Application of low-temperature resistant strain YW-1 for preventing and treating rice seedling blight

Abstract

The application discloses a kind of low temperature resistant strain YW-1 for preventing and treating rice seedling wilt, and belongs to the field of plant protection.The low temperature resistant strain YW-1 is a pseudo-odor class fungus, and the application proves that the strain YW-1 can maintain normal growth and metabolism in a low temperature environment of 15 DEG C, and can effectively destroy the hyphal structure of the wilt pathogen by secreting cellulase and chitinase, thereby inhibiting the occurrence of the disease from the source.The strain YW-1 has no antagonism with the commonly used functional fungus Trichoderma in the seedling raising substrate, and the two can be compounded and coexist, thereby realizing the synergistic effect of functional microorganisms.The application overcomes the defects of the existing biocontrol strain, such as reduced activity in a low temperature environment and unstable prevention effect, and provides a stable and reliable biological control scheme for factory seedling raising in a low temperature season without increasing the amount of chemical pesticides, thereby significantly improving the quality of seedlings and the seedling raising success rate.

Description

Technical Field

[0001] This invention relates to the field of plant protection, and in particular to the application of a low-temperature resistant strain YW-1 for the prevention and control of rice seedling wilt disease. Background Technology

[0002] Factory-style rice seedling raising is an intensive agricultural technology that utilizes modern facilities and automated equipment to produce standardized, large-scale seedlings in a controlled environment. It is now widely used in my country's major rice-producing areas. This technology significantly improves seedling quality and production efficiency through mechanized sowing, constant-temperature germination, and precise water and fertilizer management, providing crucial support for the mechanized production of rice throughout its entire process. In the research and development of rice seedling raising substrates, functional substrates incorporating beneficial microorganisms have become an important direction in current green production technologies. By combining functional microorganisms such as Bacillus and Trichoderma with soilless substrates, the dual effects of substrate improvement and biological regulation can be achieved, demonstrating significant advantages in improving seedling quality and reducing the use of chemical pesticides.

[0003] However, existing live-bacterial rice seedling substrates still face key technical bottlenecks in practical applications. In the Jianghuai region, rice seedling raising is concentrated in May each year, a period characterized by unstable temperatures, large diurnal temperature variations, and average nighttime temperatures of only 10-15℃. This low-temperature environment severely restricts the activity of beneficial microorganisms added to the seedling substrate, leading to unstable biocontrol effects. A more prominent problem is that under low-temperature and high-humidity conditions, rice seedling wilt disease is particularly severe, often causing the entire seedling tray to die, seriously affecting the quality and efficiency of factory-style seedling raising. Currently, the control of rice seedling diseases mainly relies on chemical agents such as hymexazol and carbendazim, but chemical control carries risks of pesticide residues and resistance.

[0004] To address the aforementioned problems, screening biocontrol strains that can maintain high activity under low-temperature conditions, effectively control seedling wilt, and coexist synergistically with functional microorganisms (such as Trichoderma) in existing seedling substrates has become a pressing technical challenge in this field. The difficulties faced by researchers in the screening process lie in the fact that biocontrol strains not only need to possess strong antagonistic capabilities against pathogens, but also must tolerate the low-temperature stress during the seedling stage, while simultaneously not exhibiting antagonistic effects with beneficial microorganisms already added to the substrate. These multiple screening conditions significantly increase the difficulty of obtaining functional strains. Summary of the Invention

[0005] The purpose of this invention is to provide an application of the low-temperature resistant strain YW-1 for the prevention and control of rice seedling wilt disease, thereby addressing the problems existing in the prior art. This invention reveals for the first time a novel application of the odor-mimicking fungus YW-1 in controlling this disease at a low temperature of 15℃. This strain exhibits strong activity under low-temperature conditions, destroying the pathogenic mycelium through enzyme production, and is compatible with Trichoderma. Greenhouse experiments show that adding YW-1 during low-temperature seedling cultivation prevented wilt disease, resulting in robust seedlings, providing a stable biological control solution for factory-scale seedling cultivation.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a pseudo-odorant fungus ( Myroides odoratiminus The application of YW-1 in the prevention and control of rice seedling wilt disease, wherein the preservation number of the pseudo-odorant fungus YW-1 is CGMCC NO.20620.

[0007] Optionally, the odor-mimicking fungus YW-1 can control rice seedling wilt disease under low temperature conditions.

[0008] Optionally, the low temperature condition is 15℃-16℃.

[0009] This invention also provides a pseudo-odorant fungus ( Myroides odoratiminus The application of YW-1 in the preparation of biocontrol agents for use in combination with Trichoderma, wherein the preservation number of the pseudo-odor fungus YW-1 is CGMCC NO.20620.

[0010] This invention also provides a pseudo-odorant fungus ( Myroides odoratiminus The application of YW-1 or a biocontrol agent containing the pseudo-odorant fungus YW-1 in the preparation of rice seedling substrate, wherein the preservation number of the pseudo-odorant fungus YW-1 is CGMCC NO.20620, and the biocontrol agent contains Trichoderma.

[0011] This invention also provides a method for controlling rice seedling wilt under low-temperature conditions, comprising using fungi containing odor-mimicking fungi (… Myroides odoratiminus The step involves adding the biocontrol agent YW-1 to the rice seedling substrate for rice seedling cultivation; the preservation number of the pseudo-odorant fungus YW-1 is CGMCC NO.20620; the low temperature condition is 15℃-16℃.

[0012] Optionally, the biocontrol agent contains Trichoderma.

[0013] Optionally, the concentration of the odor-mimicking fungus YW-1 in the biocontrol agent is 10. 7 CFU / mL.

[0014] The present invention discloses the following technical effects: This invention addresses the technical challenge of high incidence of Fusarium wilt during rice seedling cultivation due to nighttime low temperatures (16℃ and below). It reveals for the first time a novel application of the preserved odor-mimicking fungus YW-1 (CGMCC NO.20620) in controlling Fusarium wilt in rice seedlings under low-temperature conditions. Experiments show that this strain can maintain normal growth and metabolism at 15℃ and can effectively disrupt the mycelial structure of the Fusarium wilt pathogen by secreting cellulase and chitinase, inhibiting disease occurrence at its source. This overcomes the shortcomings of existing biocontrol strains, such as reduced activity and unstable efficacy under low-temperature conditions.

[0015] Furthermore, this invention confirms that strain YW-1 has no antagonistic effect with Trichoderma, a commonly used functional microorganism in seedling substrates, and the two can coexist in combination, achieving synergistic effects of functional microorganisms. Greenhouse seedling raising experiments showed that after adding YW-1 biocontrol agent, under seedling raising conditions with an average nighttime temperature of 16℃, rice seedlings did not develop wilt disease and grew vigorously, while the control group showed typical seedling death symptoms. This invention provides a stable and reliable biological control solution for factory-scale seedling raising in low-temperature seasons without increasing the amount of chemical pesticides used, significantly improving seedling quality and seedling survival rate. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 These are morphological images of the pathogen causing rice seedling wilt; where A is a colony image; B is a microscopic image of a large conidium; and C is a microscopic image of a small conidium. Figure 2 This is a screening diagram of antagonistic strains of the pathogen causing Fusarium wilt; where A and B are the results of plate confrontation culture, A is the treatment group and B is the control group; C and D are the mycelial morphology of the pathogen, where C is the treatment group and D is the control group. Figure 3 The image shows the enzyme activity detection results of strain YW-1; where A is the chitin detection plate, strain number 20 is YW-1; B is the cellulose detection plate, the strain at the top is YW-1. Figure 4 The low-temperature growth performance of strain YW-1; Figure 5 The images show the compatibility test results between the tested strain and Trichoderma; where A is a plate confrontation culture image and B is a Trichoderma culture image. Figure 6The diagram shows a rice seedling raising experiment; where A is the treatment group with added biocontrol agent YW-1, and B is the control group without added biocontrol agent YW-1. Detailed Implementation

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] Example 1: Isolation and Identification of the Pathogen of Rice Seedling Wilt Rice seedlings exhibiting typical symptoms of Fusarium wilt were collected from the seedling nursery of Huai'an Chaimihe Agricultural Technology Co., Ltd. in Huai'an City, Jiangsu Province. Diseased tissue at the junction of diseased and healthy tissue at the stem base was selected. The surface was rinsed three times with sterile water, then immersed in 75% ethanol for 30 seconds in a clean bench for surface disinfection, followed by rinsing three times with sterile water. The surface moisture was then blotted dry with sterile filter paper. Small pieces of diseased tissue, approximately 5mm × 5mm in size, were cut from the stem base using a sterile scalpel and placed on PDA culture plates, with four pieces evenly distributed per plate. The plates were incubated at 25℃ for 2-7 days, with colony growth observed daily.

[0024] After typical fungal colonies have grown around the diseased tissue, mycelia at the colony edge are picked using an inoculation needle and transferred to a new PDA plate for purification culture. Purification is repeated 2-3 times until a single colony is obtained. The purified strain is inoculated onto PDA slant medium and incubated at 25°C for 5 days, then stored at 4°C for later use. The culture of the purified strain is used to pick surface spores, and the morphological characteristics of the conidia are observed and recorded under an optical microscope. Results are as follows: Figure 1 As shown. Based on morphological characteristics, the pathogen was preliminarily identified as Fusarium oxysporum.

[0025] Example 2: Screening of rice wilt antagonistic strains Using the rice wilt pathogen isolated in Example 1 as the target bacterium, antagonistic bacteria were screened from the strain library preserved in the Key Laboratory of Ecological Agriculture Biotechnology around Hongze Lake, Huaiyin Normal University, using the plate confrontation culture method. Activated pathogens were punched into bacterial cakes using a 0.5cm perforator and inoculated upside down in the center of PDA medium plates, and incubated at 28℃ for 48h. The test strains were streaked on LB medium for activation and incubated at 28℃ for 24h. Single colonies were picked up with sterile toothpicks and spotted 2cm from the edge of the pathogen colony. Four test strains were symmetrically spotted per plate, with three replicates for each strain. Uninoculated pathogen plates served as controls. After inoculation, the plates were incubated at 28℃ for 48h, and the width of the inhibition zone was measured to screen for strains with significant antagonistic effects.

[0026] The selected antagonistic strains were inoculated into 100 mL of beef extract peptone culture medium and cultured at 28 °C and 180 rpm for 24 h with shaking. 1 mL of the culture medium was centrifuged at 4 °C and 8000 rpm for 5 min, and the supernatant was collected. The supernatant was filtered through a 0.22 μm microporous membrane to remove bacteria, and sterile fermentation supernatant was obtained and stored at 4 °C for later use.

[0027] The effect of the supernatant of the antagonistic strain on the mycelia of the pathogen was detected using the slide method. Mycelial cakes of the pathogen were punched and inoculated upside down in the center of a PDA plate. After incubation at 28°C for 24 hours, a sterile coverslip was inserted obliquely at a 45° angle into the culture medium 1 cm from the edge of the colony. Incubation continued at 28°C for another 48 hours to allow mycelia to grow onto the coverslip. The coverslip with mycelia was removed, and 50 μL of the sterile fermentation supernatant of the antagonistic bacteria prepared in Example 2 was added dropwise onto the mycelia on the coverslip. The plate was then incubated at 28°C for 12 hours. Sterile water was added as a control. The coverslip was removed, and the mycelial morphology was observed and photographed under an optical microscope. The results showed that strain YW-1 had a significant antagonistic effect on the pathogen of rice wilt. The mycelia in the fermentation supernatant treatment group showed breakage and deformities, while the mycelia in the control group were normal in morphology, uniform in thickness, and vigorous in growth (e.g., ...). Figure 2 (As shown in the image). This indicates that strain YW-1 can secrete active substances that inhibit the growth of pathogens, and has the potential to be used as a biocontrol bacterium.

[0028] Strain YW-1 is a pseudo-odorant fungus ( Myroides odoratiminus The strain was deposited on September 9, 2020, at the China General Microbiological Culture Collection Center (address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Microbiology), with accession number CGMCC NO.20620. This strain has been published in the invention patent application number 202011229617.2, publication number CN112358984A.

[0029] Example 3: Detection of enzyme production activity of antagonistic strain YW-1 The strain YW-1 screened in Example 2 was inoculated into LB liquid medium and cultured at 28°C with shaking at 180 rpm for 20 h as the seed culture. The extracellular enzyme production capacity of strain YW-1 was determined using cellulase activity assay plates and chitinase activity assay plates, respectively.

[0030] Cellulase activity assay: Seed culture of strain YW-1 was spotted onto cellulase activity assay plates (10g peptone, 10g yeast extract, 10g sodium carboxymethyl cellulose, 5g sodium chloride, 1g potassium dihydrogen phosphate, 18g agar, diluted to 1000mL with distilled water, pH 7.0), with 3 spots per plate, and incubated at 28℃ for 48h. The plates were then removed, stained with 1g / L Congo red for 1h, the stain was discarded, and the plates were soaked in 1mol / L NaCl solution for 1h. The diameter of the transparent zone was observed and measured.

[0031] Chitinase activity assay: Seed culture of strain YW-1 was spotted onto chitinase activity assay plates (NH4H2PO4 1.0g, KCl 0.2g, MgSO4·7H2O 0.2g, colloidal chitin 1% (w / v) diluted to 1000mL, pH 7.0, agar 20g), with 3 spots per plate. The plates were incubated at 30℃ for 3 days, and the diameter of the clear zone was observed and measured. Each treatment was repeated in triplicate.

[0032] The results showed that strain YW-1 formed a distinct transparent hydrolysis zone on the cellulase detection plate, with a radius of 7 mm; it also formed a clear hydrolysis zone on the chitinase detection plate, with a radius of 6 mm (e.g., ...). Figure 3 (As shown). This demonstrates that strain YW-1 has the ability to produce cellulase and chitinase. These extracellular enzymes can decompose cell wall components of pathogenic fungi, which is one of the important mechanisms of its antagonistic effect.

[0033] Example 4: Determination of the low-temperature resistance of antagonistic strain YW-1 Strain YW-1 was inoculated into LB liquid medium and cultured at 28°C with shaking at 180 rpm for 20 h to obtain the seed culture. The seed culture was then inoculated into 100 mL of LB liquid medium at a 1% (v / v) inoculation rate, with three replicates for each treatment. The inoculated cultures were placed in shakers at 28°C and 15°C with shaking at 180 rpm for 24 h, respectively. The absorbance (OD) of each treatment culture was measured at 600 nm using a UV spectrophotometer. 600 Simultaneously, the culture medium was serially diluted 10-fold, and 10... -5 10 -6 10 -7 Three dilutions of 100 μL each were spread on LB plates, with three replicates for each dilution. After incubation at 28°C for 24 h, colony forming units (CFU / mL) were counted.

[0034] The results showed that after strain YW-1 was cultured at 15℃ for 24 hours, the OD... 600 The value reached approximately 1.5, and the viable bacterial count reached approximately 8.5 × 10⁻⁶. 8 CFU / mL; after incubation at 28℃ for 24 h, OD 600 Value approximately 1.8, viable count approximately 9.5 × 10⁻⁶. 9 CFU / mL. Statistical analysis showed that the viable cell count at 15℃ and 28℃ were on the same order of magnitude, with no significant difference (P>0.05). Figure 4 (As shown in the image). This indicates that strain YW-1 has good low-temperature growth characteristics, and can still maintain normal growth and metabolism at a low temperature of 15℃, making it suitable for the low-temperature environment that may occur during rice seedling cultivation.

[0035] Example 5: Compatibility test of antagonistic strain YW-1 with Trichoderma. The plate confrontation culture method was used to detect the difference between strain YW-1 and Trichoderma, a commonly used functional fungus in rice seedling substrate. Trichoderma Interactions between *Trichoderma* strains (spp.) were investigated. Activated *Trichoderma* mycelial cakes were punched using a 0.5cm punch and inoculated upside down into the center of a PDA plate, then incubated at 28°C for 48 hours. Single colonies of activated strain YW-1 were picked up with a sterile toothpick and spot-inoculated 2cm from the edge of the *Trichoderma* colony. Each treatment was repeated three times. *Trichoderma* plates without *S. YW-1* served as a control. After inoculation, the plates were incubated at 28°C for 48 hours to observe whether there was an antagonistic region of mutual inhibition between strain YW-1 and *Trichoderma*.

[0036] The results showed that during the plate confrontation culture, the colonies of strain YW-1 and Trichoderma could grow and extend normally. No obvious inhibition zone or growth inhibition zone appeared at the point where the two met. Mycelial growth was normal at the colony interface, and there was no mutual antagonism (e.g., Figure 5 (As shown). This indicates that there is no antagonistic relationship between strain YW-1 and Trichoderma, and the two can coexist symbiotically. Therefore, strain YW-1 can be used in combination with Trichoderma, and is suitable for functional rice seedling substrates with added Trichoderma.

[0037] Example 6: Preparation of biocontrol agent of strain YW-1 and its application in greenhouse seedling raising. The activated strain YW-1 was inoculated into LB liquid medium and cultured at 28°C with shaking at 180 rpm for 20 h to achieve a bacterial concentration of 10. 7 CFU / mL or higher. Centrifuge the culture medium at 4℃ and 4000 rpm for 5 min, collect the bacterial pellet, resuspend it in an equal volume of sterile water, wash twice, centrifuge again, and finally resuspend it in sterile water to the original volume to obtain a cell concentration exceeding 10⁻⁶ CFU / mL. 7 YW-1 biocontrol agent with CFU / mL.

[0038] A rice seedling raising experiment was conducted in May 2025 at the seedling raising greenhouse of Huai'an Chaimihe Agricultural Technology Co., Ltd. Commercially available rice seedling raising substrate (containing Trichoderma functional bacteria) was used. Two treatments were included: the treatment group received seedling raising substrate with added YW-1 biocontrol agent, with 1L of YW-1 biocontrol agent evenly sprayed per 25kg of substrate, thoroughly mixed, and then sown in trays; the control group received seedling raising substrate without added YW-1 biocontrol agent, sprayed with an equal volume of sterile water. Each treatment was replicated three times, with 10 seedling trays per replicate. Ambient temperature was recorded during the seedling raising period, with an average daytime temperature of 25℃ and an average nighttime temperature of 16℃. Rice seedling growth and the occurrence of Fusarium wilt were investigated 25 days after sowing.

[0039] The results showed that the rice seedlings in the treatment group with added YW-1 biocontrol agent grew vigorously, with dark green leaves and well-developed root systems, and no wilt disease was observed throughout the seedling raising period; the control group showed typical wilt disease symptoms in the later stages of seedling raising, and some seedling trays showed large areas of seedling death (e.g. Figure 6 (As shown). This demonstrates that the biocontrol agent prepared by strain YW-1 has a significant control effect on rice seedling wilt under low-temperature seedling raising conditions, and can effectively ensure the quality of seedlings in factory-style seedling raising.

[0040] 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 mushroom that mimics the aroma of fragrances ( Myroides odoratiminus The application of YW-1 in the control of rice seedling wilt disease is characterized by, The preservation number of the pseudo-odor fungus YW-1 is CGMCC NO.20620.

2. The application according to claim 1, characterized in that, The described odor-mimicking fungus YW-1 can control rice seedling wilt disease under low temperature conditions.

3. The application according to claim 2, characterized in that, The low temperature condition is 15℃-16℃.

4. A mushroom that mimics the aroma of fragrances ( Myroides odoratiminus The application of YW-1 in the preparation of biocontrol agents for use in combination with Trichoderma is characterized by, The preservation number of the pseudo-odor fungus YW-1 is CGMCC NO.20620.

5. A mushroom that mimics the aroma of fragrances ( Myroides odoratiminus The application of YW-1 or a biocontrol agent containing the aforementioned odor-mimicking fungus YW-1 in the preparation of rice seedling substrate is characterized by, The preservation number of the pseudo-odor fungus YW-1 is CGMCC NO.20620, and the biocontrol agent contains Trichoderma.

6. A method for controlling rice seedling wilt disease under low temperature conditions, characterized in that, Including those containing odor-mimicking fungi ( Myroides odoratiminus The step involves adding the biocontrol agent YW-1 to the rice seedling substrate for rice seedling cultivation; the preservation number of the pseudo-odorant fungus YW-1 is CGMCC NO.20620; the low temperature condition is 15℃-16℃.

7. The method according to claim 6, characterized in that, The biocontrol agent contains Trichoderma.

8. The method according to claim 6, characterized in that, The concentration of the odor-mimicking fungus YW-1 in the biocontrol agent was 10. 7 CFU / mL.

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