Trichoderma for efficiently preventing and controlling tobacco root rot and preparation method thereof

Abstract

The invention discloses trichoderma for efficiently preventing and controlling tobacco root rot and a culture method thereof, which are characterized in that tobacco root rot biocontrol bacteria are separated from tobacco rhizosphere soil, tobacco root rot pathogenic bacteria share fusarium F. Commune as indicator bacteria, and bacterial strains with bacteriostatic action on the growth of fusarium solani are screened to obtain the trichoderma LB8-12-2; the method can effectively inhibit the tobacco from being infected by fusarium pathogenic bacteria in the growth process, and can greatly improve the survival rate and quality of the tobacco in the growth process when being applied to the tobacco growth process. Experimental data show that after tobacco is treated by the trichoderma LB8-12-2, the relative control effect is 95.51%, and it is indicated that the trichoderma LB8-12-2 has a good control effect on the tobacco fusarium root rot, so that the trichoderma LB8-12-2 has remarkable advantages in the aspects of protecting the environment, reducing pesticide residues and improving the safety of tobacco leaves when being used for controlling the tobacco fusarium root rot, and the application prospect is wide.

Description

Technical Field

[0001] This invention belongs to the field of strain technology, specifically relating to a highly effective Trichoderma for controlling tobacco root rot and its preparation method. Background Technology

[0002] Root rot is a fungal disease that causes root decay, gradually weakening the plant's ability to absorb water and nutrients, eventually leading to the death of the entire plant. The main symptoms are yellowing and wilting of the leaves. This disease can be caused by various pathogens, including Pythium, Fusarium, and Phytophthora. The pathogens overwinter in the soil or on diseased plant debris, becoming the primary source of infection the following year. The pathogens enter through wounds in the rhizome or roots and spread via rainwater or irrigation water. Low-lying areas, poor drainage, waterlogging, continuous cropping, leaky greenhouses, and damaged plant roots are more susceptible to the disease. Root rot is a very common and serious disease in agricultural production, and many important crops are easily infected.

[0003] Tobacco, as an important economic crop, is mainly distributed in Yunnan, Guizhou, Henan, Hunan, Sichuan, and Fujian provinces. Tobacco root rot is a soil-borne fungal disease caused by Fusarium. After infection, tobacco leaves wilt and lose their green color, the edges turn brown or black, the vascular bundles turn black, become blocked and deformed, and in severe cases, the entire plant wilts or even dies. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a highly efficient Trichoderma for controlling tobacco root rot and its preparation method. The Trichoderma LB8-12-2 described in this invention can effectively inhibit infection by Fusarium wilt pathogens during tobacco growth. Applying Trichoderma LB8-12-2 to tobacco growth significantly improves the survival rate and quality of tobacco plants.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A type of Trichoderma that effectively controls tobacco root rot has been classified and named Trichoderma viride The accession number is CCTCC No: M 20242896, the accession date is December 24, 2024, the depositary institution is the China Center for Type Culture Collection, and the address of the depositary institution is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0006] The ITS-rDNA sequence of the Trichoderma strain is shown in SEQ ID No. 1.

[0007] The application of the highly effective Trichoderma for controlling tobacco root rot in the control of tobacco root rot.

[0008] The method for cultivating Trichoderma for efficient control of tobacco root rot includes the following steps: (1) Isolation of biocontrol bacteria for tobacco root rot from tobacco rhizosphere soil Tobacco rhizosphere soil was collected and water was added to prepare a soil suspension. After settling, the supernatant was taken, spread on a culture medium plate, and cultured for a period of time. The mycelial cake was then collected and purified on a PDA plate to obtain the biocontrol bacteria strain to be screened. (2) Screening of strains Fusarium, the pathogen of tobacco root rot F. commune As indicator bacteria, strains that inhibit the growth of Fusarium solani were screened to obtain the Trichoderma.

[0009] In step (1), take 10g of tobacco rhizosphere soil, add 100ml of water, and shake in a shaker at 150r / min and 25℃ for 120min to obtain soil suspension.

[0010] In step (1), the supernatant is taken and serially diluted to 10. -1 -10 -3 Take 100 μL of each gradient solution, spread it evenly on a culture medium plate, and incubate at 25°C for 48 h.

[0011] In step (1), the culture medium plate is a Bengal Red culture medium plate.

[0012] The raw material composition of the Bengal Red culture medium plate is as follows: 5g peptone, 10g glucose, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, and 0.033g Bengal Red.

[0013] In step (2), the inhibition diameter of Fusarium spp. was 1.27 ± 0.2 cm.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a highly effective Trichoderma for controlling tobacco root rot. It involves first isolating a biocontrol agent for tobacco root rot from the rhizosphere soil, and then using Fusarium, the pathogen of tobacco root rot, as a shared fungus. F. commune As an indicator bacterium, strains exhibiting inhibitory effects against Fusarium solani were screened, resulting in the Trichoderma LB8-12-2. This strain effectively inhibits infection by Fusarium spores during tobacco growth. Its application in tobacco growth significantly improves the survival rate and quality of the tobacco. Experimental data show that the relative control efficacy of Trichoderma LB8-12-2 treated with this invention is 95.51%, indicating that Trichoderma LB8-12-2 has good control efficacy against tobacco Fusarium root rot. Therefore, the use of Trichoderma LB8-12-2 for the prevention and control of tobacco Fusarium root rot has significant advantages in environmental protection, pesticide residue reduction, and improved tobacco leaf safety, and has broad application prospects. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art 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.

[0016] Figure 1 The image shows the antibacterial effect of the biocontrol strain LB8-12-2. Figure 2 The colony morphology of the biocontrol strain LB8-12-2; Figure 3 Phylogenetic tree of biocontrol strain LB8-12-2; Figure 4 Physiological and biochemical characteristics of biocontrol strain LB8-12-2; where A represents carbon source utilization and B represents nitrogen source utilization.

[0017] Figure 5 The study investigated the growth-promoting effect of biocontrol agents on tobacco; where A was the sterile water control and B was the treatment with LB8-12-2 biocontrol agent.

[0018] Figure 6 The control effect of biocontrol agents on tobacco is shown; where A is the pathogen treatment and B is the LB8-12-2 biocontrol agent treatment. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0020] The above technical solution will be described in detail below with reference to specific embodiments.

[0021] Example 1: Preparation of Culture Medium Bengal Red Culture Medium: Take 5 g of peptone, 10 g of glucose, 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate and 0.033 g of Bengal Red in sequence, and make up to 1 L with distilled water.

[0022] PDB medium: 200g peeled potatoes, cut into pieces and boiled for 20min, filtered twice through 8 layers of gauze and the filtrate was collected. 20g glucose was added and the volume was adjusted to 1000mL with distilled water. Sterilized at 121℃ for 30min.

[0023] PDA medium: The preparation method is the same as that of PDB medium. Specifically, take 200g of peeled potatoes, cut them into pieces and boil for 20 minutes. Filter the liquid twice through 8 layers of gauze and collect the filtrate. Add 20g of glucose and 20g of agar powder, add water to make up to 1L, and sterilize at 121℃ for 30 minutes.

[0024] Example 2: Isolation and screening of Trichoderma LB8-12-2 Biocontrol bacteria for tobacco root rot were isolated from tobacco rhizosphere soil. Soil samples were collected from tobacco fields in Chenzhou City, Hunan Province (112°14′E, 24°54′N) during the tobacco growing season. The specific steps for strain isolation and screening are as follows: (1) Isolation and purification of strains Tobacco rhizosphere soil was collected and water was added to prepare a soil suspension. After settling, the supernatant was taken, spread on a culture medium plate, and cultured for a period of time. The mycelial cake was then collected and purified on a PDA plate to obtain the biocontrol bacteria strain to be screened. After the soil samples were allowed to air dry at room temperature, 10 g of soil sample was randomly weighed and added to an Erlenmeyer flask containing 90 ml of sterile water. The mixture was then incubated at 150 r·min. -1 Soil suspension was obtained by shaking in a shaker at 25 ℃ for 120 min. After settling, 1 mL of the supernatant was serially diluted to 10. -1 -10 -3 Take 100 μL of each gradient solution and spread it evenly on a Bengal Red agar plate using a sterile spreader (add 10 mg streptomycin to 100 mL of sterilized Bengal Red agar when cooled to 60℃ and mix well). Incubate at 25℃ for 48 h. After that, take 0.6 mm bacterial cakes and transfer them to PDA plates for 1-2 purifications to obtain the biocontrol bacteria strains to be screened. (2) Screening of strains Fusarium, the pathogen of tobacco root rot, is shared by the fungus. F. commune Using *Trichoderma* as an indicator bacterium, a confrontation culture method was used to screen for strains with highly effective inhibitory effects on the growth of *Fusarium solani*. Eight-mm mycelial blocks were collected from the edge of activated pathogen colonies and inoculated with seven different strains of the target strain, each with a diameter of 8 mm. The mixtures were incubated at 25°C, with PDA plates without *Trichoderma* mycelial blocks serving as a control. When the mycelial radius of the control pathogen reached approximately 5 cm, the colony radius (mm) of the treated group was measured, and the inhibition rate of the biocontrol bacteria against the pathogen was calculated.

[0025] like Figure 1 As shown, the Trichoderma strain with strain number LB8-12-2 obtained by the above method had an inhibition diameter of 1.27±0.2 cm against the shared falcatum, and an inhibition rate of 81.64%.

[0026] Example 3 Identification of Trichoderma viride LB8-12-2 The selected antagonistic strains were inoculated onto PDA medium for 24 hours for activation, and then their morphology was observed. Figure 2 As shown, the colonies of strain LB8-12-2 are pale green, dry, and tightly bound to the culture medium. The hyphae are transparent and septate, with conidiophores that are repeatedly branched and irregularly whorled. The apical sterigmata are flask-shaped and irregularly curved. The conidia are green with smooth walls and aggregate on the flask sterigmata to form spherical spore heads.

[0027] The ITS gene sequence of strain LB8-12-2 was analyzed for homology using the NCBI BLAST database. Reference sequences were selected based on literature review and database data, and a phylogenetic tree was constructed using MEGA 11. The phylogenetic tree is shown below. Figure 3 As shown, LB8-12-2 and Trichoderma viride They clustered together, therefore, strain LB8-12-2 was identified as *Trichoderma viride*. Trichoderma viride ).

[0028] The 16S rDNA sequence of the *Trichoderma* strain LB8-12-2 is as follows (SEQ ID No. 1): .

[0029] This strain was deposited on December 24, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number CCTCC No: M 20242896. The depository is the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, within the campus of Wuhan University.

[0030] Experimental Example 1: Physiological and biochemical properties of Trichoderma viride LB8-12-2 The strains were identified with reference to Wei Jingchao's "Handbook of Fungal Identification" and Zhang Zhongyi's "Flora of Chinese Fungi", as follows: The selected antagonistic strain LB8-12-2 was inoculated into PDA medium for 24 hours for activation, and then inoculated into: A: Carbon source growth medium was used. After inoculation, the medium was placed in a 28℃ incubator for 7 days. The changes in the medium were observed. PAD medium was used as a blank control.

[0031] B: Nitrogen source growth medium. After inoculation, the medium was placed in a 28°C incubator for 7 days. The changes in the medium were observed. PAD medium was used as a blank control.

[0032] like Figure 4 Figure A shows that the *Trichoderma viride* strain LB8-12-2 can utilize starch, maltose, glucose, lactose, and sucrose as carbon sources for growth. In the figure, *af* represents CK (blank control), starch, maltose, glucose, lactose, and sucrose, respectively, as carbon sources. Figure 4 Figure B shows that strain *Trichoderma viride* LB8-12-2 can grow with lysine, proline, and aspartic acid as nitrogen sources; gi in the figure represents control, glycine, arginine, lysine, proline, and aspartic acid, respectively.

[0033] The carbon source utilization and nitrogen source utilization of the strains are shown in Table 1 and Table 2, respectively.

[0034] Table 1 - Carbon source utilization of strain LB8-12-2

[0035] Table 2 - Nitrogen source utilization of strain LB8-12-2

[0036] 2. The growth-promoting effect of biocontrol agent (Trichoderma LB8-12-2) on tobacco. Tobacco seeds (Yunyan 87) were sown in seedling pots filled with substrate (peat moss:vermiculite = 3:1, by weight) and allowed to grow until the cross-shaped stage. Uniformly growing seedlings were selected and divided into 4 treatment groups, with 3 replicates per group and 15 seedlings per group.

[0037] Trichoderma spore suspension: Fresh Trichoderma LB8-12-2 mycelial cakes were collected and placed in PD at 25℃ and 160 rpm. -1 After 5 days of shaking culture, the spore solution was obtained by gauze filtration and the concentration was adjusted to 1×10⁻⁶ using a hemocytometer. 7 CUF / mL.

[0038] Treatment A: Water treatment; Treatment B: Biocontrol agent; Experimental method: Tobacco plants were treated with root irrigation 5 days after transplanting. 10 mL was applied to the roots of each plant, once every 5 days, for a total of 3 times.

[0039] Two treatments, A and B, were applied by root irrigation, with 10 mL applied to the roots of each plant. Treatment A served as the control.

[0040] Survey method: After 7 days, various physiological indicators of tobacco plants were measured, including plant height, stem length, root length, fresh weight, dry weight, maximum leaf length, and maximum leaf width.

[0041] The test results are shown in Table 3 and Figure 5 .

[0042] Table 3 - Growth-promoting effects of Trichoderma viride LB8-12-2 on tobacco

[0043] 3. The effect of biocontrol agent (Trichoderma LB8-12-2) on control Fusarium spore suspension: Fresh Fusarium mycelium cakes were collected and placed in a PD at 25°C and 160 r·min. -1 After 3 days of shaking culture, the spore solution was obtained by gauze filtration and the concentration of the spore solution was adjusted to 1×10⁻⁶ using a hemocytometer. 6 Add the bacterial solution (CUF / mL) to the sterilized wheat grain culture medium.

[0044] Trichoderma spore suspension: Fresh Trichoderma LB8-12-2 mycelial cakes were collected and placed in a PD incubator at 25℃ and 160 r·min. -1 After 5 days of shaking culture, the spore solution was obtained by gauze filtration and the concentration was adjusted to 1×10⁻⁶ using a hemocytometer. 7 CUF / mL.

[0045] Tobacco seeds (Yunyan 87) were sown in seedling pots filled with substrate (peat moss:vermiculite = 3:1, by weight) and allowed to grow until the cross-shaped stage. Uniformly growing seedlings were selected and divided into four treatment groups, with three replicates per group and 15 seedlings per group.

[0046] The application method of this biocontrol agent is as follows: After the seedlings reach the cross-shaped stage, inoculate the roots with Fusarium spore solution and simultaneously inoculate with biocontrol agent spore solution, applying 10 mL to the roots of each plant. Tobacco plants inoculated only with the pathogen serve as a control. After 14 days, observe the disease incidence and calculate the disease index to determine the control effect of the biocontrol agent on tobacco root rot.

[0047] Treatment A: Clean water + pathogens; Treatment B: Biocontrol agent + pathogen; Experimental method: Two treatments, A and B, were applied by root irrigation, with 10 mL applied to the roots for each treatment. Treatment A served as the control.

[0048] Survey method: Calculate the disease index.

[0049] The test results are shown in Table 4 and Figure 6 .

[0050] Table 4 - Effects of Trichoderma viride LB8-12-2 on root rot control

[0051] The results showed that the relative control efficacy of Trichoderma chlorophyllin LB8-12-2 on tobacco was 95.51%. The experimental results indicate that Trichoderma chlorophyllin LB8-12-2 has a good control effect on tobacco sickle root rot. Therefore, the promotion and application of Trichoderma chlorophyllin LB8-12-2 is expected to replace chemical pesticides in the control of tobacco sickle root rot, protect the environment, reduce pesticide residues, improve the safety of tobacco leaves, and has broad application prospects.

[0052] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A Trichoderma sp. for efficient control of tobacco black root rot, characterized in that, Classification name is Trichoderma viride , the accession number is CCTCC No: M 20242896, the preservation date is December 24, 2024, and the preservation unit is China Typical Culture Collection Center, and the address of the preservation unit is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province.

2. The Trichoderma spp. for efficiently preventing and controlling tobacco black root rot according to claim 1, characterized in that, The ITS-rDNA sequence of the Trichoderma strain is shown as SEQ ID No.

1.

3. The Trichoderma for efficiently preventing and controlling tobacco black root rot according to any one of claims 1-2, for use in preventing and controlling tobacco black root rot.

4. The method for cultivating Trichoderma for efficient control of tobacco root rot according to any one of claims 1-2, characterized in that, The method comprises the following steps: (1) isolating a tobacco black root rot biocontrol bacterium from tobacco rhizosphere soil Take tobacco rhizosphere soil, add water to obtain a soil suspension; after standing and sedimentation, take the supernatant, spread on a culture medium plate, culture for a period of time, then take the bacterial cake to a PDA plate for purification to obtain a biocontrol bacterium for screening strains; (2) screening of strains Fusarium shared with the pathogen of tobacco root rot F. commune As an indicator bacteria, strains with antibacterial effect on the growth of F. solani were screened, and the Trichoderma was obtained.

5. The culture method according to claim 4, characterized by, In step (1), 10 g of tobacco rhizosphere soil is taken, 100 ml of water is added, and the mixture is shaken at 150 r / min and 25°C for 120 min to obtain a soil suspension.

6. The culturing method according to claim 4, wherein In step (1), take the supernatant, gradient dilute to 10 -1 -10 -3 times, take 100 μL of each gradient solution, spread evenly on the medium plate, and then incubate at 25°C for 48 h.

7. The culturing method according to claim 4, wherein In step (1), the culture medium plate is a Bengal red culture medium plate.

8. The culturing method according to claim 4, wherein The raw material composition of the Bengal red culture medium plate is: 5 g of proteose peptone, 10 g of glucose, 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, and 0.033 g of Bengal red.

9. The culturing method according to claim 4, wherein In step (2), the antibacterial diameter of the shared Fusarium is 1.27±0.2 cm.

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