Application of a strain of suillus Su100 in promoting melatonin synthesis and disease resistance of poplar

Abstract

This invention provides the application of a flocculent fungus, Su100, in promoting melatonin synthesis and disease resistance in poplar trees, relating to the fields of microbial resource development and forestry cultivation technology. This flocculent fungus, Su100, can activate the activity of key enzymes in melatonin synthesis within poplar trees, significantly increasing melatonin content. Melatonin, as an endogenous antioxidant and multifunctional signaling molecule in plants, plays a central role in plant stress resistance, growth regulation, quality improvement, and ecological restoration. Flocculent fungi have high colonization efficiency in plants, and using flocculent fungal inoculation technology, a symbiotic system can be formed with poplar trees, exerting a long-term effect. The application of this invention can significantly reduce agricultural and forestry production costs, and is particularly suitable for crop cultivation under adverse conditions such as drought, salinity, and disease, reducing the application of chemical fertilizers and pesticides, and laying the foundation for building an efficient, green, and sustainable forestry planting and cultivation system.

Description

Technical Field

[0001] This invention relates to the fields of microbial resource development and forest tree cultivation technology, and in particular to the application of a flocculent fungus Su100 in promoting melatonin synthesis and disease resistance in poplar trees. Background Technology

[0002] Melatonin, also known as pineal hormone, is an indole heterocyclic hormone compound widely distributed in animals, plants, and microorganisms. As a multifunctional signaling molecule, melatonin plays multiple beneficial roles in plant growth, promoting growth and development, enhancing stress resistance, and increasing crop yield and quality. Specifically, it promotes plant growth by enhancing root development and photosynthetic efficiency; it increases fruit yield and quality by prolonging flowering time, improving fruit set rate, and delaying fruit ripening; and it mitigates damage to plants caused by stresses such as salinity, low temperature, drought, heavy metals, ultraviolet radiation, and diseases by scavenging reactive oxygen species (ROS), reducing malondialdehyde (MDA) accumulation, and deeply participating in the regulation of plant hormones and ion channels. Therefore, the synthesis of melatonin helps plants form a balanced system of growth, development, and stress response.

[0003] Populus davidiana × P. bolleana, a superior variety of poplar, is a fast-growing tree species in northern my country known for its rapid growth and excellent wood quality. It is an important raw material for timber processing and pulp and papermaking, and a preferred species for urban greening. Populus davidiana plays a vital role in windbreak and sand fixation, soil and water conservation, ecosystem improvement, and contributing to carbon neutrality goals, with wide applications in ecological restoration and industrial timber. Furthermore, it is a hot research topic in forestry genetics and breeding due to its high conversion efficiency. Genetic improvement can be used to cultivate new varieties with faster growth, stronger stress resistance, and superior wood quality, promoting sustainable forestry development. However, under natural conditions, Populus davidiana exhibits low melatonin synthesis efficiency, particularly under abiotic stresses such as drought and high UV radiation, leading to problems like bark cracking and xylem oxidative damage. These issues can be addressed by applying exogenous melatonin.

[0004] However, current methods for utilizing melatonin involve cumbersome chemical synthesis, complex purification processes, and poor environmental friendliness. While extracting melatonin from plant sources is environmentally friendly, the yield is low and the overall production cost is high, making it difficult to meet large-scale agricultural demand. Furthermore, melatonin is easily degraded under high temperature and humidity conditions, requiring cold chain transportation and light-proof storage, which also increases usage costs. In agriculture, melatonin is mainly applied exogenously through seed soaking, foliar spraying, and root irrigation, but due to limitations in production costs and technological maturity, it remains in the exploratory stage and has not yet achieved large-scale promotion and widespread adoption in agricultural applications. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the application of a flocculent fungus, Su100, in promoting melatonin synthesis in poplar trees. By adding Su100, the efficiency of endogenous melatonin synthesis in poplar trees is improved, thereby enhancing the poplar's tolerance to abiotic stresses, strengthening its defense against pathogenic microorganisms, increasing afforestation survival rate and tree growth, and comprehensively improving the environmental adaptability and resource utilization efficiency of poplar trees, effectively promoting the development of eco-friendly forestry.

[0006] In order to achieve the objective of this invention, the following technical solution is adopted:

[0007] This invention provides the application of a floppy disk fungus Su100 in promoting melatonin synthesis in poplar trees.

[0008] Furthermore, the floppy disk fungus Su100 was deposited at the China General Microbiological Culture Collection Center on May 28, 2024, with accession number CGMCC No. 41247, and the floppy disk fungus Su100 was classified as Mollisia sp. Su100.

[0009] Furthermore, the poplar mentioned is *Populus tomentosa*.

[0010] The present invention also provides a microbial preparation comprising the floppy disk fungus Su100 used in the above applications.

[0011] Furthermore, the microbial preparation is one of an aerosol, liquid, or solid preparation.

[0012] The present invention also provides a product comprising the above-mentioned microbial preparation.

[0013] The present invention also provides a method for increasing the melatonin content in poplar trees, the method using the floppy disk fungus Su100 mentioned above, or the microbial preparations mentioned above, or the products mentioned above.

[0014] The present invention has the following technical effects:

[0015] The floppy disk fungus Su100 can activate the activity of key enzymes in melatonin synthesis in poplar trees, significantly increasing melatonin content and disease resistance. Melatonin, as an endogenous antioxidant and multifunctional signaling molecule in plants, plays a central role in plant stress resistance, growth regulation, quality improvement, and ecological restoration. This floppy disk fungus has high colonization efficiency in plants, and by utilizing floppy disk fungal inoculation technology, it can form a symbiotic system with poplar trees, exerting its effects over a long period.

[0016] The application of this invention can significantly reduce agricultural and forestry production costs, and is especially suitable for crop cultivation and ecological restoration under adverse conditions such as drought, salinity, and disease. It reduces the use of chemical fertilizers and pesticides, and lays the foundation for building an efficient, green, and sustainable forestry planting and cultivation system. Attached Figure Description

[0017] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0018] Figure 1 The determination of melatonin content in the fermentation broth of floppy disk fungus Su100 in Example 1;

[0019] Figure 2 The following is a bar chart showing the effect of the floppy disk fungal inoculant Su100 on the growth of potted Populus tomentosa in Example 3. A represents the effect on growth vigor; B represents the effect on plant height; C represents the effect on root morphology; and D represents the effect on root surface area.

[0020] Figure 3 The effect of floppy disk fungus Su100 treatment on melatonin content in Populus tomentosa leaves in Example 4;

[0021] Figure 4 The effect of floppy disk fungus Su100 treatment on the resistance of Populus to leaf blight in Populus tomentosa in Example 5. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0023] In a first aspect, the present invention provides the application of a floppy disk fungus Su100 in promoting melatonin synthesis in poplar trees.

[0024] The isolation, screening, detection, and identification of this floppy disk fungus Su100 were identical to the procedures used in the previously published CN118685278A strain of floppy disk fungus Su100, biological agents containing floppy disk fungus Su100, and their applications. This means it is the floppy disk fungus Su100 screened in the aforementioned literature. This floppy disk fungus Su100 was deposited on May 28, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 41247. The taxonomic name of this floppy disk fungus Su100 is Mollisia sp. Su100.

[0025] In some embodiments, the poplar is *Populus tomentosa*.

[0026] Secondly, the present invention also provides a microbial preparation comprising the floppy disk fungus Su100 used in the above applications.

[0027] In some embodiments, the microbial preparation is one of an aerosol, a liquid, or a solid formulation.

[0028] Thirdly, the present invention also provides a product comprising the above-mentioned microbial preparation.

[0029] Fourthly, the present invention also provides a method for increasing the melatonin content in poplar trees, the method using the floppy fungus Su100 mentioned above, or the microbial preparation mentioned above, or the product mentioned above.

[0030] The following is a detailed explanation using specific embodiments:

[0031] Example 1: Determination of melatonin content in fermentation broth of floppy disk fungus Su100

[0032] A piece of Su100 mycelial growth was taken from a PDA medium plate stored at 4℃ and inoculated into PDB medium. The mixture was then cultured in a constant temperature shaking incubator at 28℃ and 150-250 rpm for 5-7 days. During this period, a large number of mycelial balls were suspended in the PDB medium, yielding the Su100 fermentation broth. This Su100 fermentation broth was then sonicated at 200-400W for 30 min, centrifuged at 12000 rpm for 10 min, and 1 mL of the supernatant was collected. 4 mL of methanol was added, and the mixture was vortexed and incubated at 4℃ for 1 h. It was then centrifuged at 12000 rpm for 5 min at 4℃, and the supernatant was filtered through a 0.45 μm filter to obtain the final sample. The melatonin content in the sample was then determined using high-performance liquid chromatography (HPLC). Uninoculated PDB medium was used as a control, and the melatonin content was determined under the same conditions.

[0033] The melatonin content in the fermentation broth of the floppy disk fungus Su100 was determined to be 285.6 μg / L. Figure 1The results indicate that the floppy disk fungus Su100 successfully synthesized melatonin during fermentation, which confirms the potential of this strain as a source of melatonin production and that it can exert multiple beneficial effects on plants and can be developed into a microbial agent.

[0034] Example 2: Preparation of Su100 fungal agent for floppy disks

[0035] Take a piece of Su100 mycelial growth from a PDA medium plate stored at 4℃, inoculate it into PDB culture medium, and incubate it in a constant temperature shaking incubator at 28℃ and 150-250 rpm for 5-7 days. During this period, a large number of mycelial balls will be suspended in the PDB culture medium, thus obtaining the Su100 fermentation broth. This Su100 fermentation broth is then sonicated at 200-400W for 30 minutes, and finally diluted 5-10 times with distilled water for later use.

[0036] Chitosan was dissolved in acetic acid solution and magnetically stirred until completely dissolved. The pH of the solution was adjusted to 5.0-6.0, and the chitosan concentration was 1%-2%. The above-mentioned Su100 fungal fermentation broth was mixed with the chitosan solution at a mass ratio of 1:5-1:10, and the mixture was thoroughly shaken and evenly dispersed to obtain a mixture. A 5% NaOH solution was slowly added dropwise to the mixture, and the pH was adjusted to 9.0-10.0. The mixture was allowed to stand for at least 2 hours to gel, ensuring the gel structure was stable.

[0037] Preparation of PDB culture medium: Weigh 200g of peeled potatoes, cut them into small pieces, add distilled water and boil for 10 minutes. Filter the juice through double-layer gauze, add 20g of glucose, stir well, and bring the volume to 1L. Sterilize by autoclaving at 120℃ for 20 minutes.

[0038] PDA medium is prepared by adding 20 g / L of agar to PDB culture medium to form a solid culture medium.

[0039] Example 3: Effects of Su100 inoculant on the growth of poplar potted seedlings

[0040] Inoculant Treatment: Healthy, uniformly grown poplar seedlings with a height of 30±2cm were selected as experimental materials and transplanted into sterilized vermiculite substrate plastic pots. Poplars inoculated with the Su100 inoculant prepared in Example 2 were used as the treatment group, while poplars treated with uninoculated PDB culture solution were used as the control group. Each plant in the treatment group was irrigated with 10mL of the Su100 inoculant, while the control group was irrigated with an equal amount of PDB culture solution. Ten pots were replicated for each group. During the cultivation period, distilled water was applied every 3 days to maintain a substrate humidity of 60%. The light intensity was 2000-3000 lx, the photoperiod was 16h / 8h, and the cultivation temperature was (25±2)℃. Results were observed after one month of cultivation.

[0041] Experimental results showed that after one month of cultivation, the growth indicators of *Populus alba* bonsai seedlings treated with the *Su100* fungal agent were significantly higher than those of the control group, including plant height and diameter at root. Specifically, the *Populus alba* bonsai seedlings treated with the *Su100* fungal agent exhibited more vigorous growth of the above-ground stems and leaves, and were taller and stronger, showing significantly better growth than the control group. Figure 2 A), while root development is significantly superior, the number of lateral root branches increases significantly, the root system is denser, and the overall biomass increases ( ). Figure 2 C). Bar chart analysis showed that the average plant height of the treatment group increased by 29.9% compared with the control group. Figure 2 B), the root surface area increased by 21.8% compared to the control group. Figure 2 D) All of these are statistically significant differences. This indicates that the flocculated fungus Su100 can significantly enhance the growth vigor of *Populus tomentosa* bonsai seedlings by promoting stem elongation and root biomass. Its mechanism of action may be related to stimulating the plant to produce growth-regulating substances. Even in a limited nutrient environment with only distilled water irrigated in a sterilized vermiculite substrate, flocculated fungus Su100 still has a positive promoting effect on the growth of *Populus tomentosa* bonsai seedlings, showing that in poplar seedling cultivation, the flocculated fungus Su100 in this inoculant forms a symbiotic system with the poplar and exerts a long-term effect, indicating potential application value.

[0042] Example 4: Effects of floppy disk fungus Su100 on melatonin biosynthesis in poplar leaves

[0043] Take the top leaves of the potted Populus tomentosa seedlings cultured in the treatment group and control group in Example 3, weigh 0.5g of leaves, grind them with liquid nitrogen, add 5mL of methanol solution, sonicate for 10min, centrifuge at 12000rpm for 10min, take the supernatant and filter it through a 0.45μm filter membrane to obtain Populus tomentosa leaf extract, and then use high performance liquid chromatography (HPLC) to determine the melatonin content in Populus tomentosa leaf extract.

[0044] Compared with the control group, the melatonin content determination results showed that the melatonin content of poplar leaves treated with the Su100 inoculant was significantly increased, reaching 1.56 ng / gFW, which was 11.7 times that of the control group. Figure 3 Therefore, it can be seen that the floppy disk fungal inoculant Su100 can promote the endogenous melatonin biosynthesis of poplar trees, effectively promoting plant growth and morphogenesis.

[0045] Example 5: Effect of Su100 inoculant on the disease resistance of Populus tomentosa

[0046] Healthy, uniformly grown poplar seedlings with a height of 30±2cm were selected and randomly divided into two groups (10 seedlings per group): the treatment group was inoculated with 200mL of the Su100 fungal agent prepared in Example 2 into the rhizosphere of each seedling, while the control group was inoculated with an equal volume of sterilized PDB culture medium. On the 7th day after treatment, a spore suspension of Alternaria alternata (concentration 1×10⁻⁶) was evenly sprayed onto the leaves of all plants. 6 CFU / mL), 5 mL per plant, cover with plastic film for more than 24 hours after spraying and maintain high humidity, culture temperature 25±1℃, humidity 80%±5%, light intensity 2000-3000lx, photoperiod 16h / 8h, observe the results after 1 month.

[0047] Experimental results showed that, compared with the untreated control group, *Populus alba* treated with the flocculant Su100 significantly improved resistance to poplar leaf blight pathogens. Simultaneously, the number of diseased leaves in the treated group was significantly reduced, and the area and extent of diseased spots were significantly smaller than those in the control group, with higher leaf integrity; while in the control group, numerous leaves fell off, the entire plant became sparse, leaf spots expanded rapidly with severe tissue damage, and leaves withered and curled. Figure 4 This indicates that the floppy disk fungus Su100 can enhance the plant's active defense against pathogens by promoting the synthesis of melatonin in the plant, through metabolic regulation and activation of signaling pathways, thereby effectively inhibiting pathogen infection and significantly improving the resistance of Populus tomentosa leaf blight.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. A strain of floppy disk fungus ( Mollisia sp. The application of Su100 in promoting melatonin synthesis in poplar and resisting poplar leaf blight is characterized by, The soft disk fungus Su100 is preserved in China General Microbiological Culture Collection Center on May 28, 2024, and the preservation number is CGMCC No.41247. The poplar is Populus deltoids.

2. A method of increasing the content of melatonin in Populus simonii, characterized in that, The method comprises inoculating the soft disk fungus Su100 in the application of claim 1 into Populus deltoids. The poplar is Populus deltoids. The method comprises inoculating the soft disk fungus Su100 in the application of claim 1 into Populus deltoids.

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