A chryseobacterium strain and its application in improving stability of mushroom residue substrate

Abstract

The application belongs to the field of agricultural microbial technology, and discloses a golden yellow bacillus strain and application thereof in mushroom residue substrate stability improvement. Chryseobacterium The golden yellow bacillus strain is a golden yellow bacillus (sp.) strain JYT19. The strain is isolated from coconut dregs material, and is preserved on February 2, 2026, with a preservation number of CGMCC No. 37640. Inoculation of the strain into mushroom residue substrate can significantly improve the physical structure stability of the substrate, delay collapse and hardening, and prolong the service life. Meanwhile, the strain has beneficial effects on plant growth and quality improvement. The application provides an environment-friendly and efficient microbial technical solution for solving the problems of fast degradation and poor stability of mushroom residue substrate, and has important application value in the fields of facility agriculture soilless culture and organic waste resource utilization.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, specifically to a strain of *Aureobacillus* and its application in improving the stability of mushroom residue substrate. Background Technology

[0002] Soilless cultivation is a crucial technology in the protected horticulture industry. Substrate cultivation effectively improves the utilization rate of organic waste by combining abundant organic solid waste with soilless cultivation. However, existing substrates generally suffer from problems such as short service life, heavy continuous investment, frequent replacement or disinfection, and significant environmental costs. This "high investment, short cycle, and heavy pollution" model has become a prominent bottleneck restricting the sustainable development of organic substrate cultivation.

[0003] Mushroom residue, the waste culture medium after harvesting edible fungi, is rich in organic matter and fiber. Through high-temperature fermentation (45-60 days) and physicochemical conditioning, it can be transformed into a high-quality cultivation substrate, partially replacing peat moss for vegetable and fruit seedling cultivation and hydroponics, reducing production costs by more than 60%. Mushroom residue substrate, as a cultivation substrate for vegetables, fruits, flowers, and other crops, has promising application prospects in the protected horticulture industry, especially in hydroponics. However, the following are the main problems in the practical application of mushroom residue substrate:

[0004] 1. Unstable physical structure: Mushroom residue is prone to further decomposition, collapse, and compaction during use, resulting in reduced aeration and drainage, which is detrimental to plant root growth.

[0005] 2. Rapid biodegradation and short service life: The weight of existing mushroom substrate increases significantly and the total porosity decreases significantly during a cultivation cycle, requiring frequent replacement and increasing production costs.

[0006] Therefore, there is an urgent need to develop an environmentally friendly, efficient, and sustainable biostabilization technology for mushroom residue substrate. Summary of the Invention

[0007] To address the aforementioned problems in existing technologies, the present invention aims to provide a strain of *Aureobacterium* with the function of stabilizing mushroom substrate and its application, thereby solving the technical problems of easy collapse of the physical structure, rapid biodegradation, and poor stability of existing mushroom substrates.

[0008] The specific technical solution of the present invention is as follows:

[0009] The first objective of this invention is to provide a Chlorella strain JYT19, which has the accession number CGMCC No. 37640 and the accession date of February 2, 2026.

[0010] A second objective of the present invention is to provide a cultivation substrate composition comprising a base substrate and the aforementioned *Aureobacterium chrysogenum* strain JYT19, wherein the base substrate is a mushroom residue substrate or a composite substrate containing mushroom residue.

[0011] In a specific embodiment, the mushroom residue substrate is mushroom residue, and the mushroom residue composite substrate is a mixture of mushroom residue, peat moss, perlite and vermiculite in a ratio of 3:3:2:2.

[0012] The third objective of this invention is to provide a method for improving the physical structural stability of a cultivation substrate by inoculating the aforementioned *Aureobacillus* strain JYT19 into a mushroom residue substrate or a composite substrate containing mushroom residue.

[0013] Furthermore, the inoculation method involves mixing the bacterial solution with the substrate evenly.

[0014] Furthermore, the inoculation concentration was 1×10⁻⁶. 8 CFU / g matrix.

[0015] A fourth objective of this invention is to provide the application of the aforementioned Chlorella strain JYT19 in improving the physical stability of cultivation substrates and / or increasing crop yield and / or improving crop quality.

[0016] Furthermore, the improved physical structural stability of the cultivation substrate includes at least one of the following:

[0017] a. Reduce the bulk density of the substrate;

[0018] b. Slow down changes in total matrix porosity;

[0019] c. Increase the aeration porosity of the substrate;

[0020] d. Reduce the water-holding porosity of the substrate;

[0021] e. Increase the matrix size-to-void ratio;

[0022] f. Delays matrix collapse;

[0023] g. Delays the collapse and compaction of the matrix.

[0024] Furthermore, improving crop quality means increasing vitamin C content.

[0025] A fifth objective of this invention is to provide the use of the aforementioned cultivation substrate composition in increasing crop yield and / or improving crop quality.

[0026] Furthermore, improving crop quality means increasing vitamin C content.

[0027] The beneficial effects of this invention are as follows:

[0028] (1) Significantly improves the physical structure stability of mushroom residue substrate

[0029] like Figure 3-7 As shown, after treatment with the strain JYT19 of this invention for 40 days, the bulk density of the substrate with the addition of strain JYT19 decreased by 7.06% compared with the mushroom residue substrate without the addition of the inoculant (control), the change in total porosity slowed down by 1.23%, the aeration porosity increased by 61.49%, the water-holding porosity decreased by 13.95%, and the ratio of large to small pores increased by 74.45%. This indicates that Chlorella vulgaris JYT19 has an improving effect on the stability of the substrate during use, effectively delays the collapse and compaction of the substrate, significantly extends the service life of the substrate, and reduces the frequency of substrate replacement and production costs.

[0030] (2) Improve plant yield and quality through substrate improvement

[0031] Mushroom residue substrate inoculated with strain JYT19 was used for the cultivation of non-heading Chinese cabbage 'Shanghai Green'. After 40 days, compared with the uninoculated control, the fresh weight of the aboveground parts of the plants increased by 42.54%, the dry weight increased by 31.55%, the leaf area increased by 18.96%, the vitamin C content of the leaves increased by 34.01%, the plant yield increased, and the quality was improved.

[0032] (3) The process is simple and the cost is low, making it suitable for large-scale application.

[0033] The strain of this invention has simple culture conditions, convenient inoculation operation, no need for complicated equipment, and can be directly applied to the existing mushroom residue substrate production process. Attached Figure Description

[0034] Figure 1 The colony morphology of strain JYT19 on tryptone soybean agar solid medium.

[0035] Figure 2 This is a phylogenetic tree of the 16S rDNA gene of strain JYT19.

[0036] Figure 3 A comparison of the bulk density changes of mushroom residue composite substrate before and after treatment with strain JYT19.

[0037] Figure 4 A comparison of the changes in total porosity of the mushroom residue composite substrate before and after treatment with strain JYT19.

[0038] Figure 5 A comparison of the changes in the aeration pore size of the mushroom residue composite substrate before and after treatment with strain JYT19.

[0039] Figure 6 A comparison of the changes in water-holding porosity of the mushroom residue composite substrate before and after treatment with strain JYT19.

[0040] Figure 7A comparison of the changes in the size-to-void ratio of the mushroom residue composite substrate before and after treatment with strain JYT19.

[0041] Figure 8 Comparison of the fresh weight of aboveground parts of plants cultivated in mushroom residue composite substrate treated with strain JYT19.

[0042] Figure 9 Comparison of aboveground dry weight of plants grown in mushroom residue composite substrate treated with strain JYT19.

[0043] Figure 10 A comparison of leaf area of ​​plants cultivated in mushroom residue composite substrate treated with strain JYT19.

[0044] Figure 11 A comparison of vitamin C content in plants cultivated on mushroom residue composite substrate treated with strain JYT19.

[0045] Preservation information:

[0046] Strain name: JYT19;

[0047] Category Naming: Chryseobacterium sp.;

[0048] Accession number: CGMCC No. 37640;

[0049] Preservation period: February 2, 2026;

[0050] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections;

[0051] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0052] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0053] Example 1

[0054] Chrysogenum ( Chryseobacterium Isolation, purification and identification of sp.)JYT19.

[0055] 1.1 Isolation and purification of Chlorella JYT19:

[0056] The *Chlorella vulgaris* strain JYT19 of this invention was isolated from coconut coir material. The isolation method was a dilution-coating method. The specific steps are as follows:

[0057] 10 g of coconut coir was quickly placed in a 250 mL Erlenmeyer flask containing 100 mL of sterile distilled water and shaken on a shaker (180 rpm / min) for 2 h. Then, 0.1 mL of the coconut coir suspension was added to a new centrifuge tube and diluted with 0.9 mL of sterile distilled water, and this serial dilution was repeated. Finally, 200 μL of each dilution was plated onto tryptone-soybean agar plates and incubated at 30°C for 2 days. After single colonies appeared on the plates, streak plating was performed three times to isolate and purify the culture, ultimately obtaining a pure culture of *Cyclophorus* JYT19.

[0058] 1.2 Identification of Chlorella vulgaris strain JYT19

[0059] (1) Microbiological characteristics

[0060] like Figure 1 As shown, after culturing this strain on tryptone soybean agar solid medium plates at 30°C for 2 days, the single colonies are round, golden yellow and opaque, with a size of 1-2 mm, smooth and glossy surface, and regular edges.

[0061] (2) Molecular biological characteristics

[0062] Strain DNA was extracted and used as a template for 16S rDNA sequence amplification using primers 27F and 1492R. The PCR amplification reaction system was 25 μL, including 12.5 μL 2×Taq enzyme, 1 μL 27F primer, 1 μL 1492R primer, 2 μL bacterial culture, and 8.5 μL ddH2O. The amplification conditions were: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 5 s, 57℃ annealing for 10 s, 72℃ extension for 45 s, 32 cycles, followed by a 72℃ extension for 5 min. The amplified products were stored at 4℃. The amplified products were separated and identified by 1% agarose gel electrophoresis, and the PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for bidirectional sequencing.

[0063] Primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.2);

[0064] Primer 1492R: 5'-TACGGTTACCTTGTTACGACTT-3' (SEQ ID NO.3).

[0065] The 16S rDNA of strain JYT19 is shown in SEQ ID NO.1:

[0066] SEQ ID NO.1:

[0067] JYT19 16S rDNA

[0068]

[0069] The sequencing results were input into the NCBI database (http: / / www.ncbi.nlm.nih.gov) for BLAST homology alignment. The 16S rDNA gene sequences of strains with high sequence similarity and homology were selected as references. A phylogenetic tree was constructed using the neighbor-joining method with MEGAX software. The phylogenetic tree constructed based on the 16S rDNA gene sequences is shown below. Figure 2 As shown, strain JYT19 and Chryseobacterium wanjuense They clustered together, and their 16S rDNA gene sequences were similar to those of *Chlorella vulgaris*. Chryseobacterium wanjuense The strain showed 98.19% homology with strain DSM 17724(T). This strain was identified as... Chryseobacterium A strain of the genus, named *Cryptobacter yunnanensis* ( Chryseobacterium sp.)JYT19 was deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No.37640.

[0070] Example 2 Chlorella vulgaris ( Chryseobacterium Verification of the Functional Effect of JYT19 Stabilized Mushroom Residue Substrate

[0071] 2.1 Preparation of bacterial culture

[0072] The tested strain was *Cyclocarya paliurus* ( Chryseobacterium (sp.)JYT19. Single colonies of strain JYT19 were picked and inoculated into Erlenmeyer flasks containing 400 mL of tryptone soybean broth. The flasks were incubated overnight with shaking at 30°C and 200 rpm / min. The enriched bacterial culture was then diluted to OD0.05. 600 = 0.6, then centrifuged at 5000 rpm / min for 10 min, removed the supernatant and preserved the bottom bacterial cells. The strain was resuspended in sterile distilled water to prepare the bacterial suspension for use.

[0073] 2.2 Inoculation Cultivation

[0074] The prepared bacterial solution was prepared at a ratio of 1×10 8 The concentration of CFU / g was mixed with the mushroom residue composite substrate. In this experiment, the mushroom residue composite substrate was a mixture of mushroom residue, peat moss, perlite, and vermiculite in a ratio of 3:3:2:2. Two treatments were set up: the original mushroom residue composite substrate (CK) and the substrate modified with Aureobacterium chrysogenum JYT19 (T1). Non-heading Chinese cabbage 'Shanghai Bok Choy' was sown. Samples were taken every 10 days until the end of the cultivation cycle.

[0075] The physical properties of each formulation of the mixed substrate, such as bulk density, total porosity, aeration porosity, water-holding porosity, and the ratio of large to small pores, were determined according to the method of Guo Shirong (Guo Shirong. Soilless Cultivation [M]. Beijing: China Agriculture Press, 2003: 423.).

[0076] 2.3 Results Analysis

[0077] like Figure 3-7 As shown, after 40 days of cultivation, the bulk density of CK increased significantly by 11.87%, while that of T1 increased by only 4.81%, significantly lower than that of CK, and the change in bulk density was 7.06% slower than that of CK; the total porosity of CK increased significantly by 4.35%, while that of T1 increased by 3.12%, and the change in total porosity was 1.23% slower than that of CK; the aeration porosity of CK decreased significantly by 54.59%, while the aeration porosity of T1 increased by 6.90%, significantly higher than that of CK; the water-holding porosity of CK increased significantly by 14.60%, while that of T1 was not significant, increasing by only 0.66%, and was 13.95% lower than that of CK; the size ratio of CK decreased significantly by 62.66%, while the size ratio of T1 increased by 11.80%, which was 74.45% higher than that of CK, indicating that *Chlorella vulgaris* JYT19 has a modifying effect on the stability of the substrate during use.

[0078] Example 3 Chlorella vulgaris ( Chryseobacterium sp.) JYT19 Stable Mushroom Residue Substrate Plant Growth and Quality

[0079] 3.1 Plant Cultivation

[0080] The improved substrate was prepared using the method in Example 2. Plump and undamaged non-heading Chinese cabbage 'Shanghai Bok Choy' seeds were selected and placed on petri dishes containing moistened filter paper to germinate for 1 day. 'Shanghai Bok Choy' seeds were then sown in 15-well trays under natural light and normal management. After 40 days, the fresh weight of the aboveground parts of the plants was measured using a balance. After drying in an electric hot air drying oven, the dry weight of the aboveground parts of the plants was measured. The leaf area was measured using a leaf area scanner, and the vitamin C content was determined using the phosphomolybdic acid colorimetric method.

[0081] 3.2 Results Analysis

[0082] like Figure 8-11 As shown, after 40 days, the plants treated with the fungicide (T1) grew faster than the plants without fungicide (CK). The aboveground fresh weight of the plants was significantly increased, with aboveground fresh weight, aboveground dry weight and leaf area increasing by 42.54%, 31.55% and 18.96%, respectively. The vitamin C content was also significantly increased, by 34.01% compared to the CK plants. This indicates that the JYT19 fungicide can improve the physicochemical properties of mushroom residue substrate, promote the growth of non-heading Chinese cabbage plants and increase the vitamin C content in the leaves.

[0083] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Chlorella strain ( Chryseobacterium sp.)JYT19, characterized in that, The *Aureobacterium* strain JYT19 has the accession number CGMCC No. 37640 and the accession date is February 2, 2026.

2. A cultivation substrate composition, characterized in that, The cultivation substrate contains a base substrate and the *Aureobacterium chrysogenum* strain JYT19 as described in claim 1, wherein the base substrate is a composite substrate containing mushroom residue.

3. A method for improving the physical structural stability of a cultivation substrate, characterized in that, The *Aureobacterium* strain JYT19 of claim 1 was inoculated into a composite substrate containing mushroom residue.

4. The method according to claim 3, characterized in that, The inoculation method involves mixing the bacterial solution with the substrate evenly.

5. The method according to claim 4, characterized in that, Inoculation concentration: 1×10 8 CFU / g composite cultivation substrate.

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