in available phosphorus content in the experimental group was significantly greater than that in the control group, especially between 13-17 days, when the available phosphorus content in the experimental group increased considerably. This period coincides with the formation of *Buttonella spp.* fruiting bodies. *Pseudomonas putida* has the ability to dissolve phosphates; after its addition, it can convert more inorganic phosphorus into available phosphorus that can be absorbed and utilized by plants.

[0092] 2. Determination of available potassium content in soil

[0093] The available potassium content in the soil was determined using ammonium acetate extraction-flame photometry. The results are as follows: Figure 11 As shown, the available potassium content increased throughout the entire process of Agaricus bisporus cultivation, with the experimental group showing a greater increase than the control group. While *Pseudomonas putida* typically does not affect the available potassium content in the soil, the exogenous addition of bacterial solution increased its metabolic activity, potentially indirectly affecting available potassium production. Similar to available phosphorus, the increase was rapid between 13 and 17 days, indicating high metabolic activity of soil microorganisms during this stage.

[0094] In summary, the *Pseudomonas putida* AT130 strain described in this invention is easy to cultivate, has strong survival ability, and in the cultivation of *Button Mushroom*, it has the characteristics of increasing the protein content, soluble sugar content, and organic acid content of *Button Mushroom* fruiting bodies, and can advance the fruiting time by about 48 hours. It is an excellent strain for enhancing *Button Mushroom* cultivation.

[0095] The 16S rDNA sequence of *Pseudomonas putida* AT130, with a length of 1477 bp, is as follows:

[0096]

[0097] The above description is merely a preferred embodiment for explaining the present invention and is not intended to limit the present invention in any way. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included within the scope of protection intended by the present invention.

Claims

1. A strain of *Pseudomonas putida* ( Pseudomonas putida AT130, characterized in that: The aforementioned *Pseudomonas putida* AT130 has been deposited at the China Center for Type Culture Collection (CCTCC) on September 5, 2023, with the accession number CCTCC NO: M20231607.

2. The application of *Pseudomonas putida* AT130 as described in claim 1 in a biological agent for promoting the growth of *Agaricus bisporus*, characterized in that: A biological agent made from Pseudomonas putida AT130 was sprayed onto the casing soil of button mushroom cultivation.

3. The application of *Pseudomonas putida* according to claim 2 in a biological agent for promoting the growth of *Agaricus bisporus*, characterized in that, The bacterial content of *Pseudomonas putida* in the biological agent is 10. 8 -10 9 cfu / ml.

Images