Application of Micrococcus WR1 with rough-walled surface in drought-resistant chestnut cultivation

Abstract

This invention discloses the application of *Micrococcus rubra* WR1 in drought-resistant chestnut cultivation, belonging to the field of agricultural microbial application and drought-resistant planting technology. The application includes the following steps: (1) fermenting and culturing *Micrococcus rubra* WR1 to obtain WR1 fermentation broth, and using the WR1 fermentation broth to prepare WR1 inoculum; (2) applying the WR1 inoculum prepared in step (1) to the soil where chestnuts are planted. Based on the characteristics of chestnut's deep root system and high tannin rhizosphere environment, this invention obtains a plant endophytic fungus WR1 with good symbiotic effect with chestnuts through extensive screening, and applies it to chestnut drought-resistant cultivation. Through symbiosis between this strain and chestnuts, the nutrient absorption of chestnuts can be improved, chestnut growth can be promoted, and the drought resistance of chestnuts can be improved by enhancing photosynthetic physiology and antioxidant defense system, reducing oxidative damage and metabolic disorders under drought stress.

Description

Technical Field

[0001] This invention relates to the application of Micrococcus WR1 in drought-resistant chestnut cultivation, belonging to the field of agricultural microbial application and drought-resistant planting technology. Background Technology

[0002] Drought is a limiting factor for chestnuts ( Castanea mollissima The main environmental stressors in chestnut cultivation not only reduce yield by directly inhibiting the physiological metabolism of chestnut plants, but also damage the systemic stability of chestnut plants in the long term, exacerbating the pressure on sustainable development of the industry.

[0003] Existing drought-resistant technologies mainly include physical and chemical methods. While physical methods such as mulching can effectively retain moisture in the short term, long-term application can lead to decreased soil permeability, plastic residue, and excessive pollution. Chemical methods typically involve the application of water-retaining agents. Although chemical water-retaining agents can rapidly absorb hundreds of times their own weight in water through strong hydrophilic groups and form a gel, significantly inhibiting soil moisture evaporation and improving soil water-holding capacity, they can effectively alleviate drought stress and increase crop yields in arid or semi-arid regions. However, their application is environmentally dependent. High salinity and strong acid / alkalinity conditions can damage the structure of water-retaining agent molecules, leading to a decrease in their water absorption capacity. Long-term, large-scale use can also alter the soil microbial community structure, cause soil compaction, and pose risks of ecological damage.

[0004] Against this backdrop, microbial inoculants for drought resistance have become a crucial direction in drought resistance research and application. The applied microbial inoculants form a symbiotic relationship with plants, effectively promoting root development, enhancing antioxidant capacity and leaf photosynthetic rate, thereby significantly improving the plant's water absorption and utilization efficiency and enhancing its survival ability under drought conditions. Furthermore, microbial inoculants are environmentally friendly, leaving no chemical residues after use, helping to reduce the environmental risks and production costs associated with traditional chemical drought-resistant agents and agricultural films. More importantly, microbial inoculants can also optimize the soil microecological environment, promoting carbon and nitrogen cycling, thus enhancing crop drought resistance while maintaining soil ecological health, providing an effective pathway for the healthy and sustainable development of agriculture and forestry.

[0005] Currently, the application of microbial inoculants is mostly concentrated in the field of agricultural crops, while the research and development of specialized strains for woody plants is relatively lagging behind. Chestnut is an important woody food tree species. Its cupules and leaves are rich in tannins, which decompose in the rhizosphere soil after decaying, significantly affecting the soil chemical environment. This high-tannin rhizosphere microenvironment not only seriously affects the colonization conditions of microorganisms but also restricts the application effect of conventional inoculants, making existing microbial inoculants unsuitable for chestnut cultivation.

[0006] Chinese patent CN120349917A discloses a compound phosphate-solubilizing agent for chestnuts and its application. This compound phosphate-solubilizing agent can increase the growth rate of chestnut seedlings, promote root growth, and enhance phosphorus absorption by the plant. However, existing technologies have not yet disclosed microbial agents suitable for drought-resistant cultivation of chestnuts. Therefore, screening for specialized strains with strong tannin tolerance and adaptability to the rhizosphere environment of chestnuts has become an urgent task in current chestnut drought resistance research. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the purpose of this invention is to provide an application of Micrococcus WR1 in drought-resistant chestnut cultivation, which can significantly improve the drought resistance of chestnuts.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0009] Application of a rough-walled micrococcus WR1 in drought-resistant chestnut cultivation, the application includes the following steps:

[0010] (1) Micrococcus WR1 was fermented to obtain WR1 fermentation broth, and WR1 bacterial agent was prepared using the WR1 fermentation broth;

[0011] (2) Apply the WR1 inoculant prepared in step (1) to chestnut cultivation.

[0012] Based on the characteristics of chestnut's deep root system and high tannin rhizosphere environment, this invention obtained a plant endophytic fungus WR1 with good symbiotic effects with chestnut through extensive screening. WR1 establishes a "symbiotic interaction" with chestnut, which can significantly enhance the drought resistance of chestnut. Furthermore, through metabolomics analysis of its fermentation broth, it was revealed for the first time that fungus WR1 enhances the drought resistance of chestnut through a metabolic regulation mechanism mediated by unsaturated fatty acids, arginine and proline, and diterpenoids. This fills the gap in the field of drought-resistant microbial technology for chestnut, a deep-rooted woody crop, and is in line with the global development trend of green agriculture.

[0013] Furthermore, in step (1), the *Micrococcus rubrum* WR1 is a plant endophytic fungus, and its classification name is... Minutisphaera aspera It was deposited on April 11, 2017, at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, with accession number CGMCC No. 13884. For ease of use, the Chinese name of strain WR1 is defined as *Micrococcus rubra* WR1.

[0014] Furthermore, in step (1), the WR1 microbial agent includes one of WR1 liquid formulation and WR1 solid formulation.

[0015] Furthermore, the preparation method of the WR1 liquid formulation is as follows: first, dilute the WR1 fermentation broth with sterile water to a WR1 cell concentration of 1×10⁻⁶. 7 -1×10 8 The concentration of CFU / mL was increased, and then 2% glycerol was added to obtain the WR1 liquid formulation. Glycerol acts as a stabilizer to maintain the stability of the WR1 liquid formulation.

[0016] Furthermore, the preparation method of the WR1 solid dosage form is as follows: dilute the WR1 fermentation broth with sterile water to a WR1 cell concentration of 1×10⁻⁶. 8 The concentration of CFU / mL was then mixed with sterilized diatomaceous earth at a ratio of 1 mL: 2 g. After thorough mixing, the mixture was dried at a low temperature of 40 °C to obtain WR1 solid dosage form.

[0017] Furthermore, the preparation method of the WR1 fermentation broth is as follows: Micrococcus roughiformis WR1 is inoculated onto potato dextrose agar (PDA) plates and incubated upside down at 28 ℃ for 7 days; then, a 1×1 cm square piece of bacterial growth is scooped out and inoculated into potato dextrose broth (PDB) culture medium, and cultured with shaking at 28 ℃ and 180 rpm for 10-15 days.

[0018] Furthermore, the method for preparing the potato dextrose agar medium (PDA) plates is as follows: 0.6% potato extract powder, 2% glucose, and 2% agar are added to water by mass percentage, and the mixture is autoclaved at 120 ℃ for 20 min. After sterilization, the mixture is cooled to 60 ℃, poured into petri dishes, cooled and solidified, and then stored upside down. The method for preparing the potato dextrose broth (PDB) culture medium is as follows: 0.6% potato extract powder and 2% glucose are added to water by mass percentage, and the mixture is autoclaved at 120 ℃ for 20 min.

[0019] Furthermore, in step (2), the application method of the WR1 microbial agent is as follows: soak chestnut seeds and / or soak chestnut seedlings and / or irrigate the soil around chestnut trees with the WR1 liquid preparation; or mix the WR1 solid preparation with the soil and / or apply it in trenches or holes.

[0020] Furthermore, the method for soaking chestnut seeds with the WR1 liquid preparation is as follows: dilute the WR1 liquid preparation with sterile water to a WR1 cell concentration of 1×10⁻⁶. 6 Soak chestnut seeds in CFU / mL solution for 4-6 hours, then remove and air-dry before sowing. The method for soaking chestnut seedlings in the WR1 liquid preparation is as follows: Before transplanting the chestnut seedlings, dilute the WR1 liquid preparation with sterile water to a WR1 cell concentration of 1×10⁻⁶. 6Soak the roots of chestnut seedlings in CFU / mL solution for 20-30 minutes before transplanting. The method for watering the soil around the chestnut trees with the WR1 liquid formulation is as follows: After planting the chestnut trees, dilute the WR1 liquid formulation with sterile water to a WR1 bacterial concentration of 1×10⁻⁶. 6 Apply CFU / mL to the soil around the roots of the chestnut tree at a dosage of 100-200 mL per tree.

[0021] Furthermore, the method for mixing the WR1 solid preparation with the soil is as follows: In the planting holes of chestnut trees, mix the WR1 solid preparation with the soil at a dosage of 10-20 g per hole, and then use this mixture for planting chestnut seedlings or seeds. The method for applying the WR1 solid preparation in trenches or holes is as follows: Dig trenches or holes around the root system of mature chestnut trees to a depth of 10-15 cm, apply the WR1 solid preparation at a dosage of 50-100 g per tree into the trenches or holes, cover with soil, and water. When applied to the soil for chestnut planting, the WR1 solid preparation can be directly applied to the root zone, promoting fungal colonization.

[0022] Furthermore, the WR1 solid dosage form can also be diluted with sterile water or deionized water to a WR1 bacterial concentration of 1×10⁻⁶. 6 The CFU / mL suspension is administered in the same manner as the WR1 liquid formulation.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] 1. This invention applies Micrococcus wr.1 to the drought-resistant cultivation of chestnuts. Through the symbiosis between this strain and chestnuts, the absorption of nutrients by chestnuts is improved, the growth of chestnuts is promoted, and the photosynthetic physiology and antioxidant defense system of chestnuts are enhanced. The oxidative damage and metabolic disorders under drought stress are reduced, thereby improving the drought resistance of chestnuts.

[0025] 2. This invention prepares *Micrococcus roughus* WR1 into a microbial agent for drought resistance in chestnuts. Compared to traditional drought-resistant measures, this invention is a biological regulation method that does not require large amounts of water resources and avoids chemical pollution of soil and fruit. It meets the needs of green agriculture development, reduces cultivation costs, and improves ecological and economic benefits. This will provide a microbial solution for drought-resistant cultivation of chestnuts and other deep-rooted woody crops. Attached Figure Description

[0026] Figure 1 The effect of WR1 inoculant on the growth of chestnut potted seedlings under drought stress is shown in the figure. A is the control group of chestnut potted seedlings without WR1 inoculant, and B is the treatment group of chestnut potted seedlings with WR1 inoculant.

[0027] Figure 2The effects of WR1 inoculant on growth indicators of potted chestnut seedlings under drought stress are shown in Figure A, B, and C. In Figure C, WR1 inoculant affects stem length, stem diameter, and biomass of potted chestnut seedlings under drought stress. CK is the control group and WR1 is the treatment group.

[0028] Figure 3 The effect of WR1 inoculant on photosynthetic indicators of chestnut potted seedlings under drought stress is shown in Figure A, which shows the effect of WR1 inoculant on chlorophyll content of chestnut potted seedlings under drought stress, and B shows the effect of WR1 inoculant on net photosynthetic rate of chestnut potted seedlings under drought stress. CK is the control group and WR1 is the treatment group.

[0029] Figure 4 The effects of WR1 inoculant on the stress resistance index of chestnut potted seedlings under drought stress are shown in the figure. A in the figure shows the effect of WR1 inoculant on malondialdehyde content in chestnut potted seedlings under drought stress, B shows the effect of WR1 inoculant on proline content in chestnut potted seedlings under drought stress, C shows the effect of WR1 inoculant on peroxidase activity in chestnut potted seedlings under drought stress, and D shows the effect of WR1 inoculant on catalase activity in chestnut potted seedlings under drought stress. CK is the control group, and WR1 is the treatment group. Detailed Implementation

[0030] In the following examples and experimental cases, *Micrococcus roughiformis* WR1 is a plant endophytic fungus, and its classification name is... Minutisphaera aspera It was deposited on April 11, 2017 at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, with accession number CGMCC No. 13884.

[0031] Example 1

[0032] The application of a rough-walled micrococcus WR1 in drought-resistant chestnut cultivation includes the following steps:

[0033] (1) Prepare WR1 liquid formulation by fermentation culture of Micrococcus roughaena WR1. The specific method is as follows:

[0034] Micrococcus rough-walled WR1 was inoculated onto potato dextrose agar (PDA) plates and incubated upside down at 28°C for 7 days. A 1×1 cm square of bacterial growth was then collected and inoculated into potato dextrose broth (PDB) and cultured with shaking at 28°C and 180 rpm for 10–15 days to obtain the WR1 fermentation broth. The WR1 fermentation broth was then diluted with sterile water to a WR1 cell concentration of 1×10⁻⁶. 7 -1×10 8 CFU / mL, then add 2% glycerol as a stabilizer to obtain WR1 liquid formulation;

[0035] (2) Dilute the WR1 liquid preparation obtained in step (1) with sterile water to a WR1 cell concentration of 1×10⁻⁶. 6 Soak chestnut seeds at CFU / mL for 4-6 hours, then remove and air-dry before sowing.

[0036] Example 2

[0037] The application of a rough-walled micrococcus WR1 in drought-resistant chestnut cultivation includes the following steps:

[0038] (1) Prepare WR1 liquid formulation by fermentation culture of Micrococcus roughaena WR1. The specific method is as follows:

[0039] Micrococcus rough-walled WR1 was inoculated onto potato dextrose agar (PDA) plates and incubated upside down at 28°C for 7 days. A 1×1 cm square of bacterial growth was then collected and inoculated into potato dextrose broth (PDB) and cultured with shaking at 28°C and 180 rpm for 10–15 days to obtain the WR1 fermentation broth. The WR1 fermentation broth was then diluted with sterile water to a WR1 cell concentration of 1×10⁻⁶. 7 -1×10 8 CFU / mL, then add 2% glycerol as a stabilizer to obtain WR1 liquid formulation;

[0040] (2) Before transplanting chestnut seedlings, dilute the WR1 liquid preparation obtained in step (1) to a WR1 cell concentration of 1×10⁻⁶. 6 Soak the roots of chestnut seedlings in CFU / mL solution for 20-30 minutes, then transplant them.

[0041] Example 3

[0042] The application of a rough-walled micrococcus WR1 in drought-resistant chestnut cultivation includes the following steps:

[0043] (1) Prepare WR1 liquid formulation by fermentation culture of Micrococcus roughaena WR1. The specific method is as follows:

[0044] Micrococcus roughiflora WR1 was inoculated onto potato dextrose agar (PDA) plates and incubated upside down at 28°C for 7 days. A 1×1 cm square of bacterial growth was then collected and inoculated into potato dextrose broth (PDB) and cultured with shaking at 28°C and 180 rpm for 10–15 days to obtain the WR1 fermentation broth. The WR1 fermentation broth was then diluted with sterile water to a bacterial concentration of 1×10⁻⁶. 7 -1×10 8 CFU / mL, with the addition of 2% glycerol as a stabilizer, yields WR1 liquid formulation;

[0045] (2) After planting the chestnut trees, dilute the WR1 liquid preparation obtained in step (1) to a WR1 cell concentration of 1×10⁻⁶. 6 Apply CFU / mL to the soil around the roots of the chestnut tree at a dosage of 100-200 mL per tree.

[0046] Example 4

[0047] The application of a rough-walled micrococcus WR1 in drought-resistant chestnut cultivation includes the following steps:

[0048] (1) Micrococcus roughaena WR1 was fermented and cultured to prepare WR1 solid formulation, specifically as follows:

[0049] Micrococcus rough-walled WR1 was inoculated onto potato dextrose agar (PDA) plates and incubated upside down at 28°C for 7 days. A 1×1 cm square of bacterial growth was then collected and inoculated into potato dextrose broth (PDB) and cultured with shaking at 28°C and 180 rpm for 10–15 days to obtain the WR1 fermentation broth. The WR1 fermentation broth was then diluted with sterile water to a WR1 cell concentration of 1×10⁻⁶. 8 CFU / mL, it was mixed with sterilized diatomaceous earth at a ratio of 1mL:2g, and after thorough mixing, it was dried at a low temperature of 40℃ to obtain WR1 solid dosage form;

[0050] (2) In the chestnut tree planting hole, mix the WR1 solid preparation obtained in step (1) with the soil at a dosage of 10-20 g per hole and then use it for planting chestnut seedlings.

[0051] Example 5

[0052] The application of a rough-walled micrococcus WR1 in drought-resistant chestnut cultivation includes the following steps:

[0053] (1) Prepare WR1 solid formulation by fermentation culture of Micrococcus roughaena WR1. The specific method is as follows:

[0054] Micrococcus rough-walled WR1 was inoculated onto potato dextrose agar (PDA) plates and incubated upside down at 28°C for 7 days. A 1×1 cm square of bacterial growth was then collected and inoculated into potato dextrose broth (PDB) and cultured with shaking at 28°C and 180 rpm for 10–15 days to obtain the WR1 fermentation broth. The WR1 fermentation broth was then diluted with sterile water to a WR1 cell concentration of 1×10⁻⁶. 8 CFU / mL, it was mixed with sterilized diatomaceous earth at a ratio of 1mL:2g, and after thorough mixing, it was dried at a low temperature of 40℃ to obtain WR1 solid dosage form;

[0055] (2) Dig trenches or holes around the roots of mature chestnut trees, with a depth of 10-15 cm. Apply the WR1 solid preparation obtained in step (1) into the trenches or holes at a dosage of 50-100 g per tree, cover with soil and water.

[0056] Example 6

[0057] The application of a rough-walled micrococcus WR1 in drought-resistant chestnut cultivation includes the following steps:

[0058] (1) Prepare WR1 solid formulation by fermentation culture of Micrococcus roughaena WR1. The specific method is as follows:

[0059] Micrococcus rough-walled WR1 was inoculated onto PDA plates and incubated upside down at 28°C for 7 days. A 1×1 cm square of bacterial growth was then collected and inoculated into PDB culture medium, and cultured with shaking at 28°C and 180 rpm for 10-15 days to obtain the WR1 fermentation broth. The WR1 fermentation broth was then diluted with sterile water to a WR1 cell concentration of 1×10⁻⁶. 8 CFU / mL, it was mixed with sterilized diatomaceous earth at a ratio of 1mL:2g, and after thorough mixing, it was dried at a low temperature of 40℃ to obtain WR1 solid dosage form;

[0060] (2) Dilute the WR1 solid dosage form obtained in step (1) with sterile water or deionized water to a concentration of 1×10⁻⁶. 6 Apply a CFU / mL suspension to the soil around the roots of the chestnut tree at a rate of 100-200 mL per tree.

[0061] Experimental Example 1: Metabolomics Analysis of Micrococcus WR1 Fermentation Broth

[0062] 1. Preparation of fermentation broth of Micrococcus roughiflora WR1:

[0063] (1) Activation of strain: A small amount of mycelium was picked up from the slant culture of Micrococcus WR1 and transferred to a PDA plate. It was then incubated at 25-28 ℃ for 2-5 days until a strong single colony grew on the plate or a uniform mycelial ball was formed in the liquid culture medium. The activated WR1 mycelium was obtained.

[0064] The method for preparing the potato dextrose agar medium (PDA plate) is as follows: add 0.6% potato extract powder and 2% glucose by mass to water, autoclave at 120°C for 20 min, cool to 60°C after sterilization, pour into petri dishes, and store upside down after cooling and solidification.

[0065] (2) Seed culture: The activated WR1 mycelium obtained in step (1) was inoculated into 100 mL of seed culture medium and cultured with shaking at 25-28℃ and 150-200 rpm for 3-5 days to obtain seed liquid;

[0066] The seed culture medium consists of: 20 g glucose, 5 g peptone, 3 g yeast extract, 3 g potassium dihydrogen phosphate, 1.5 g magnesium sulfate, and distilled water to a final volume of 1 L, with a pH of 6.5-7.5.

[0067] (3) Fermentation culture: The seed liquid obtained in step (2) is inoculated into 1 L of fermentation medium at a volume ratio of 5-10%, and cultured with shaking at a temperature of 25-28 ℃ and a rotation speed of 180-220 rpm for 7-10 days to obtain the fermentation broth of Micrococcus wr.

[0068] The fermentation medium consists of: 20 g glucose, 5 g peptone, 3 g yeast extract, 3 g potassium dihydrogen phosphate, 1.5 g magnesium sulfate, and distilled water to a final volume of 1 L, with a pH of 6.5-7.5.

[0069] 2. Metabolomics analysis of Micrococcus WR1 fermentation broth:

[0070] (1) Take the fermentation broth of Micrococcus WR1 obtained above, centrifuge at 4℃ and 12000 rpm for 10 min, filter the supernatant through a 0.45 μm filter membrane, take 100 μL of the filtrate sample into a 1.5 mL centrifuge tube, add 400 μL of extraction solution (acetonitrile:methanol=1:1, v / v), vortex mix for 30 s, extract by low temperature ultrasonication for 30 min (5℃, 40 KHz), let stand at -20℃ for 30 min, then centrifuge at 4℃ and 13000 g for 15 min, transfer the supernatant, blow dry with nitrogen, reconstitute with 100 µL of reconstitution solution (acetonitrile:water=1:1, v / v), extract by low temperature ultrasonication for 5 min (5℃, 40 KHz), centrifuge at 4℃ and 13000 g for 10 min to obtain the supernatant.

[0071] (2) Transfer the supernatant obtained in step (1) into a vial with an inner tube for analysis. Metabolites were detected using liquid chromatography-mass spectrometry (LC-MS). The LC-MS analysis was performed using ultra-high performance liquid chromatography-tandem Fourier transform mass spectrometry (Thermo Fisher Scientific), with positive and negative ion scanning modes and a mass scan range of m / z = 70-1050. The raw data obtained by LC-MS were imported into the metabolomics processing software Progenesis QI (Waters Corporation, Milford, USA) for baseline filtering, peak identification, alignment, and normalization preprocessing. Finally, the mass spectrometry information was compared with databases such as HMDB and KEGG for structural identification and pathway enrichment analysis to reveal the information and dynamic changes of metabolites during microbial fermentation.

[0072] KEGG metabolic pathway and metabolite analysis revealed that the metabolites of *Micrococcus rubrum* WR1 contain multiple drought-related substances, indicating that *Micrococcus rubrum* WR1 exhibits multiple biosynthetic and metabolic pathways related to drought-resistant metabolites:

[0073] (1) Metabolism of unsaturated fatty acids:

[0074] The metabolites of *Micrococcus roughis* WR1 contain unsaturated fatty acids such as oleic acid, linoleic acid, and linolenic acid. Unsaturated fatty acids are important components of cell membrane phospholipids. Under drought stress, the unsaturated fatty acids secreted by the fungus can participate in the synthesis of plant cell membranes, enhance membrane fluidity, and prevent drought-induced membrane damage. Furthermore, unsaturated fatty acids are precursors for the synthesis of antioxidants (such as glutathione), promoting glutathione synthesis, increasing the activity of antioxidant enzymes, scavenging reactive oxygen species (ROS), and reducing membrane lipid peroxidation damage. In addition, WR1 metabolites also contain precursors for the biosynthesis of jasmonic acid and linoleic acid derivatives (11-hydroperoxylinolenic acid), which can also participate in stress signal transduction and enhance plant drought resistance.

[0075] (2) Arginine and proline metabolism:

[0076] The metabolites of *Micrococcus roughiflora* WR1 not only contain classic compatibility osmotic regulators such as arginine and proline, but also novel proline and arginine derivatives such as 4-acetamidobutyric acid and arginylvaline. Arginine and proline metabolism can maintain and ensure basic physiological metabolic activities. Proline, a highly soluble neutral amino acid, accumulates in large quantities in plant cells, significantly reducing cell water potential to maintain water absorption. It also acts as an antioxidant and molecular chaperone, scavenging reactive oxygen species and stabilizing protein structures and biomembranes, thereby directly protecting cells from drought damage. Arginine, on the other hand, is a precursor to the synthesis of polyamines and nitric oxide. Through its conversion into polyamines and nitric oxide, it stabilizes nucleic acid and membrane structures, regulates stomata, maintains ion balance, and enhances antioxidant defense, preventing membrane damage and severe water loss caused by drought.

[0077] (3) Metabolism of diterpenoids:

[0078] Micrococcus roughiflora WR1 metabolites contain abundant diterpenoids, including gibberellin A. 14 Diterpenoid plant hormones; diterpenoid alkaloids such as aminopicrin; and a series of unique diterpenoid compounds such as thymol. Diterpenoids are important secondary metabolites in plant stress responses. Under drought stress, these substances can activate the expression of plant defense genes, enhance osmotic regulation, improve membrane stability, and participate in antioxidant defense. Some diterpenoid hormones (such as gibberellins) participate in stomatal closure regulation, reduce water transpiration, and activate the plant's antioxidant defense system to mitigate reactive oxygen species damage. They also promote root development to enhance water absorption capacity, thereby helping to maintain a balance between growth and defense under drought conditions. In addition, endophytic fungi can also alter the diterpenoid composition of root exudates through symbiosis with plants, reshaping the rhizosphere microbial community structure and enhancing plant drought resistance.

[0079] In summary, Micrococcus wr. 1 contains multiple biosynthetic and metabolic pathways related to drought-resistant metabolites. Applying it to chestnut drought resistance can significantly improve the drought resistance of chestnuts through metabolic regulation mechanisms mediated by unsaturated fatty acids, arginine and proline, and diterpenoids.

[0080] Example 2: Effects of WR1 inoculant inoculation on chestnut potted seedlings under drought stress

[0081] Healthy chestnut seedlings were used as experimental materials and planted in a sterilized substrate (vermiculite and perennial chestnut rhizosphere soil mixed at a 1:1 mass ratio). Five potted chestnut seedlings were used as the treatment group (WR1) and five as the control group (CK) for the treatment group (100-200 mL per plant). 6A CFU / mL diluted WR1 liquid formulation was applied to the soil around the chestnut tree roots; the control group received an equal volume of sterilized WR1 liquid formulation diluted to the soil around the chestnut tree roots. After thorough watering, the potted chestnut seedlings in each group underwent routine management for 30 days. Then, drought stress treatment was initiated for both groups. Drought stress was considered to have been reached when the weight of the potted chestnut seedlings reached 40% of their saturated water holding capacity. The seedlings were then kept under natural light at a temperature of 18-25℃, without further watering. The growth of the potted chestnut seedlings was observed after 20 days (see [link to chestnut seedling growth information]). Figure 1 The study measured growth indicators of potted chestnut seedlings, including plant height, stem diameter, and biomass, as well as physiological and biochemical indicators such as net photosynthetic rate (Pn), chlorophyll (Chl) content, proline (Pro) content, malondialdehyde (MDA) content, peroxidase (POD) activity, and catalase (CAT) activity. Pn was measured using a photosynthesis meter, Chl content was determined using 95% ethanol extraction, and Pro, MDA, POD, and CAT activities were measured using reagent kits from Beijing Solarbio Science & Technology Co., Ltd. All indicators for the treatment and control groups are expressed as the average values ​​of the five potted chestnut seedlings in each group.

[0082] Experimental results are as follows Figure 1-4 As shown.

[0083] from Figure 1 It can be seen that under drought stress, the chestnut potted seedlings in the control group without WR1 inoculant had lighter-colored leaves with some yellowing, more leaves with browning at the tips, and were shorter, with fewer leaves and wilting and drooping. In contrast, the chestnut potted seedlings in the treatment group with WR1 inoculant had dark green leaves, were taller, had more and more leaves, no obvious wilting, and grew more robustly overall. This indicates that the application of WR1 inoculant can promote the growth of chestnut potted seedlings under drought stress, enhance their tolerance to drought stress, and improve their drought resistance.

[0084] Depend on Figure 2 It can be seen that under drought stress, the chestnut potted seedlings treated with WR1 microbial agent showed significantly better growth indicators such as plant height, stem diameter and biomass than the control group. This indicates that WR1 microbial agent can alleviate the inhibition of plant growth by promoting cell elongation or differentiation, and can increase plant biomass by enhancing vascular tissue development, improving water transport capacity and mechanical support strength, thereby improving the drought resistance of chestnut potted seedlings.

[0085] from Figure 3It can be seen that, under the same drought stress, the chlorophyll content and net photosynthetic rate of chestnut potted seedlings treated with WR1 inoculant were significantly better than those of the control group. This indicates that WR1 inoculant can significantly increase the chlorophyll content and net photosynthetic rate of chestnut leaves, suggesting that in terms of photosynthetic physiology, WR1 inoculant can improve the nutrient absorption of chestnut potted seedlings and promote growth through symbiosis with them. At the same time, it can alleviate the damage to the photosynthetic system caused by drought, maintain a high photosynthetic capacity, and promote the growth of chestnut potted seedlings by increasing the accumulation of organic matter through promoting the photosynthetic rate.

[0086] from Figure 4 It can be seen that, compared with the control group, the application of WR1 bacterial agent in the treatment group can significantly reduce the content of membrane lipid peroxidation product MDA, increase the content of Pro osmotic regulator, and enhance the activity of POD and CAT. MDA is the end product of membrane lipid peroxidation, and its content is a key indicator for measuring the degree of drought damage to plant cell membranes. Increased MDA content indicates that drought stress triggers reactive oxygen species (ROS) to attack cell membrane lipids, leading to membrane structure damage. Therefore, decreased MDA content indicates that the WR1 inoculant effectively alleviates the oxidative damage to chestnut potted seedling cell membranes caused by drought stress. Under drought stress, plant leaves maintain osmotic balance by accumulating protease (Pro), which is an important osmotic regulation mechanism. As a soluble solute, increased Pro content can effectively increase intracellular solute concentration, thereby increasing cell osmotic pressure and preventing cell dehydration. Therefore, increased Pro content indicates improved drought adaptability of chestnut potted seedlings. POD and CAT are both important enzymes in the plant's antioxidant defense system, capable of decomposing hydrogen peroxide. Their synergistic effect removes excess ROS generated under drought stress, reducing oxidative damage and MDA accumulation caused by drought. Enhanced POD and CAT activity can improve the drought resistance of chestnuts. Therefore, the application of the WR1 inoculant of this invention can significantly improve the drought resistance of chestnuts.

[0087] In summary, under drought stress, *Micrococcus roughus* WR1 effectively alleviated the inhibitory effect of drought stress on potted chestnut seedlings by promoting plant growth, enhancing photosynthetic performance, improving antioxidant defense levels and osmotic regulation capabilities, and reducing cell membrane damage, thus demonstrating a significant drought resistance effect in chestnuts. This is because the WR1 fungus can improve nutrient absorption and promote chestnut plant growth through symbiosis with chestnuts, while also enhancing the chestnut's photosynthetic physiology and antioxidant defense system, reducing oxidative damage and metabolic disorders under drought stress, and improving the chestnut's drought resistance. This invention utilizes the WR1 inoculant for drought resistance in chestnuts, providing a microbial solution for drought-resistant cultivation of chestnuts and other deep-rooted woody crops.

Claims

1. The application of a type of rough-walled micrococcus WR1 in drought-resistant chestnut cultivation, characterized in that: The application includes the following steps: (1) Micrococcus WR1 was fermented to obtain WR1 fermentation broth, and WR1 bacterial agent was prepared using the WR1 fermentation broth; WR1 microbial agent includes one of WR1 liquid formulation and WR1 solid formulation; the preparation method of WR1 liquid formulation is as follows: first, dilute WR1 fermentation broth with sterile water to a WR1 cell concentration of 1×10⁻⁶. 7 -1×10 8 The concentration of CFU / mL was increased, and then 2% glycerol was added to obtain the WR1 liquid formulation. The preparation method of the WR1 solid formulation was as follows: the WR1 fermentation broth was diluted with sterile water to a WR1 cell concentration of 1×10⁻⁶. 8 CFU / mL, then mixed with sterilized diatomaceous earth at a ratio of 1mL:2g, and dried at 40℃ after thorough mixing to obtain WR1 solid dosage form; (2) Apply the WR1 inoculant prepared in step (1) to chestnut cultivation; The application methods for WR1 inoculant are as follows: soak chestnut seeds and / or soak chestnut seedlings in WR1 liquid formulation and / or irrigate the soil around chestnut trees; or mix WR1 solid formulation with soil and / or apply it in trenches or holes. The method for soaking chestnut seeds with the WR1 liquid preparation is as follows: Dilute the WR1 liquid preparation with sterile water to a WR1 bacterial concentration of 1×10⁻⁶. 6 Soak chestnut seeds in CFU / mL solution for 4-6 hours, then remove and air-dry before sowing. The method for soaking chestnut seedlings in the WR1 liquid preparation is as follows: Before transplanting the chestnut seedlings, dilute the WR1 liquid preparation with sterile water to a WR1 cell concentration of 1×10⁻⁶. 6 Soak the roots of chestnut seedlings in CFU / mL solution for 20-30 minutes before transplanting. The method for watering the soil around the chestnut trees with the WR1 liquid formulation is as follows: After planting the chestnut trees, dilute the WR1 liquid formulation with sterile water to a WR1 bacterial concentration of 1×10⁻⁶. 6 CFU / mL, apply 100-200 mL per tree to the soil around the roots of the chestnut tree; The method of mixing the WR1 solid preparation with the soil is as follows: In the planting hole of chestnut tree, mix the WR1 solid preparation with the soil at a dosage of 10-20 g per hole and use it for planting chestnut seedlings or chestnut seeds; The method of applying the WR1 solid preparation in trenches or holes is as follows: Dig trenches or holes around the root system of mature chestnut trees, with a depth of 10-15 cm, apply the WR1 solid preparation at a dosage of 50-100 g per tree into the trench or hole, cover with soil and water. In step (1), *Micrococcus rubra* WR1 is a plant endophytic fungus, and its classification name is... Minutisphaera aspera It was deposited on April 11, 2017 at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, with accession number CGMCC No. 13884.

2. The application according to claim 1, characterized in that: The *Micrococcus wr. 1* can grow in the high-tannin rhizosphere environment of chestnut and induce chestnut to produce metabolites including unsaturated fatty acids, arginine and proline derivatives, and diterpenoids, thereby enhancing the drought resistance of chestnut.

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