A bacterial agent for efficiently preventing and treating rhizoctonia solani and a preparation method thereof

Abstract

This invention relates to the field of agricultural microbiology, and discloses a highly effective fungal agent for controlling ginger stem base rot and its preparation method. The agent includes *Trichoderma harzianum* (accession number CCTCC NO:M 20253070), which is first inoculated onto potato dextrose agar to obtain plates covered with fresh spores. Then, wheat bran and soybean meal are mixed to adjust the initial moisture content and initial pH to prepare a solid fermentation substrate. A diluted spore suspension is uniformly inoculated into the substrate for fermentation to obtain a fermentation product. The fermentation product is mixed with an extract and shaken, then filtered through sterile gauze to obtain a crude spore solution, which is finally diluted to obtain the final product. This invention achieves control by using *Trichoderma harzianum* to antagonize the pathogen and disrupt its cell structure. The use of wheat bran and soybean meal to construct a microenvironment with a high carbon-to-nitrogen ratio yields a fermentation product with high spore production. The combination of extract elution and gauze filtration to intercept residues and prepare a crude spore solution improves the purity of the fungal agent.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, specifically to a highly effective fungal agent for controlling ginger stem base rot and its preparation method. Background Technology

[0002] Ginger is a perennial herbaceous plant belonging to the genus *Zingiber* of the family Zingiberaceae. Its rhizome is the main economic part, serving as both an important edible condiment and a widely used medicinal material in traditional Chinese medicine. Ginger stem base rot primarily affects the base of the ginger stem and underground tubers. In the early stages, yellowish-brown to dark brown lesions appear at the stem base, gradually expanding and encircling the stem base, leading to yellowing and wilting of the above-ground leaves and even the death of the entire plant, while the underground tubers turn brown and rot. Soil diversity analysis and isolation verification have identified *Fusarium solani* as the main pathogenic fungus causing ginger stem base rot.

[0003] Currently, the main methods for controlling ginger stem base rot are divided into chemical control and biological control. Chemical control is difficult to reach and eradicate deep-seated pathogens after soil-borne diseases occur, and long-term use can easily lead to soil pollution and pesticide residues. In contrast, biological control uses microbial agents derived from nature, which are easily degraded in the environment and do not accumulate residues. Beneficial microorganisms fight pathogens by competing for nutrients and space, secreting antibacterial substances, directly infecting pathogens, and stimulating the ginger plant's own immune defense, making it difficult for pathogens to quickly develop resistance. At the same time, biocontrol bacteria can colonize and reproduce in the soil, forming a beneficial microbial community and improving the micro-ecological environment unfavorable to the survival of pathogens.

[0004] Existing biocontrol technologies for ginger stem base rot still have shortcomings in practical application and production. Conventional biocontrol agents have limited antagonistic effects against the pathogen causing ginger stem base rot, and improper application concentrations can easily lead to excessive nutrient consumption among the same fungal species in the early stages of colonization. In industrial production, existing solid fermentation substrates are unable to provide suitable oxygen exchange channels and carbon-nitrogen ratio microenvironments for Trichoderma growth, resulting in low sporulation yield of the fermentation product. Furthermore, existing extraction processes are unable to reduce the surface tension of Trichoderma hydrophobic spores, making it difficult to effectively elute spores from the mycelial network and solid residues, and it is also difficult to achieve residue interception and dialysis separation of monodisperse spores, resulting in low purity of the final extracted agent. Therefore, developing a high-purity, high-spore-yielding, and highly efficient agent for controlling ginger stem base rot and its preparation method are problems that need to be solved in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a highly efficient microbial agent for controlling ginger stem base rot and its preparation method. This invention solves the problems of limited antagonistic efficacy of existing conventional biocontrol agents against the pathogen causing ginger stem base rot, excessive nutrient consumption among the same microbial cells during colonization and application, and low spore production during fermentation and low purity during extraction and elution during the preparation process.

[0006] To address the above problems, the present invention provides the following technical solution:

[0007] This invention provides a highly effective fungicide for controlling stem base rot in ginger, employing the following technical solution:

[0008] A highly effective fungal agent for controlling stem base rot in ginger, comprising *Trichodermuafroharzianum*; the *Trichodermuafroharzianum* species is deposited at the China Center for Type Culture Collection (CCTCC) on December 31, 2025, with accession number CCTCC NO: M 20253070.

[0009] By employing the above technical solution, and using *Trichoderma harzianum* (CCTCC NO: M 20253070) as the core antibacterial component, this strain exhibits specific targeted antagonistic activity against the pathogen causing ginger stem base rot. The mechanism is as follows: after entering the soil and rhizosphere environment, the strain demonstrates an advantage in nutrient and spatial competition, seizing rhizosphere niches; simultaneously, it can hyperparasitize the mycelium of the pathogen, dissolving and destroying the pathogen's cell structure by secreting cell wall degrading enzymes such as chitinase and glucanase; furthermore, the strain's secondary metabolites can induce systemic resistance in ginger plants. These multiple mechanisms work synergistically, thus achieving a significant and long-lasting effect in controlling ginger stem base rot.

[0010] Preferably, the concentration of the bacterial agent is 1×10⁻⁶. 6 cfu / mL to 1×10 7 cfu / mL.

[0011] By adopting the above technical solution, the concentration of the bacterial agent is defined as 1×10⁻⁶. 6 cfu / mL to 1×10 7 Within the cfu / mL range, the number of viable bacteria at the specified concentration is sufficient to break the homeostasis of the original soil microbial population in the early stages of application, ensuring that the Trichoderma genus Trichoderma harzianum establishes a population advantage in the field; at the same time, it avoids the internal consumption of nutrients and waste of resources due to excessive concentration, thus ensuring a balance between efficacy and application economy.

[0012] This invention provides a method for preparing a highly effective fungal agent for controlling ginger stem base rot, using the following technical solution:

[0013] A method for preparing a highly effective fungal agent for controlling stem base rot in ginger includes the following steps:

[0014] Trichoderma harzianum with accession number CCTCC NO: M 20253070 was inoculated onto potato dextrose agar medium and cultured until the potato dextrose agar medium was covered with fresh spores, thus obtaining a plate covered with fresh spores.

[0015] Wheat bran and soybean meal powder are provided. The wheat bran and soybean meal powder are mixed to obtain a mixture. The initial moisture content and initial pH of the mixture are adjusted. The mixture is then sterilized at 121°C under high pressure and cooled to obtain a solid fermentation substrate.

[0016] In a sterile environment, sterile water is injected into the plate covered with fresh spores to prepare a spore suspension. The spore suspension is then diluted to obtain a diluted spore suspension. The diluted spore suspension is then uniformly inoculated into the solid fermentation substrate and placed in a constant temperature incubator for fermentation to obtain the fermentation product.

[0017] The fermentation product is mixed with the extract, shaken on a shaker, and filtered with sterile gauze to remove mycelia and residues to obtain a filtrate. The filtrate is then adjusted with sterile water to obtain a crude spore solution.

[0018] The crude spore liquid is diluted with water or a solution containing a protectant to prepare the highly effective fungal agent for controlling ginger stem rot.

[0019] By adopting the above technical solution, a two-stage solid-state fermentation system is used. First, initial activation and propagation are performed using the potato dextrose agar medium to obtain fresh spores as the primary seed source. Then, the diluted spore suspension is inoculated into a solid-state fermentation substrate composed of wheat bran and soybean meal powder for solid-state fermentation. The porous physical structure of the wheat bran provides oxygen exchange channels and attachment surface area for aerobic Trichoderma fungi, while the added soybean meal powder supplements the organic nitrogen source, creating a carbon-nitrogen ratio microenvironment conducive to the transition of fungal cells from vegetative growth to reproductive growth during sporulation. After fermentation, a shaking elution and physical filtration process is used to separate the mycelium, solid substrate, and mature spores. Therefore, a fungal agent with high sporulation, high purity, and good batch-to-batch stability is obtained, meeting the needs of industrial production.

[0020] Preferably, when inoculating the potato dextrose agar medium for culture, the solid culture conditions are 28°C and the culture time is 3 to 5 days.

[0021] By adopting the above technical solution, 28℃ is the suitable physiological temperature for the activity of the Trichoderma genus Trichoderma harzianum enzyme system. The culture cycle of 3 to 5 days can ensure that the colonies on the plate complete the sporulation cycle and have not entered the decay period. The harvested spores have a high germination rate and vitality, providing an inoculum source for subsequent fermentation culture.

[0022] Preferably, the method for preparing the potato dextrose agar medium is as follows: weigh 39g of potato agar powder, dissolve the potato agar powder in 1000mL of distilled water, then sterilize at 115℃ for 30min, and obtain the potato dextrose agar medium after cooling.

[0023] By adopting the above technical solution and using the autoclaving parameter of 115℃ for 30 minutes, while killing bacteria and spores, the caramelization destruction or degradation of reducing sugars, heat-sensitive vitamins and growth factors in potato extract is avoided, thus ensuring the nutritional integrity of the potato glucose agar substrate.

[0024] Preferably, in the mixture, the mass of the wheat bran is 15g, the mass of the soybean meal powder is 1% of the mass of the wheat bran; the initial moisture content is 60%, the initial pH is 7; and the solid fermentation substrate is prepared in a 250mL volumetric flask.

[0025] By adopting the above technical solution, the addition of 1% soybean meal powder regulates the carbon-nitrogen ratio of the system, avoiding excessive growth of mycelium without spore production caused by excessive nitrogen source; the initial moisture content of 60%, combined with the space left by the 15g bottling volume in the 250mL volumetric flask, creates a suitable ratio of gas and liquid phases within the substrate, which satisfies the free water required for spore germination and ensures the dissolved oxygen supply required by the deep mycelium; the initial pH of 7 is conducive to the initial metabolic initiation of the strain, and the spontaneous acid production of the mycelium then adjusts the environment to a slightly acidic state conducive to spore production.

[0026] Preferably, the autoclaving time is 1 to 2 hours.

[0027] By adopting the above technical solution, since the solid fermentation substrate has pores and its heat conduction efficiency is lower than that of pure liquid, high-pressure sterilization for 1 to 2 hours can ensure that high-pressure steam penetrates the core of the solid material, inactivates the heat-resistant bacterial spores inside, and provides a sterile environmental barrier for pure solid fermentation.

[0028] Preferably, the concentration of the diluted spore suspension is 1×10⁻⁶. 7 The concentration of cfu / mL is specified. The diluted spore suspension is obtained by diluting the spore suspension under a microscope using a hemocytometer. The inoculum amount of the diluted spore suspension in the solid fermentation substrate is 15%. The temperature of the constant temperature incubator is 28°C, and the fermentation time is 7 days.

[0029] By adopting the above technical solution, the concentration is 1×10⁻⁶. 7The combined inoculation strategy of diluting the spore suspension to cfu / mL and using an inoculum of 15% allows Trichoderma spores to establish a significant numerical advantage in the early stages of fermentation, encapsulating and degrading the material and shortening the lag phase. The 7-day fermentation cycle matches the growth and metabolic curve of the strain on the substrate. During this period, the carbon and nitrogen sources in the substrate are consumed, and the stress environment promotes the large-scale conversion of hyphae into chlamydospores or conidia, thereby increasing the yield.

[0030] Preferably, the extract is sterile water or contains a surfactant with a mass fraction of 0.05% Tween 80; the mixing ratio of the fermentation product to the extract is 1:10.

[0031] By adopting the above technical solution, a surfactant containing 0.05% Tween 80 by mass is selected as the extraction liquid. Its properties can significantly reduce the surface tension of the hydrophobic spores of Trichoderma, break the spore aggregation effect, and make them better eluted from the hyphal network and the surface of solid particles. The 1:10 mixing ratio forms a suitable osmotic pressure and hydrodynamic environment, which ensures elution efficiency while avoiding excessive viscosity of the filtrate.

[0032] Preferably, the oscillation time on the shaker is 15 to 30 minutes, and the sterile gauze consists of 4 to 8 layers of sterile gauze; the spore content in the crude spore solution is not less than 8 × 10⁻⁶. 9 cfu / mL.

[0033] By employing the above technical solution, 15 to 30 minutes of oscillation dissociates the attached spores without damaging the cell walls; 4 to 8 layers of sterile gauze construct a three-dimensional physical interception network, blocking the wheat bran residue and interwoven mycelia while allowing the monodisperse spores to pass through. Through this series of extraction and separation methods, a spore content of not less than 8 × 10⁻⁶ spores is prepared. 9 The crude spore solution of cfu / mL enhances the biopotency of the product.

[0034] This invention provides a highly effective fungal agent for controlling ginger stem base rot and its preparation method. It has the following beneficial effects:

[0035] 1. This invention utilizes *Trichoderma harzianum* (accession number CCTCC NO: M 20253070) to prepare a highly effective fungal agent for controlling ginger stem base rot. This agent antagonizes the pathogen causing ginger stem base rot and disrupts its cellular structure, thereby achieving the desired control. Simultaneously, the highly effective fungal agent is diluted and adjusted to a suitable application concentration, allowing *Trichoderma harzianum* to break through soil homeostasis limitations and establish population dominance in the initial application stage, thus avoiding excessive nutrient consumption caused by excessively high concentrations.

[0036] 2. This invention obtains a solid fermentation substrate by mixing wheat bran and soybean meal powder, and appropriately adjusting the initial moisture content and initial pH of the mixture. Wheat bran provides the necessary oxygen exchange channels and attachment surface area for Trichoderma harzianum, and soybean meal powder supplements the organic nitrogen source to construct a carbon-nitrogen ratio microenvironment conducive to the sporulation process. After the diluted spore suspension is uniformly inoculated into the solid fermentation substrate and fermented in a constant temperature incubator, a fermentation product with high sporulation yield can be obtained.

[0037] 3. This invention reduces the surface tension of the hydrophobic spores of Trichoderma by mixing the fermentation product with a surfactant-containing solution as the extract and continuously oscillating it on a shaker. This allows the spores to be fully washed away from the mycelial network and the surface of the residue. Subsequently, the mycelium and residue are removed by filtration with sterile gauze to obtain the filtrate. The filtrate intercepts the wheat bran residue and the intertwined mycelium while allowing the monodisperse spores to pass through, thus preparing a crude spore liquid with a high spore content. This improves the extraction purity of the fungal agent for the efficient control of ginger stem base rot. Attached Figure Description

[0038] Figure 1 This is a colony morphology diagram of the strain *Trichoderma harzianum* from one embodiment of the present invention, after being cultured on PDA solid medium at 28°C for 5 days.

[0039] Figure 2 This is a phylogenetic tree diagram constructed based on the ITS rRNA gene sequence of the strain Trichoderma harzianum according to an embodiment of the present invention;

[0040] Figure 3 A comparative diagram of the salt tolerance of two antagonistic Trichoderma strains according to an embodiment of the present invention;

[0041] Figure 4 This is a comparative graph showing the resistance of two antagonistic Trichoderma strains to low and high temperature stress according to an embodiment of the present invention.

[0042] Figure 5 This is a comparison chart of the antibacterial rates of metabolites from two antagonistic Trichoderma strains according to an embodiment of the present invention.

[0043] Figure 6 This is a disease index graph of ginger under various treatments according to an embodiment of the present invention;

[0044] Figure 7 This is a diagram showing the growth of ginger under various treatments after 40 days, according to one embodiment of the present invention. Detailed Implementation

[0045] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] Preparation Examples 1-6:

[0047] Preparation Example 1:

[0048] This preparation example provides a potato dextrose agar (PDA) medium, including the following steps:

[0049] Accurately weigh 39g of potato agar powder (BD Difco, USA) and dissolve it completely in 1000mL of distilled water. Then, sterilize the solution at 115℃ for 30min, and after cooling, potato dextrose agar (PDA) medium is obtained.

[0050] Preparation Example 2:

[0051] This preparation example provides a potato glucose medium (PDB), which includes the following steps:

[0052] Accurately weigh 24g of potato glucose powder (BD Difco, USA) and dissolve it completely in 1000mL of distilled water. Then, sterilize the solution at 115℃ for 30min, and after cooling, potato glucose medium (PDB) is obtained.

[0053] Preparation Example 3:

[0054] This preparation example provides a NA solid culture medium, including the following steps:

[0055] Weigh out 10g of glucose, 5g of peptone, 3g of beef extract, 0.5g of yeast powder, and 2g of agar powder. Mix the above components and add deionized water to a final volume of 1000mL. Then, sterilize at 105℃ for 30min and cool to obtain NA solid culture medium.

[0056] Preparation Example 4:

[0057] This preparation example provides a NA solid culture medium, including the following steps:

[0058] Weigh out 10g of glucose, 5g of peptone, 3g of beef extract, 0.5g of yeast powder, and 2.5g of agar powder. Mix the above components and add deionized water to a final volume of 1000mL. Then, sterilize at 105℃ for 30min and cool to obtain NA solid culture medium.

[0059] Preparation Example 5:

[0060] This preparation example provides a NA solid culture medium, including the following steps:

[0061] Weigh out 10g of glucose, 5g of peptone, 3g of beef extract, 0.5g of yeast powder, and 3g of agar powder. Mix the above components and add deionized water to a final volume of 1000mL. Then, sterilize at 105℃ for 30min and cool to obtain NA solid culture medium.

[0062] Preparation Example 6:

[0063] This preparation example provides a NA liquid culture medium, including the following steps:

[0064] Weigh out 10g of glucose, 5g of peptone, 3g of beef extract, and 0.5g of yeast powder. Mix the above components and add deionized water to a final volume of 1000mL. Then, sterilize at 105℃ for 30min and cool to obtain NA liquid culture medium.

[0065] Examples 1-3:

[0066] Example 1:

[0067] This embodiment provides a highly effective fungicide for controlling stem base rot in ginger, comprising the following steps:

[0068] S1. Trichoderma afroharzianum with accession number CCTCC NO: M 20253070 was inoculated into the PDA solid medium prepared in Preparation Example 1 and cultured at 28°C for 3 days until fresh spores were grown on the medium.

[0069] S2. Using a 250ml volumetric flask as the standard, add 15g of wheat bran and an additional 1% soybean meal powder to adjust the initial moisture content to 60% and the initial pH to 7. Sterilize at 121℃ for 1 hour to kill miscellaneous bacteria. After cooling, use it as a solid fermentation substrate for later use.

[0070] S3. Under aseptic conditions, inject an appropriate amount of sterile water into the plate contaminated with fresh spores from step S1 to prepare a spore suspension, and dilute the concentration to 1×10⁻⁶ using a hemocytometer under a microscope. 7The inoculum was 15% cfu / ml and uniformly inoculated into the solid fermentation substrate prepared in step S2. The substrate was then placed in a constant temperature incubator at 28℃ and cultured for 7 days for fermentation. Fermentation was carried out when the substrate was covered with dense green spores inside and out.

[0071] S4. After fermentation, mix the fermentation product with sterile water at a ratio of 1:10, shake thoroughly on a shaker for 15 minutes, filter through 4 layers of sterile gauze to remove mycelia and residue, and the resulting filtrate is the crude spore solution. The spore content in the bacterial suspension obtained by adjusting the crude spore solution with sterile water should be ≥8×10⁻⁶. 9 cfu / ml;

[0072] S5. Dilute the above crude spore solution with clean water or a solution containing a protectant to a field application concentration of 1×10⁻⁶. 6 The bacterial agent is obtained by measuring cfu / ml.

[0073] Example 2:

[0074] This embodiment provides a highly effective fungicide for controlling stem base rot in ginger, comprising the following steps:

[0075] S1. Trichoderma afroharzianum with accession number CCTCC NO: M 20253070 was inoculated into the PDA solid medium prepared in Preparation Example 1 and cultured at 28°C for 4 days until fresh spores were grown on the medium.

[0076] S2. Using a 250ml volumetric flask as the standard, add 15g of wheat bran and an additional 1% soybean meal powder to adjust the initial moisture content to 60% and the initial pH to 7. Autoclave at 121℃ for 1.5 hours to kill miscellaneous bacteria. After cooling, use it as a solid fermentation substrate for later use.

[0077] S3. Under aseptic conditions, inject an appropriate amount of sterile water into the plate contaminated with fresh spores from step S1 to prepare a spore suspension, and dilute the concentration to 1×10⁻⁶ using a hemocytometer under a microscope. 7 The inoculum was 15% cfu / ml and uniformly inoculated into the solid fermentation substrate prepared in step S2. The substrate was then placed in a constant temperature incubator at 28℃ and cultured for 7 days for fermentation. Fermentation was carried out when the substrate was covered with dense green spores inside and out.

[0078] S4. After fermentation, the fermentation product is mixed with a surfactant containing 0.05% Tween 80 at a ratio of 1:10. The mixture is shaken thoroughly for 22.5 minutes on a shaker. The mixture is then filtered through six layers of sterile gauze to remove mycelia and residue. The resulting filtrate is the crude spore solution. The spore content in the bacterial suspension obtained by adjusting the crude spore solution with sterile water should be ≥8×10⁻⁶. 9 cfu / ml;

[0079] S5. Dilute the above crude spore solution with clean water or a solution containing a protectant to a field application concentration of 5.5 × 10⁻⁶. 6 The bacterial agent is obtained by measuring cfu / ml.

[0080] Example 3:

[0081] This embodiment provides a highly effective fungicide for controlling stem base rot in ginger, comprising the following steps:

[0082] S1. Trichoderma afroharzianum with accession number CCTCC NO: M 20253070 was inoculated into the PDA solid medium prepared in Preparation Example 1 and cultured at 28°C for 5 days until the medium was covered with fresh spores.

[0083] S2. Using a 250ml volumetric flask as the standard, add 15g of wheat bran and an additional 1% soybean meal powder to adjust the initial moisture content to 60% and the initial pH to 7. Sterilize at 121℃ for 2 hours to kill miscellaneous bacteria. After cooling, use it as a solid fermentation substrate for later use.

[0084] S3. Under aseptic conditions, inject an appropriate amount of sterile water into the plate contaminated with fresh spores from step S1 to prepare a spore suspension, and dilute the concentration to 1×10⁻⁶ using a hemocytometer under a microscope. 7 The inoculum was 15% cfu / ml and uniformly inoculated into the solid fermentation substrate prepared in step S2. The substrate was then placed in a constant temperature incubator at 28℃ and cultured for 7 days for fermentation. Fermentation was carried out when the substrate was covered with dense green spores inside and out.

[0085] S4. After fermentation, mix the fermentation product with sterile water at a ratio of 1:10, shake thoroughly on a shaker for 30 minutes, filter through 8 layers of sterile gauze to remove mycelia and residue, and the resulting filtrate is the crude spore solution. The spore content in the bacterial suspension obtained by adjusting the crude spore solution with sterile water should be ≥8×10⁻⁶. 9 cfu / ml;

[0086] S5. Dilute the above crude spore solution with clean water or a solution containing a protectant to a field application concentration of 1×10⁻⁶. 7 The bacterial agent is obtained by measuring cfu / ml.

[0087] Comparative Examples 1-3:

[0088] Comparative Example 1:

[0089] Compared with Example 2, the difference is that the strain inoculated in step S1 is another common Trichoderma strain with a certain antagonistic effect, instead of Trichoderma harzianum of the Trichoderma genus with preservation number CCTCC NO: M 20253070. All other parameters and steps are the same.

[0090] Comparative Example 2:

[0091] Compared with Example 2, the difference is that the preparation of Trichoderma agent was not carried out in steps S1 to S5. Instead, the traditional antidote commonly used for ginger stem base rot was used directly as the control agent. The other application parameters and steps in the pot control experiment were the same.

[0092] Comparative Example 3:

[0093] Compared with Example 2, the difference is that no bacterial agent or disease prevention agent was added. It was used as a blank treatment containing diseased soil and organic fertilizer. The other culture parameters and steps in the pot control experiment were the same.

[0094] Test Example 1-3:

[0095] Test Example 1:

[0096] Experimental description:

[0097] This experiment aimed to isolate and screen functional strains that antagonize Fusarium solani, the main pathogen of ginger stem rot, from soil samples in areas severely affected by ginger diseases, and to perform morphological and molecular biological identification on the selected functional strains.

[0098] Experimental steps:

[0099] The isolation and purification process involved collecting soil samples from a depth of 0 to 40 cm in May 2025 in an area severely affected by ginger disease in Weifang, Shandong Province. After removing surface stones and weeds, a five-point sampling method was used to collect mixed rhizosphere soil samples from both diseased and healthy ginger plants. 10 g of soil sample was accurately weighed and added to an Erlenmeyer flask containing 100 mL of sterile water. The flask was then incubated at 28°C with shaking at 170 rpm for 2 hours. After standing for 10 minutes, 2 mL of the suspension was added to a selection medium and incubated at 28°C with shaking for 2 days. Subsequently, the enriched culture was extracted and diluted with sterile water to a concentration of 10 g / mL. -2 10 -3 and 10 -4 Three gradients were used. 100 μL of each dilution was spread onto plates, with three replicates for each gradient. The plates were incubated upside down at 28°C for 3 to 4 days. Well-grown colonies were repeatedly transferred or washed to remove spores before spreading, until a single strain was obtained through purification.

[0100] The antagonistic ability test was conducted using the plate confrontation method. The antagonistic fungal discs to be tested were symmetrically inoculated on both sides of the PDA plate at a distance of 2.5 cm from the center, with the pathogenic fungus *Fusarium solani*, the causative agent of ginger stem rot, inoculated at the center. Plates inoculated solely with the pathogenic fungus served as controls, with each treatment repeated three times. The plates were incubated in the dark at 28°C. When the pathogenic fungal colonies in the control group grew to near the edge of the plate, the diameter of the pathogenic fungal colonies was measured using the cross-sectional method, and the inhibition rate was calculated using the formula: Inhibition rate (%) = (Difference between control colony diameter and treated colony diameter) / Control colony diameter × 100%. The strain exhibiting significant antagonistic effect was selected and named WF2.

[0101] The identification and preservation process involved picking purified WF2 colonies to observe their color and texture characteristics, and extracting genomic DNA for PCR amplification. After passing 1% agarose gel electrophoresis, the amplified products were sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were then submitted to the GenBank database of the National Center for Biotechnology Information (NCBI) for comparison and confirmation. After identification, 1 mL of the bacterial culture was added to an equal volume of 50% glycerol, mixed thoroughly, and stored at -80°C for later use.

[0102] Experimental conclusion:

[0103] Through the above isolation, screening, and identification steps, the biocontrol strain WF2, successfully isolated and screened from the rhizosphere soil of ginger disease-causing areas in Weifang, Shandong, exhibits significant antagonistic activity against the fungus *Fusarium solani*, the pathogen causing ginger stem rot. Figure 1 As shown, the obtained strain, after being inoculated onto PDA solid medium and cultured at 28°C for 5 days, produced dense green spores exhibiting typical morphological characteristics of Trichoderma fungi. Figure 2 As shown, a phylogenetic tree was constructed based on the ITS rRNA gene sequence of strain WF2. Sequencing alignment results confirmed that this strain belongs to the genus *Trichoderma*, specifically *Trichoderma harzianum*. Therefore, this strain was identified as *Trichoderma harzianum*, and it was deposited at the China Center for Type Culture Collection (CCTCC) on December 31, 2025, with accession number CCTCC NO: M20253070. It is a suitable strain for preparing biological agents to control ginger stem rot.

[0104] Test Example 2:

[0105] Experimental description:

[0106] This experiment aims to compare and evaluate the performance of the strain prepared in Example 2 of this invention with that of Trichoderma in Comparative Example 1 by testing salt tolerance, resistance to temperature stress, and the antibacterial rate of metabolites, thereby demonstrating that the selected Trichoderma harzianum WF2 has significant advantages.

[0107] Experimental steps:

[0108] The salt tolerance comparison process involved using potato dextrose agar as the basal medium. After sterilization and cooling to approximately 50°C, sterile sodium chloride stock solution was added to prepare salt-containing plates with final sodium chloride concentrations of 1%, 3%, and 5%. Plates with an equal volume of sterile water were used as controls, and each treatment was replicated in triplicate. Using a sterile punch with a diameter of 5 mm, mycelial blocks from the edge of *Trichoderma* colonies (comparative example 1) and activated cultured strains from Example 2 were inoculated into the center of the salt-containing and control plates, respectively. The inoculated plates were then inverted and incubated in the dark at 28°C. Mycelial growth was measured every 12 hours after inoculation, and growth curves were plotted.

[0109] The comparison of resistance to temperature stress was conducted using potato dextrose agar as the basal medium. A 5mm diameter sterile punch was used to inoculate the activated mycelial culture from Example 2 and the edge hyphae of *Trichoderma* colonies from Comparative Example 1 into the center of a plate. The plates were then incubated upside down in incubators at 15℃, 25℃, 28℃, 32℃, and 37℃ in the dark. Mycelial growth was measured every 12 hours after inoculation, and growth curves were plotted.

[0110] The process for comparing the antibacterial rates of the metabolites was as follows: The two *Trichoderma* strains were inoculated into PDB liquid medium and cultured with shaking at 28°C and 120 rpm for 5 to 7 days. The culture was filtered through four layers of sterile gauze to remove mycelia. The filtrate was centrifuged at 8000 rpm for 15 min, and the supernatant was collected. The supernatant was concentrated to one-tenth of its original volume using a rotary evaporator at a temperature not exceeding 45°C, or freeze-dried to obtain a crude extract. Before use, the crude extract was redissolved in an appropriate amount of sterile distilled water or a solvent for dissolving the medium, such as 1% dimethyl sulfoxide, to prepare a stock solution of the desired concentration. This stock solution was then filtered through a 0.22 μm microporous membrane for sterilization. Sterilized PDA medium cooled to approximately 50°C was mixed with the sterile crude extract solution at a volume ratio of 9:1, resulting in final concentrations of 1 ppm, 10 ppm, and 100 ppm of the crude extract in the plates. The mixture was quickly shaken and poured into sterile petri dishes, approximately 10 mL per dish. A plate containing only an equal volume of solvent was set up as a negative control. Using a sterile punch with a diameter of 5 mm, collect the edge mycelial discs of pathogenic bacteria activated and cultured on PDA plates and inoculate them into the center of both drug-containing and control plates. Invert the inoculated plates and incubate at the optimal temperature of 25-28℃. When the control group colonies have grown to near the edge of the plate, measure the colony diameter of the pathogens in each treatment group using the cross-sectional method. Each treatment has three replicates. Calculate the inhibition rate based on the measurement data using the formula: Inhibition rate (%) = (Control colony diameter - Treatment colony diameter) / Control colony diameter × 100%.

[0111] Experimental conclusion:

[0112] The comparison of salt tolerance showed that the strain prepared in Example 2 of this invention had a significantly better salt tolerance than Trichoderma in Comparative Example 1. Figure 3 As shown in the figure, the horizontal axis Time (h) represents the culture time, and the vertical axis represents the colony growth radius. After being inoculated on salt plates of different concentrations and cultured for 72 hours, the strains prepared in Example 2 showed obvious salt tolerance growth ability.

[0113] The comparison of resistance to temperature stress showed that the strain prepared in Example 2 of this invention exhibited significantly better resistance to both low and high temperatures than Trichoderma in Comparative Example 1. Figure 4 As shown, the horizontal and vertical axes have the same meaning. Figure 3 After being cultured for 72 hours at different temperature gradients from 15°C to 37°C, the strains prepared in Example 2 showed significant temperature adaptability and growth viability.

[0114] The comparison of the antibacterial rates of the metabolites showed that the antibacterial effect of the bacterial metabolites prepared in Example 2 of this invention against the pathogen of ginger stem rot was significantly better than that of Trichoderma in Comparative Example 1. Figure 5 As shown in the figure, CK is the control group in the above steps, and the vertical axis is the colony radius of the pathogen. Under different concentrations of crude extract treatment, the colony radius of the pathogen in the bacterial metabolite treatment group prepared in Example 2 is significantly smaller than that of CK and Comparative Example 1, and all show obvious antibacterial rate.

[0115] In summary, the African Trichoderma harzianum WF2 strain screened by this invention has significant advantages in salt tolerance, temperature stress resistance, and antibacterial activity of its metabolites, demonstrating that this preferred strain has obvious superiority in stress resistance and antibacterial and disease prevention functions.

[0116] Test Example 3:

[0117] Experimental description:

[0118] This experiment aims to study the actual control effect of the fungal agent prepared in Example 2 of this invention on ginger stem base rot using a pot experiment.

[0119] Experimental steps:

[0120] Soil samples for the pot experiment were collected from a severely diseased ginger area in Weifang, Shandong Province, to simulate the local disease-causing environment. The experiment was conducted in a greenhouse incubator at the Ark Laboratory of the Nanjing National Agricultural Innovation Center. Each treatment group contained 6 ginger seedlings, for a total of 4 treatment groups. On day 40 after transplanting, the disease incidence of the ginger seedlings in each treatment was recorded, and disease index and other indicators were calculated. The specific settings for each treatment group are shown in Table 1.

[0121] Experimental data:

[0122] Table 1. Experimental setup of ginger pot plants for each treatment

[0123] Example 2: Microbial Agent Treatment Group (T2) Diseased soil plus organic fertilizer plus Trichoderma harzianum agent Joint processing group (T3) Diseased soil plus organic fertilizer plus traditional detoxifier plus Trichoderma harzianum Comparative Example 2 (T1) Diseased soil plus organic fertilizer plus traditional detoxifying agent Comparative Example 3 (CK) Add organic fertilizer to diseased soil

[0124] Experimental conclusion:

[0125] Combination Figure 6 The disease index of ginger under each treatment was obtained. In the figure, CK (Comparative Example 3) showed the most severe disease, with ginger plants dying and a disease index reaching 100%. T1 (Comparative Example 2) had a disease index lower than CK after the addition of a traditional antidote, but it was still significantly higher than the treatment group treated with the Trichoderma fungicide. T2 (Example 2) and T3 (Combined Treatment Group) were both treated with the Trichoderma fungicide of this invention, and their disease indices decreased significantly, to 40.39% and 40.88%, respectively. Figure 7 As shown, observations of ginger growth after 40 days revealed that the CK group plants died and withered, the T1 group showed average growth, while the T2 and T3 groups, which received the Trichoderma harzianum fungicide, exhibited better growth. In conclusion, the fungal agent prepared using Trichoderma harzianum WF2 of this invention has a significant inhibitory effect on ginger stem rot and can significantly promote ginger plant growth, demonstrating significant application value in the prevention and control of soil-borne diseases.

Claims

1. A highly effective fungicide for controlling stem base rot in ginger, characterized in that, Including the genus *Trichoderma* afroharzianum; The aforementioned *Trichoderma afroharzianum* is deposited at the China Center for Type Culture Collection (CCTCC) on December 31, 2025, with accession number CCTCC NO: M 20253070.

2. The fungal agent for the efficient control of ginger stem base rot according to claim 1, characterized in that, The concentration of the bacterial agent is 1×10⁻⁶. 6 cfu / mL to 1×10 7 cfu / mL.

3. A method for preparing a highly effective fungal agent for controlling ginger stem base rot, characterized in that, The preparation of a highly effective fungal agent for controlling ginger stem base rot as described in claim 1 or 2 includes the following steps: Trichoderma harzianum with accession number CCTCC NO: M 20253070 was inoculated onto potato dextrose agar medium and cultured until the potato dextrose agar medium was covered with fresh spores, thus obtaining a plate covered with fresh spores. Wheat bran and soybean meal powder are provided. The wheat bran and soybean meal powder are mixed to obtain a mixture. The initial moisture content and initial pH of the mixture are adjusted. The mixture is then sterilized at 121°C under high pressure and cooled to obtain a solid fermentation substrate. In a sterile environment, sterile water is injected into the plate covered with fresh spores to prepare a spore suspension. The spore suspension is then diluted to obtain a diluted spore suspension. The diluted spore suspension is then uniformly inoculated into the solid fermentation substrate and placed in a constant temperature incubator for fermentation to obtain the fermentation product. The fermentation product is mixed with the extract, shaken on a shaker, and filtered with sterile gauze to remove mycelia and residues to obtain a filtrate. The filtrate is then adjusted with sterile water to obtain a crude spore solution. The crude spore liquid is diluted with water or a solution containing a protectant to prepare the fungal agent.

4. The method for preparing a highly effective fungal agent for controlling ginger stem base rot according to claim 3, characterized in that, When inoculated into the potato dextrose agar medium for culture, the solid culture conditions are 28°C and the culture time is 3 to 5 days.

5. The method for preparing a highly effective fungal agent for controlling ginger stem base rot according to claim 3, characterized in that, The method for preparing the potato dextrose agar medium is as follows: Weigh 39g of potato agar powder, dissolve the potato agar powder in 1000mL of distilled water, then sterilize it at 115℃ for 30min, and obtain the potato dextrose agar medium after cooling.

6. The method for preparing a highly effective fungal agent for controlling ginger stem base rot according to claim 3, characterized in that, In the mixture, the mass of wheat bran is 15g, and the mass of soybean meal powder is 1% of the mass of wheat bran; the initial moisture content is 60%, and the initial pH is 7; the solid fermentation substrate is prepared in a 250mL volumetric flask.

7. The method for preparing a highly effective fungal agent for controlling ginger stem base rot according to claim 3, characterized in that, The autoclaving time is 1 to 2 hours.

8. The method for preparing a highly effective fungal agent for controlling ginger stem base rot according to claim 3, characterized in that, The concentration of the diluted spore suspension is 1×10⁻⁶. 7 cfu / mL, wherein the diluted spore suspension is obtained by diluting the spore suspension under a microscope using a hemocytometer; The inoculation amount of the diluted spore suspension into the solid fermentation substrate is 15%; the temperature of the constant temperature incubator is 28°C, and the fermentation time is 7 days.

9. The method for preparing a highly effective fungal agent for controlling ginger stem base rot according to claim 3, characterized in that, The extract is sterile water or contains a surfactant with a mass fraction of 0.05% Tween 80; The mixing ratio of the fermentation product to the extract is 1:

10.

10. The method for preparing a highly effective fungal agent for controlling ginger stem base rot according to claim 9, characterized in that, The oscillation time on the oscillator is 15 min to 30 min, and the sterile gauze is 4 to 8 layers of sterile gauze. The crude spore solution contains no less than 8 × 10⁻⁶ spores. 9 cfu / mL.

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