Tea tree culture medium and application thereof

Abstract

The invention discloses a tea tree culture medium and application thereof, and relates to the technical field of biology. The tea tree culture medium comprises a basic culture medium and a microorganism combination, the microbial composition comprises bacillus niger and trichoderma longibrachiatum. The culture medium with air permeability, water-retaining property and fertility is constructed through scientific proportioning, and meanwhile, microorganisms are uniformly colonized in the medium by adding a microorganism combination, so that growth and diffusion of root cancer pathogenic bacteria can be continuously inhibited. Experimental data show that the survival rate of the tea trees cultivated by adopting the tea tree cultivation medium is effectively improved, the root activity is remarkably enhanced, and the dual purposes of disease prevention and control and high quality and high yield of the tea trees are achieved. According to the technical scheme, microbial ecological regulation and control and substrate physical and chemical improvement are combined, and a green and sustainable solution is provided for tea tree planting.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a tea tree cultivation substrate and its application. BACKGROUND

[0002] Tea tree root cancer disease is a bacterial disease caused by Agrobacterium tumefaciens, mainly harming the roots of tea trees. The roots of diseased plants initially form light brown spherical protrusions, which gradually develop into brown woody tumor-like structures, causing the plants to weaken or die. The pathogen can be transmitted through infected seedlings, soil, and irrigation water. The pathogen is a gram-negative short rod that thrives at temperatures between 25-30°C. It invades the cortical tissue of tea trees through wounds and stimulates abnormal cell proliferation, forming tumors. It mainly damages the roots. The main roots and lateral roots initially produce light brown spherical protrusions at the diseased area, which later expand into tumor-like structures, small like grains, and large like broad beans. Multiple small tumors aggregate to form large tumors, and the tumors become brown and woody in the later stages, with a rough surface and hard texture. The infected seedlings of cuttings have few or no root hairs, causing the seedling plants to weaken or the leaves to turn yellow and fall off, leading to plant death.

[0003] Tea tree is an important economic crop, and the cultivation of high-quality tea seedlings is the foundation for ensuring tea yield and quality. Cuttage propagation is one of the main methods for tea seedling cultivation due to its ability to maintain the excellent traits of the mother plant, high propagation efficiency, and simple operation, and is widely used in large-scale tea garden construction and variety improvement. However, the occurrence of root cancer disease during tea seedling cuttage has always been a major problem that affects the success rate of seedling cultivation, seriously affecting the healthy development of the tea industry.

[0004] Tea tree root cancer disease is caused by the infection of Agrobacterium tumefaciens in the soil. This pathogen can invade the roots of tea seedlings through cuttage wounds, leading to abnormal cell proliferation in the root system and the formation of cancerous tumor-like structures of varying sizes. These tumors not only hinder the absorption and transport of water and nutrients by the root system, but also disrupt the normal physiological functions of the root system, causing tea seedlings to exhibit symptoms such as growth retardation, leaf yellowing, and decreased stress tolerance, and in severe cases, can lead to the death of the entire plant. Survey data shows that the incidence of root cancer disease in tea seedling cuttage can reach 15%~30%, and in some areas, it is even higher, causing significant economic losses to seedling cultivation enterprises and tea farmers.

[0005] Currently, the main control methods for tea crown gall disease include chemical control, physical control, and agricultural control. While chemical control can suppress pathogens in the short term, long-term use of chemical agents can easily lead to soil pollution, increased pathogen resistance, and decreased tea quality, contradicting the current concept of green agriculture. Physical control methods, such as high-temperature soil sterilization, can reduce the number of pathogens, but they are costly, energy-intensive, and may disrupt the soil's microecological balance, which is detrimental to the long-term growth of tea seedlings. Agricultural control measures, such as crop rotation and the selection of disease-resistant varieties, while environmentally friendly, are slow to take effect and difficult to cope with the sudden outbreaks of crown gall disease. Therefore, finding an efficient, environmentally friendly, and sustainable method for controlling crown gall disease, while also considering the rooting and growth needs of tea seedlings, has become a pressing technical challenge in the field of tea seedling cultivation.

[0006] As the carrier for tea seedling cuttings, the cultivation substrate's physicochemical properties and microbial environment directly affect the rooting rate, survival rate, and disease resistance of the cuttings. Traditional tea cutting substrates are often prepared by mixing garden soil, leaf mold, and perlite. While these provide some physical support and nutrients, their effectiveness in inhibiting pathogens is limited, making it difficult to effectively prevent crown gall disease. In recent years, with the expansion of microbial technology applications in agriculture, the idea of ​​using beneficial microorganisms to regulate soil microecology and inhibit pathogen reproduction has provided a new direction for the prevention and control of tea crown gall disease. Developing a tea cultivation substrate integrating highly efficient disease-resistant microbial agents is of significant practical importance for improving the survival rate of tea seedling cuttings and controlling the occurrence of crown gall disease. Summary of the Invention

[0007] The purpose of this invention is to provide a tea tree cultivation substrate and its application to solve the problems existing in the prior art. Using this tea tree cultivation substrate can effectively prevent and control tea tree crown gall, improve the survival rate of tea trees, and enhance root vitality, thereby facilitating the achievement of the dual goals of disease control and high-quality, high-yield tea production.

[0008] To achieve the above objectives, the present invention provides the following solution:

[0009] This invention provides a microbial combination for preventing and treating root cancer in tea trees, comprising Bacillus badius and Trichoderma longibrachiatum.

[0010] Furthermore, the ratio of viable bacteria of the *Bacillus spp.* and the *Trichoderma longifolia* is (1-9):9.

[0011] Preferably, the ratio of viable bacteria of Bacillus spp. and Trichoderma longifolia is 2:3.

[0012] The present invention also provides the application of the above-mentioned microbial combination in the prevention and treatment of tea tree crown gall disease or in the preparation of tea tree cultivation substrate for the prevention and treatment of tea tree crown gall disease.

[0013] The present invention also provides a tea tree cultivation substrate, comprising a basic cultivation substrate and the above-mentioned microbial combination.

[0014] Further, by weight, the basic cultivation substrate comprises the following components: 30-40 parts leaf mold, 10-20 parts perlite, 20-30 parts peat moss, 10-20 parts well-rotted sheep manure, 5-10 parts vermiculite, and 1-3 parts rice husk ash.

[0015] Preferably, the basic cultivation substrate comprises the following components by weight: 35 parts leaf mold, 15 parts perlite, 25 parts peat moss, 15 parts well-rotted sheep manure, 8 parts vermiculite, and 2 parts rice husk ash.

[0016] The present invention also provides a method for preparing the above-mentioned tea tree cultivation substrate, comprising the following steps:

[0017] The leaf mold, perlite, peat moss, decomposed sheep manure, vermiculite, and rice husk ash are mixed evenly according to the formula to obtain the basic cultivation substrate;

[0018] The mixed bacterial suspension was added to the basic cultivation substrate and mixed evenly to obtain the tea tree cultivation substrate;

[0019] The mixed bacterial suspension includes the aforementioned microbial combination.

[0020] Furthermore, the mixed bacterial suspension is added at 4-6% of the weight of the basic cultivation substrate.

[0021] The present invention also provides the application of the above-mentioned tea tree cultivation substrate in tea tree cultivation.

[0022] The present invention discloses the following technical effects:

[0023] This invention discovers a microbial synergy—Bacillus erythrophorus and Trichoderma longifolia—that exhibits a significant synergistic effect in controlling crown gall disease in tea trees. When the viable count ratio of Bacillus erythrophorus to Trichoderma longifolia is (1-9):9, the two synergistically inhibit pathogen infection. A 2:3 ratio further optimizes the synergy between the microorganisms, significantly reducing the incidence of crown gall disease (inhibition rate exceeding 85%) while simultaneously promoting root development and nutrient absorption in tea trees. Compared to single strains or conventional chemical agents, this synergy not only avoids chemical residues and pathogen resistance issues but also enhances the persistence of control through dynamic balance of viable bacteria, making it suitable for healthy cultivation throughout the entire growth cycle of tea trees.

[0024] Based on this microbial combination, this invention further constructs a cultivation substrate that combines breathability, water retention, and fertility through scientific formulation. Simultaneously, the addition of the microbial combination ensures uniform colonization of the microorganisms within the substrate, thereby continuously inhibiting the growth and spread of crown gall pathogens. Experimental data show that tea trees cultivated using the substrate of this invention exhibit significantly improved survival rates and enhanced root vitality, achieving the dual goals of disease control and high-quality, high-yield tea production. The technical solution of this invention combines microbial ecological regulation with substrate physicochemical improvement, providing a green and sustainable solution for tea cultivation. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] The strain information used in the following examples is as follows:

[0031] Bacillus badius ATCC 14574 was purchased from the China Industrial Microbial Culture Collection Center (CICC), product code 01008K; Trichoderma longibrachiatum was purchased from the China Industrial Microbial Culture Collection Center, strain number CICC 40340; Trichoderma harzianum was purchased from the China Industrial Microbial Culture Collection Center, strain number CICC 13010.

[0032] The culture medium formulations used in the following examples are as follows:

[0033] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract and 5 g / L NaCl, pH 7.0±0.2.

[0034] LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl and 15 g / L agar, pH 7.0±0.2.

[0035] PDA liquid culture medium: 200 g / L potato, 20 g / L sucrose, natural pH.

[0036] PDA solid medium: 200 g / L potato, 20 g / L sucrose, 20 g / L agar, natural pH.

[0037] The fermentation culture method of Bacillus thuringiensis is as follows:

[0038] (1) Inoculate Bacillus spp. into LB liquid medium and culture at 37℃ with shaking at 160 r / min for 16 h to obtain Bacillus spp. bacterial culture;

[0039] (2) The Bacillus thuringiensis bronchiseptica obtained in step (1) was streaked onto LB solid medium plates and incubated at 37°C for 48 h.

[0040] (3) Pick a single colony from the plate in step (2) and inoculate it into a shake flask containing LB liquid medium. Incubate at 37℃ and 180 r / min for 48 h to obtain Bacillus thuringiensis fermentation broth.

[0041] The fermentation culture methods for Trichoderma longifolia and Trichoderma harzianum are as follows:

[0042] (1) Select a piece of Trichoderma longifolia (or Trichoderma harzianum) and inoculate it into PDA liquid culture medium. Incubate at 30℃ and 160 r / min for 72 h to obtain the culture medium.

[0043] (2) Dilute the culture medium obtained in step (1) and spread it on PDA solid culture medium, and incubate it in an incubator at 30°C for 5 days;

[0044] (3) Pick a single colony from the plate in step (2) and inoculate it into a shake flask containing PDA liquid culture medium. Incubate at 30°C and 200 r / min for 72 h to obtain Trichoderma longifolia (or Trichoderma harzianum) fermentation broth.

[0045] The TTC method for determining root activity is as follows:

[0046] (1) Reagent preparation: Prepare 0.4% 2,3,5-triphenyltetrazolium chloride (TTC) solution (weigh 0.4g TTC and dissolve it in a small amount of distilled water, and make up to 100mL), phosphate buffer (0.1mol / L, pH 7.0), 1mol / L sulfuric acid solution (for terminating the reaction), and ethyl acetate (for extracting the chromogenic substance).

[0047] (2) Root treatment: Three tea seedlings were randomly selected from each treatment group. The soil around the roots was carefully rinsed, the surface moisture was dried with absorbent paper, and healthy roots were cut off (avoiding the cancerous parts). 0.5g of fresh roots were weighed, cut into small sections of about 0.5cm, and placed into 50mL centrifuge tubes.

[0048] (3) Construction of reaction system: Add 5 mL of 0.4% TTC solution and 5 mL of phosphate buffer to a centrifuge tube, shake gently to completely immerse the roots in the solution, and place in a 37℃ constant temperature incubator to react in the dark for 3 hours.

[0049] (4) Termination and extraction: After the reaction is completed, immediately add 2 mL of 1 mol / L sulfuric acid solution to terminate the reaction, pour out the supernatant, rinse the roots with distilled water 2-3 times, absorb the water and transfer them to a mortar, add 5 mL of ethyl acetate and grind until the roots turn white, transfer the grinding liquid to a centrifuge tube, centrifuge at 3000 r / min for 5 minutes, and take the supernatant.

[0050] (5) Measurement and Calculation: The absorbance of the supernatant was measured at a wavelength of 485 nm using a spectrophotometer, with ethyl acetate as a blank control. The content of the reduction product triphenylformazan (TTF) was calculated based on the standard curve. Root activity was expressed as the number of milligrams (mg·g) of TTC reduced per gram of fresh root per hour. -1 ·h -1 The data indicates that the root vitality enhancement rate of each group was calculated based on the negative control group.

[0051] Example 1: Detection of bacterial affinity

[0052] Using a 5mm diameter punch, mycelial blocks were taken from the edges of *Trichoderma longifolia* and *Trichoderma harzianum* colonies cultured for 2 days, and placed in the center of PDA plates. Then, activated *Bacillus oryzae* was inoculated at a distance of approximately 20mm from the center of each mycelial block. *Trichoderma longifolia* and *Trichoderma harzianum* without *Bacillus oryzae* inoculation were set up as control groups. Each strain was tested in triplicate. The cultures were co-cultured at 30℃ for 5 days, and the formation of inhibition zones between strains was observed and recorded. The results showed that no obvious inhibition zones were observed between *Bacillus oryzae* and *Trichoderma longifolia*, nor between *Bacillus oryzae* and *Trichoderma harzianum*, indicating that *Bacillus oryzae* has good symbiotic affinity with either *Trichoderma longifolia* or *Trichoderma harzianum*.

[0053] Example 2

[0054] The half-maximal effective concentrations (EC50) of *Bacillus spp.*, *Trichoderma longifolia*, and *Trichoderma harzianum* in inhibiting *Agrobacterium tumefaciens* were determined using the mycelial growth rate method. 50 The method is as follows:

[0055] After fermentation culture of the test strains, bacterial cells (Trichoderma longifolia and Trichoderma harzianum both refer to spores) were isolated. The obtained bacterial cells were resuspended in sterile physiological saline to concentrations of 10 and 10, respectively. 2 10 3 10 4 10 5 10 6 10 7 10 8 CFU / mL bacterial suspension was prepared, and then 1 mL of bacterial suspension was added to each 20 mL LB solid medium to prepare LB plates containing different concentrations of bacterial suspension. The control treatment was to add 1 mL of sterile physiological saline to the LB plate, and each treatment was repeated in triplicate.

[0056] Agrobacterium tumefaciens cultured for 2 days was inoculated into the center of LB agar plates containing different concentrations of bacterial suspension using the filter paper disc inoculation method. The plates were then incubated upside down at 30°C for 5 days. The diameter of colonies in each treatment was measured using the cross-sectional method. Based on the measurement results, the inhibition rate of each treatment against the pathogen was calculated, and the EC50 was determined. 50 value.

[0057] Inhibition rate = [(Control group colony diameter - filter paper diameter) - (Treatment group colony diameter - filter paper diameter)] / (Control group colony diameter - filter paper diameter) × 100%.

[0058] The results showed that *Bacillus churnii*, *Trichoderma longifolia*, and *Trichoderma harzianum* all had certain inhibitory effects on *Agrobacterium tumefaciens*. Among them, *Bacillus churnii* showed the highest EC50 concentration.50 3.2×10 5 CFU / mL, EC of Trichoderma longibranchii 50 It is 6.1×10 5 CFU / mL, EC of Trichoderma harzianum 50 2.5×10 5 CFU / mL.

[0059] Example 3

[0060] Take a concentration of 10 6 LB agar plates were prepared with CFU / mL suspensions of *Bacillus pumilus*, *Trichoderma longifolia*, and *Trichoderma harzianum*, using different suspension ratios. The volume ratios of *Bacillus pumilus* to *Trichoderma longifolia* (or *Trichoderma harzianum*) suspensions were 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10, resulting in 22 different compound culture media (11 ratios of *Bacillus pumilus* to *Trichoderma longifolia* and 11 ratios of *Bacillus pumilus* to *Trichoderma harzianum*). The control treatment involved adding 1 mL of sterile physiological saline to the LB agar plate. Each treatment was repeated three times. The inoculation method for *Agrobacterium tumefaciens* and the subsequent plate culture method were the same as in Example 2.

[0061] After incubating upside down in a 30℃ constant temperature incubator for 5 days, the growth diameter of colonies in each treatment was measured using the cross-sectional method. Based on the measurement results, the actual inhibition rate and theoretical inhibition rate of each treatment were calculated.

[0062] Combined toxicity synergistic calculation method:

[0063] The Horsfall method was used to calculate the synergistic effect of the combined bacteria in different proportions. The synergistic effect was determined based on the synergistic ratio (IR): synergistic effect: IR value > 1.2; additive effect: 0.8 ≤ IR value ≤ 1.2; antagonistic effect: IR value < 0.8.

[0064] IR = Actual antibacterial rate / Theoretical antibacterial rate;

[0065] Theoretical antibacterial rate = Actual antibacterial rate of strain A when used alone × Percentage of strain A in the mixture + Actual antibacterial rate of strain B when used alone × Percentage of strain B in the mixture.

[0066] The combined toxicity test results of each treatment group against *Agrobacterium tumefaciens* are shown in Table 1. The results showed that when the volume ratio of *Bacillus churnii* and *Trichoderma longicornis* suspensions was in the range of 1:(1~9), different degrees of synergistic effect were observed. The 4:6 ratio treatment group showed the best synergistic effect, with an synergistic ratio of 1.62 and an actual inhibition rate of 80.25%. The 3:7 ratio treatment group was the second best, with an synergistic ratio of 1.41 and an actual inhibition rate of 70.52%. However, no synergistic effect was observed between *Bacillus churnii* and *Trichoderma harzianum* suspensions at any volume ratio. Later, two treatment groups with better synergistic effects between *Bacillus churnii* and *Trichoderma longicornis* were selected for pot experiment on control efficacy.

[0067] Table 1. Results of combined virulence assays of each treatment group against *Agrobacterium tumefaciens*.

[0068]

[0069] Example 4

[0070] A method for preparing a tea tree cultivation substrate:

[0071] (1) Preparation of basic cultivation substrate

[0072] The basic cultivation substrate consists of the following components by weight: 35 parts leaf mold, 15 parts perlite, 25 parts peat moss, 15 parts well-rotted sheep manure, 8 parts vermiculite, and 2 parts rice husk ash.

[0073] During preparation, the leaf mold is first sieved using a 5mm mesh screen to remove impurities such as stones and twigs, ensuring uniform particle size. Perlite is soaked in water for 2 hours beforehand, then drained to reduce dust generation during subsequent mixing. Peat moss is naturally sun-dried for 1 day to control its moisture content at 30%~35% (i.e., peat moss can be formed into a clump when squeezed, but crumbles easily when lightly touched). Well-rotted sheep manure is sieved through a 60-mesh screen to remove incompletely decomposed manure particles and impurities. Vermiculite is crushed and sieved through a 3mm mesh screen to select uniformly sized particles. Rice husk ash is placed in a ventilated area to cool naturally to room temperature to avoid affecting the activity of subsequent microorganisms due to excessively high temperatures. Add the treated leaf mold, perlite, peat moss, well-rotted sheep manure, vermiculite, and rice husk ash to a horizontal mixer in the specified proportions. Set the mixing speed to 150 rpm and the mixing time to 20 minutes to ensure that all components are mixed evenly to obtain the basic cultivation substrate.

[0074] (2) Preparation of Bacillus chrysogenum suspension

[0075] After fermentation culture of Bacillus oryzae, bacterial cells were isolated and then resuspended in sterile physiological saline to obtain 10 7 A suspension of Bacillus oryzae at a concentration of CFU / mL.

[0076] (3) Preparation of Trichoderma longifolia suspension

[0077] After fermentation culture of Trichoderma longifolia, spores were isolated and then resuspended in sterile physiological saline to obtain 10 7 A suspension of Trichoderma longifolia at a concentration of CFU / mL.

[0078] (4) Preparation of tea tree cultivation substrate

[0079] Place the basic cultivation substrate prepared in step (1) into a mixer and start stirring (100 r / min). Simultaneously, measure the Bacillus erythrorhizon suspension prepared in step (2) and the Trichoderma longicornis suspension prepared in step (3) according to a bacterial volume ratio of 4:6. Slowly pour both into the same sterile container and stir with a sterile glass rod for 1 minute to ensure that the two bacterial suspensions are evenly mixed. Then, slowly spray the mixed bacterial suspension into the stirring basic cultivation substrate at a ratio of 5% of the weight of the basic cultivation substrate (i.e., add 5 kg of mixed bacterial suspension per 100 kg of basic cultivation substrate). Keep stirring during the spraying process. After spraying, continue stirring for 30 minutes to ensure that the bacterial suspension and basic cultivation substrate are fully mixed to obtain the tea tree cultivation substrate. Put the cultivation substrate into a sterile plastic bag, seal it, and place it in a 25°C environment for 24 hours to allow the microorganisms to initially colonize in the substrate, which can then be used for tea tree cultivation.

[0080] Example 5

[0081] A method for preparing a tea tree cultivation substrate:

[0082] (1) Preparation of basic cultivation substrate

[0083] The basic cultivation substrate consists of the following components by weight: 30 parts leaf mold, 20 parts perlite, 20 parts peat moss, 20 parts well-rotted sheep manure, 5 parts vermiculite, and 3 parts rice husk ash.

[0084] During preparation, the leaf mold is first sieved using a 5mm mesh screen to remove impurities such as stones and twigs, ensuring uniform particle size. Perlite is soaked in water for 2 hours beforehand, then drained to reduce dust generation during subsequent mixing. Peat moss is naturally sun-dried for 1 day to control its moisture content at 30%~35% (i.e., peat moss can be formed into a clump when squeezed, but crumbles easily when lightly touched). Well-rotted sheep manure is sieved through a 60-mesh screen to remove incompletely decomposed manure particles and impurities. Vermiculite is crushed and sieved through a 3mm mesh screen to select uniformly sized particles. Rice husk ash is placed in a ventilated area to cool naturally to room temperature to avoid affecting the activity of subsequent microorganisms due to excessively high temperatures. Add the treated leaf mold, perlite, peat moss, well-rotted sheep manure, vermiculite, and rice husk ash to a horizontal mixer in the specified proportions. Set the mixing speed to 150 rpm and the mixing time to 20 minutes to ensure that all components are mixed evenly to obtain the basic cultivation substrate.

[0085] (2) Preparation of Bacillus chrysogenum suspension

[0086] After fermentation culture of Bacillus oryzae, bacterial cells were isolated and then resuspended in sterile physiological saline to obtain 10 7 A suspension of Bacillus oryzae at a concentration of CFU / mL.

[0087] (3) Preparation of Trichoderma longifolia suspension

[0088] After fermentation culture of Trichoderma longifolia, spores were isolated and then resuspended in sterile physiological saline to obtain 10 7 A suspension of Trichoderma longifolia at a concentration of CFU / mL.

[0089] (4) Preparation of tea tree cultivation substrate

[0090] Place the basic cultivation substrate prepared in step (1) into a mixer and start stirring (100 r / min). Simultaneously, measure the Bacillus erythrorhizon suspension prepared in step (2) and the Trichoderma longicornis suspension prepared in step (3) according to a 5:5 volume ratio. Slowly pour both into the same sterile container and stir with a sterile glass rod for 1 minute to ensure uniform mixing of the two bacterial suspensions. Then, slowly spray the mixed bacterial suspension into the stirring basic cultivation substrate at a ratio of 4% of the weight of the basic cultivation substrate (i.e., 4 kg of mixed bacterial suspension per 100 kg of basic cultivation substrate). Keep stirring during the spraying process. After spraying, continue stirring for 30 minutes to ensure that the bacterial suspension and basic cultivation substrate are fully mixed to obtain the tea tree cultivation substrate. Place the cultivation substrate into a sterile plastic bag, seal it, and place it at 25°C for 24 hours to allow the microorganisms to initially colonize in the substrate, which is then ready for use in tea tree cultivation.

[0091] Example 6

[0092] A method for preparing a tea tree cultivation substrate:

[0093] (1) Preparation of basic cultivation substrate

[0094] The basic cultivation substrate consists of the following components by weight: 40 parts leaf mold, 10 parts perlite, 30 parts peat moss, 10 parts well-rotted sheep manure, 10 parts vermiculite, and 1 part rice husk ash.

[0095] During preparation, the leaf mold is first sieved using a 5mm mesh screen to remove impurities such as stones and twigs, ensuring uniform particle size. Perlite is soaked in water for 2 hours beforehand, then drained to reduce dust generation during subsequent mixing. Peat moss is naturally sun-dried for 1 day to control its moisture content at 30%~35% (i.e., peat moss can be formed into a clump when squeezed, but crumbles easily when lightly touched). Well-rotted sheep manure is sieved through a 60-mesh screen to remove incompletely decomposed manure particles and impurities. Vermiculite is crushed and sieved through a 3mm mesh screen to select uniformly sized particles. Rice husk ash is placed in a ventilated area to cool naturally to room temperature to avoid affecting the activity of subsequent microorganisms due to excessively high temperatures. Add the treated leaf mold, perlite, peat moss, well-rotted sheep manure, vermiculite, and rice husk ash to a horizontal mixer in the specified proportions. Set the mixing speed to 150 rpm and the mixing time to 20 minutes to ensure that all components are mixed evenly to obtain the basic cultivation substrate.

[0096] (2) Preparation of Bacillus chrysogenum suspension

[0097] After fermentation culture of Bacillus oryzae, bacterial cells were isolated and then resuspended in sterile physiological saline to obtain 10 7 A suspension of Bacillus oryzae at a concentration of CFU / mL.

[0098] (3) Preparation of Trichoderma longifolia suspension

[0099] After fermentation culture of Trichoderma longifolia, spores were isolated and then resuspended in sterile physiological saline to obtain 10 7 A suspension of Trichoderma longifolia at a concentration of CFU / mL.

[0100] (4) Preparation of tea tree cultivation substrate

[0101] Place the basic cultivation substrate prepared in step (1) into a mixer and start stirring (100 r / min). Simultaneously, measure the Bacillus erythrorhizon suspension prepared in step (2) and the Trichoderma longicornis suspension prepared in step (3) according to a bacterial volume ratio of 1:9. Slowly pour both into the same sterile container and stir with a sterile glass rod for 1 minute to ensure that the two bacterial suspensions are evenly mixed. Then, slowly spray the mixed bacterial suspension into the stirring basic cultivation substrate at a ratio of 6% of the weight of the basic cultivation substrate (i.e., 6 kg of mixed bacterial suspension per 100 kg of basic cultivation substrate). Keep stirring during the spraying process. After spraying, continue stirring for 30 minutes to ensure that the bacterial suspension and the basic cultivation substrate are fully mixed to obtain the tea tree cultivation substrate. Put the cultivation substrate into a sterile plastic bag, seal it, and place it in a 25°C environment for 24 hours to allow the microorganisms to initially colonize in the substrate, which can then be used for tea tree cultivation.

[0102] Comparative Example 1

[0103] Same as Example 4, except that the Trichoderma longifolia suspension is not used, and the mixed bacterial suspension in step (4) is replaced with Bacillus thuringiensis suspension.

[0104] Comparative Example 2

[0105] Same as Example 4, except that Bacillus spp. suspension is not used, and the mixed bacterial suspension in step (4) is replaced with Trichoderma longifolia suspension.

[0106] Comparative Example 3

[0107] Same as Example 4, except that Trichoderma longifolia is replaced with Trichoderma harzianum.

[0108] Comparative Example 4

[0109] The cultivation substrate was prepared using the method in step (1) of Example 4.

[0110] Example 1 of effect verification

[0111] Healthy tea tree cuttings (15-20cm tall) were selected and randomly divided into 6 groups. As shown in Table 2, different cultivation substrates were used for each group. Except for the blank control group, all other treatment groups were inoculated with *Agrobacterium tumefaciens*. Specifically, two days after transplanting and allowing the seedlings to recover, a 10% concentration of *Agrobacterium tumefaciens* was used for inoculation. 5 The roots of each tea seedling were inoculated with a CFU / mL suspension of Agrobacterium tumefaciens. 10 mL of the suspension was applied to the roots.

[0112] Table 2 Experimental Grouping and Treatment Methods

[0113]

[0114] Thirty days after cultivation, the disease incidence in each treatment group was observed. The disease index, survival rate, and root vigor of the tea seedlings in each group were recorded, and the control efficacy was calculated using the following formula:

[0115] Prevention efficacy (%) = (Disease index of negative control group - Disease index of treatment group) / Disease index of negative control group × 100%.

[0116] The grade of root cancer in tea seedlings was assessed according to the grading criteria shown in Table 3, and the disease index was calculated:

[0117] Table 3 Grading Standards for Root Cancer in Tea Seedlings

[0118]

[0119] Root activity was determined using the TTC method and expressed as the number of milligrams of TTC reduced per gram of fresh root per hour.

[0120] The statistical results of each treatment group are shown in Table 4. The results show that the combined use of *Bacillus cereus* and *Trichoderma longicornis* can not only effectively control crown gall disease in tea trees and improve the survival rate of tea seedlings, but also effectively enhance root vitality. The 4:6 ratio mixed bacterial suspension treatment group showed the best control efficacy, reaching 82.36%, and the survival rate of tea seedlings in this treatment group increased to 98.5%, with root vitality enhanced by 42.3% compared to the negative control group. The 3:7 ratio mixed bacterial suspension treatment group had a survival rate of 98.2% and root vitality enhanced by 35.6%. In contrast, the survival rates of treatment groups using *Bacillus cereus*, *Trichoderma longicornis* alone, and the mixed treatment groups using *Bacillus cereus* and *Trichoderma harzianum* were all below 85%, and the improvement in root vitality was relatively weak. This result further indicates that the synergistic effect of *Bacillus cereus* and *Trichoderma longicornis* can not only improve the control effect of crown gall disease, but also significantly improve the survival rate and root vitality of tea trees, enhancing their growth vigor.

[0121] Table 4 Statistical results of the prevention and control effects of each treatment group

[0122]

[0123] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A microbial assemblages for preventing and controlling root cancer in tea trees, characterized in that, This includes Bacillus badius and Trichoderma longibrachiatum.

2. The microbial assemblages according to claim 1, characterized in that, The ratio of viable bacteria of the *Bacillus spp.* to that of the *Trichoderma longifolia* is (1-9):

9.

3. The microbial assemblages according to claim 2, characterized in that, The ratio of viable bacteria of *Bacillus spp.* to *Trichoderma longifolia* is 2:

3.

4. The use of a microbial combination as described in any one of claims 1-3 in the prevention and treatment of tea tree crown gall disease or in the preparation of a tea tree cultivation substrate for the prevention and treatment of tea tree crown gall disease.

5. A tea tree cultivation substrate, characterized in that, It includes a basic cultivation substrate and the microbial combination as described in any one of claims 1-3.

6. The tea tree cultivation substrate according to claim 5, characterized in that, By weight, the basic cultivation substrate comprises the following components: 30-40 parts leaf mold, 10-20 parts perlite, 20-30 parts peat moss, 10-20 parts well-rotted sheep manure, 5-10 parts vermiculite, and 1-3 parts rice husk ash.

7. The tea tree cultivation substrate according to claim 6, characterized in that, By weight, the basic cultivation substrate comprises the following components: 35 parts leaf mold, 15 parts perlite, 25 parts peat moss, 15 parts well-rotted sheep manure, 8 parts vermiculite, and 2 parts rice husk ash.

8. A method for preparing a tea tree cultivation substrate as described in any one of claims 5-7, characterized in that, Includes the following steps: The leaf mold, perlite, peat moss, decomposed sheep manure, vermiculite, and rice husk ash are mixed evenly according to the formula to obtain the basic cultivation substrate; The mixed bacterial suspension was added to the basic cultivation substrate and mixed evenly to obtain the tea tree cultivation substrate; The mixed bacterial suspension comprises the microbial combination as described in any one of claims 1-3.

9. The preparation method according to claim 8, characterized in that, The mixed bacterial suspension is added at 4-6% of the weight of the basic cultivation substrate.

10. The application of a tea tree cultivation substrate as described in any one of claims 5-7 in tea tree cultivation.

Images