Talaromyces cordiformis and application thereof

Abstract

The invention relates to talaromyces cordiformis and application thereof. The talaromyces sp. Strain Dz-1118 is found to have an antagonistic inhibition effect on fusarium solani, fusarium oxysporum, fusarium pseudograminearum, fusarium verticillium, fusarium graminearum or botrytis cinerea, so that the talaromyces sp. Strain Dz-1118 can be used for preventing and treating root rot, fusarium wilt, stem rot, spike rot, gibberellic disease or gray mold.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, in particular to biocontrol microorganisms. Background Art

[0002] Fusarium spp. Fusarium There are many types of fungi, which are widely distributed in nature. They can live as facultative parasites or saprophytes. Plants infected by Fusarium often show symptoms such as wilting, root rot, and ear rot. Diseases caused by Fusarium are widely distributed and have great harm, often leading to a serious reduction in crop yields and causing significant economic losses. For example, Fusarium solani ) and other fungi is a plant fungal disease that can cause serious yield reduction and quality degradation of flue-cured tobacco.

[0003] In recent years, the implementation of continuous tobacco cropping and other farming practices has led to an increase in diseased and damaged tobacco leaves, a surge in the number of fungal sources in the field, and frequent outbreaks of tobacco root rot, posing new challenges to tobacco production. Current strategies for tobacco root rot prevention and control primarily include breeding resistant varieties, chemical control, and biological control, with chemical control being the primary approach. However, the long-term application of chemical pesticides can lead to the development of resistance in pathogens, causing environmental pollution and severely impacting the ecological environment. Biological control offers advantages such as being environmentally friendly, safe for humans and animals, having a long-lasting effect, and being easily coordinated with other plant protection measures, providing an effective approach to reducing the use of chemical pesticides. Summary of the Invention

[0004] One of the present invention provides a pseudo-stringy Basilicum ( Talaromyces pseudofuniculosus ) strain Dz-1118, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number CGMCC No.41854.

[0005] The second aspect of the present invention provides the pseudo-trapezid Talaromyces strain Dz-1118 according to one aspect of the present invention for inhibiting Fusarium ( Fusarium ) and / or Botrytis cinerea ( Botrytis ) in the application.

[0006] In one embodiment, the Fusarium is Fusarium solani ( Fusarium solani ), Fusarium oxysporum ( Fusarium oxysporum ), Pseudomonas graminearum ( Fusarium. pseudograminearum ), Fusarium spp. ( Fusarium. verticillioides ) and Fusarium graminearum ( Fusarium graminearum ) at least one of the following.

[0007] In one embodiment, the botrytis cinerea is botrytis cinerea ( Botrytis cinerea ).

[0008] The third aspect of the present invention provides the use of the Talaromyces pseudotrapeziaceus strain Dz-1118 described in one of the present invention for preventing and controlling at least one of root rot (especially tobacco root rot), wilt (especially banana wilt), stem base rot (especially wheat stem base rot), ear rot (especially corn ear rot), fusarium fusarium (especially wheat fusarium fusarium) and gray mold.

[0009] Beneficial effects of the present invention: The present invention found that the pseudo-stringy basket fungus ( Talaromyces pseudofuniculosus ) strain Dz-1118 against Fusarium spp. (e.g. Fusarium solani ( Fusarium. solani ), Fusarium oxysporum ( Fusarium oxysporum ), Pseudomonas graminearum ( Fusarium. pseudograminearum ), Fusarium spp. ( Fusarium. verticillioides graminearum ( Fusarium graminearum )) or Botrytis cinerea ( Botrytis cinerea ) has an antagonistic inhibitory effect, so it can be used to prevent and control root rot, wilt, stem base rot, ear rot, ergot or gray mold.

[0010] Bacterial culture deposit: The microorganism Dz-1118 strain screened in the present invention is deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration, with the deposit number CGMCC No. 41854 and the deposit date of March 24, 2025. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It is systematically classified as Talaromyces pseudotrapeziformis Talaromyces pseudofuniculosus . BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The plate standoff photos show the antibacterial effect of Dz-1118 strain on Fusarium solani.

[0012] Figure 2 The colony morphology of Dz-1118 on PDA plates is shown.

[0013] Figure 3 The phylogenetic tree of the Dz-1118 strain is shown.

[0014] Figure 4 The plate shows the antibacterial photos of Dz-1118 strain against different pathogens.

[0015] Figure 5 The content of flue-cured tobacco rhizosphere pathogens in the pot test in the embodiment of the present invention is shown, and different letters indicate significant differences between treatments ( P <0.05). DETAILED DESCRIPTION

[0016] The above contents of the present invention are further described in detail below in the form of preferred implementation cases, but they do not constitute a limitation of the present invention.

[0017] Unless otherwise specified, the strains and reagents in the examples of the present invention can be purchased through commercial channels.

[0018] The quantitative analysis in the following examples was repeated three times.

[0019] Fusarium solani ( Fusarium. solani ) For details, see Biological characteristics of tobacco Fusarium root rot pathogen and metabolic phenotypic characteristics of dominant species [J]; Jiangsu Agricultural Sciences; Author: Huang Yufeng, Li Fei, Wang Hancheng, etc.; 2024, 52(07):124-132.

[0020] Banana wilt pathogen Fusarium oxysporum ( Fusarium oxysporum f.sp. cubense ) For details, see Asmooth vetch (Vicia villosa var.) strain endogenous to the broad-spectrumantagonist Bacillus siamensis JSZ06 alleviates banana wilt disease[J]; Frontiers in Plant Science; Author: Ruan Y, Nong C, Jintrawet A et al; 2024, 15:1410197.

[0021] Wheat stem rot pathogen Fusarium graminearum ( Fusarium. pseudograminearum ) For details, see Sensitivity of Fusarium graminearum to epoxiconazole in wheat in Henan Province [J]; Acta Phytopathologica Sinica; Authors: Hou Ying, Xin Hewen, Zhang Xin, et al.; 2023, 53(2): 307-316.

[0022] Corn ear rot pathogen Fusarium spp. Fusarium. verticillioides ) For details, see Staining method of Fusarium oxysporum and construction of fluorescent strains [J]; Acta Agriculturae Boreali-Sinica; Authors: Kong Ming, Zhu Jinjie, Qi Xiantao, et al.; 2024, 39(S1): 252-258.

[0023] Wheat fusarium graminearum pathogen Fusarium graminearum ) For details, see Isolation and Identification of Pathogen Populations of Wheat Stem Rot and Scab in Henan Province in 2022 [J]; Journal of Triticeae Crops; Authors: Yan Shuwei, Bai Nima, Pan Xin, et al.; 2024, 44(5): 667-674.

[0024] Botrytis cinerea (Botrytis cinerea Botrytis. cinerea)For details, see Resistance of PetuniaPhenotypes to[J];HORTSCIENCE;Author: HI, Krahl, Randle et al;2019. 34(4): 690-692.

[0025] Rose Bengal medium: peptone 5.0 g / L, glucose 10.0 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate 0.5 g / L, agar 20.0 g / L, rose Bengal 0.033 g / L, chloramphenicol 0.1 g / L, and distilled water.

[0026] PDA medium: 200 g peeled potatoes, 20 g glucose, 15 g agar, 1000 ml distilled water, natural pH.

[0027] PDB medium: 200 g peeled potatoes, 20 g glucose, 1000 ml distilled water, natural pH. Example 1: Isolation of strains

[0028] Rhizosphere soil of healthy flue-cured tobacco was collected from diseased plots in the flue-cured tobacco experimental field in Longjie Town, Chengjiang City, Yunnan Province. The soil was sealed in sterile ziplock bags and brought back to the laboratory, where it was temporarily stored at 4°C for the isolation of antagonistic bacteria.

[0029] Take 10 g of soil in a 200 mL Erlenmeyer flask, add 90 mL of water, and shake at 180 rpm for 30 minutes to make a suspension. Let the suspension stand for 10 minutes, and use the supernatant as the stock solution for gradient dilution. The concentration gradient is set to 10 -3 , 10 -4 , 10 -5 and 10 -6 .

[0030] Spread 100 μl of each of the four dilutions onto Red Bengal medium. Incubate the Red Bengal plates at 30°C for 72 hours. Select individual fungal colonies with inconsistent morphology and color. Cut the edges of each colony and culture on PDA medium. Repeat this process two to three times to achieve purification. Transfer the colonies to PDA slants for storage. Example 2: Biological activity analysis of isolated strains

[0031] The plate standoff method was used to determine the activity of the isolated fungal strains against Fusarium solani ( F. solani ) antagonistic effect.

[0032] Preparation of conidia suspension of the test fungal strain: Inoculate each test fungal strain stored on a PDA slant onto a PDA plate and culture at 30°C for 3 days. Use a sterile punch to prepare an 8 mm test fungal cake, transfer it to 200 mL of PDB medium under a sterile environment, and culture it at 30°C and 180 rpm for 5 days. Filter to remove the mycelium, centrifuge the filtrate, and remove the supernatant to obtain the conidia pellet. Resuspend the conidia pellet with sterile water and dilute it to 1×10 8 CFU / mL, and obtain the conidia suspension of each fungal strain to be tested.

[0033] The Fusarium solani ( F. solani ) and the test fungal strain were inoculated onto PDA plates and incubated at 30°C for 72 hours for activation. After sufficient mycelium had grown, a 5 mm diameter borer was used to prepare a cake of each of the Fusarium solani and the test fungal strain. The Fusarium solani cake was inoculated onto the center of a new PDA plate. 50 μl of a conidia suspension of each test fungal strain was then spotted 2.5 cm from the edge of the cake. Four spots were evenly inoculated on each plate. The plate was then incubated in the dark at 30°C in a biochemical incubator for 7 days and observed for the development of an inhibition zone. A control group was also established, inoculated only with Fusarium solani.

[0034] For the fungal strains with antibacterial ability, the same operation was used for rescreening, and the width of the inhibition zone was measured after 7 days of culture. Three replicates were used for each treatment. The results showed that 10 fungal strains with antibacterial activity were isolated and purified from the rhizosphere soil of flue-cured tobacco, and 2 antagonistic strains had an inhibition zone width greater than 5 mm. Among them, the inhibition zone width of Dz-1118 was greater than 20 mm. Figure 1 .

[0035] The samples were cultured at 30°C for 7 days. While measuring the inhibition zones mentioned above, the colony radius of the pathogens treated with the Dz-1118 strain was measured, and the inhibition rate was calculated based on formula (1).

[0036] Inhibition rate = [(colony radius of control group - colony radius of treatment group) / radius of control group] × 100% Formula (1).

[0037] The inhibition rate of Dz-1118 strain against Fusarium solani was 93.2% as determined by plate confrontation method. Example 3: Classification and Identification of Dz-1118 Strain

[0038] The Dz-1118 strain was identified by combining colony morphology with molecular biology.

[0039] 1. Colony morphology of Dz-1118 strain: Cultured upright on PDA medium at 30 degrees Celsius for 6 days, the colony morphology was observed and recorded every day. Initially, the colonies of Dz-1118 strain appeared white, and over time, the color gradually changed from light green to olive green. Figure 2 .according to Figure 2 It can be seen that the colony texture of the Dz-1118 strain is velvety, the surface is relatively delicate, the edges of the colonies are usually relatively neat and regular circles, and there are some radial grooves on the surface of the colonies, spreading from the center of the colony to the edge.

[0040] 2. ITS analysis of Dz-1118 strain: The genomic DNA of Dz-1118 strain was extracted using the TSINGKE Plant DNA Extraction Kit (Universal). ITS1 (as shown in SEQ ID No. 1) and ITS4 (as shown in SEQ ID No. 2) universal primers for fungal identification were used as primers, and the extracted genomic DNA was used as a template for PCR amplification. After the amplified PCR product was detected by gel electrophoresis, the amplified product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Among them, the ITS sequence of the Dz-1118 strain is shown in SEQ ID No. 3. The sequence results were compared and analyzed on NCBI, and the results showed that the Dz-1118 strain was similar to the model strain. Talaromyces pseudofuniculosus FMR 15307 (NR 172396.1) is the closest relative. The phylogenetic tree was constructed using the Neighbor-Joining method in MEGA6.0 software with the default settings for related parameters. The phylogenetic tree is shown in Figure 3 .according to Figure 3 The phylogenetic tree shows that the Dz-1118 strain is a pseudo-trapezoidal Talaromyces Talaromyces pseudofuniculosus .

[0041] The Dz-1118 strain was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration with the deposit number CGMCC No. 41854 and the deposit date March 24, 2025. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Talaromyces pseudofuniculosus . Example 4: Determination of the Inhibitory Spectrum of Dz-1118 Strain

[0042] The antibacterial spectrum of Dz-1118 strain was determined by plate standoff method.

[0043] The preparation of conidia suspension of strain Dz-1118 was the same as that of the conidia suspension of the fungal strain to be tested in Example 2, and the concentration was also 1×10 8CFU / mL.

[0044] The preparation of pathogenic fungus cake was the same as in Example 2, and both were cultured at 30 degrees Celsius for 72 hours. The selected pathogenic fungus was banana wilt pathogen - Fusarium oxysporum ( Fusarium oxysporum f.sp. cubense )、Botrytis cinerea-Botrytis cinerea ( Botrytis cinerea ), wheat stem rot pathogen - Pseudomonas graminearum ( Fusarium. pseudograminearum )、Corn ear rot pathogen - Fusarium spp. ( Fusarium verticillioides ), wheat fusarium head blight - Fusarium graminearum ( Fusarium graminearum ).

[0045] The operation of plate confrontation was the same as in Example 2, with three replicates for each treatment. After culturing in the dark at 30 degrees Celsius in a biochemical incubator for 7 days, the colony radius was measured and the inhibition rate was calculated based on formula (1).

[0046] The photos of Dz-1118 strain's antibacterial activity against different pathogens are shown in the table below. Figure 4 shown.

[0047] The plate persistence inhibition rate of Dz-1118 strain against Fusarium oxysporum was 92.6%, and the plate persistence inhibition rate against Botrytis cinerea was 88.2%; the plate persistence inhibition rate against Fusarium graminearum was 86.5%, the plate persistence inhibition rate against Fusarium oxysporum was 90.3%, and the plate persistence inhibition rate against Fusarium graminearum was 85.8%.

[0048] The above results show that in addition to having a strong inhibitory effect on Fusarium solani, the Dz-1118 strain also has a strong inhibitory effect on Fusarium oxysporum, Botrytis cinerea, Fusarium graminearum, Fusarium spp. and Fusarium graminearum, with the lowest inhibition rate being 85.8%. Example 5: Potted experiment on the control of flue-cured tobacco root rot by Dz-1118 strain

[0049] The test soil was from the 0 to 20 cm topsoil layer of a flue-cured tobacco experimental field in Longjie Town, Chengjiang City, Yunnan Province. This paddy soil type possesses the following basic physical and chemical properties: pH 7.6, organic matter 37.1 g / kg, total nitrogen 2.2 g / kg, inorganic nitrogen 65.7 mg / kg, available phosphorus 55.5 mg / kg, and available potassium 327.0 mg / kg. Soil samples were collected, sieved through a 2 mm sieve, and refrigerated at 4°C until use.

[0050] The flue-cured tobacco variety used for the experiment was K326.

[0051] The preparation method of conidia suspension of strain Dz-1118 was the same as that in Example 4. The concentration of the obtained conidia suspension was 1×10 8 CFU / mL.

[0052] The preparation method of conidia suspension of Fusarium solani was the same as that of strain Dz-1118. The concentration of the conidia suspension was 1×10 7 CFU / mL.

[0053] Pot experiments were conducted: Healthy flue-cured tobacco seedlings with 5 to 6 true leaves were transplanted into pots containing 4 kg of sterile soil. Four treatments were set up: 1) FDz group: Immediately after transplanting the seedlings, 100 mL of Dz-1118 conidia suspension was applied to the tobacco roots, and one week after transplanting, 100 mL of Fusarium solani conidia suspension was applied to the tobacco roots; 2) Fs group: One week after transplanting, 100 mL of Fusarium solani conidia suspension was applied to the tobacco roots; 3) Dz group: Immediately after transplanting the seedlings, 100 mL of Dz-1118 conidia suspension was applied; and 4) Control group (CK): Immediately after transplanting the seedlings, 100 mL of sterile water was applied. Each treatment had 10 replicates, with one plant per replicate. Growth of the tobacco seedlings was measured every 7 days for a total of 28 days after transplanting. Water each pot with 50 ml of sterile water in the morning and evening to keep the soil moist. The incidence rate, disease index, and relative control efficacy were calculated according to formulas (2), (3), and (4). The results are shown in Table 1.

[0054] Incidence rate = ni × 100% / N (2).

[0055] Disease index = ∑ni×vi×100% / N×4 (3).

[0056] Relative control effect (%) = (FT) × 100% / F (4).

[0057] Where ni refers to the number of diseased plants at each disease level, vi = disease level (0, 1, 2, 3, 4), N refers to the total number of flue-cured tobacco plants used in each treatment, F refers to the disease index of the control group, and T refers to the disease index of the treatment group. Disease severity is graded (per plant): 0, disease-free; 1, 0 to 25% of leaves wilt; 2, 26% to 50% of leaves wilt; 3, 51% to 75% of leaves wilt; 4, 76% to 100% of leaves wilt, and the diseased plant is essentially dead.

[0058] Table 1

[0059] The results in Table 1 show that 28 days after inoculation, all tobacco seedlings treated with Fs became diseased. Compared with the Fs treatment, the FDz treatment reduced the disease incidence by 60.0%. The relative control efficacy of the FDz treatment reached 62.5%.

[0060] After 28 days, soil microbial DNA was extracted from the tobacco rhizosphere using the FastDNA Spin Kit (MP Bio, Santa Ana, CA, USA), and the pathogen content in the rhizosphere was quantified by qPCR. FFs (shown in SEQ ID No. 4) and RFs (shown in SEQ ID No. 5) were used as primers, and the rhizosphere microbial DNA was used as a template for qPCR analysis. Serial 10-fold dilutions of the plasmid containing the target gene were used as qPCR standards. Amplification efficiencies ranged from 90% to 110%, and the standard curve had a good linear relationship (R 2 >0.99). The quality of qPCR amplification was verified by melting curve analysis, and nonspecific amplification was negligible. Figure 5 .

[0061] Figure 5 The quantitative results of pathogens showed that the abundance of pathogens in the FDz treatment was significantly reduced by 80.7% compared with the Fs treatment.

[0062] The above results show that Dz-1118 has a good indoor control effect on tobacco root rot. Example 6: Field trial of Dz-1118 strain for controlling flue-cured tobacco root rot

[0063] The experiment was conducted in Houxiang Village, Longjie Town, Chengjiang City, Yunnan Province (102°53′29.418′′E 24°39′29.654′′N, 1753 m above sea level), within the Fuxian Lake watershed. The average annual temperature is 16.4°C, the average annual precipitation is 823.7 mm, the relative humidity is 76%, and the total annual sunshine hours are 1780.5 h. Soil information was the same as in Example 5.

[0064] The flue-cured tobacco variety used for the experiment was K326.

[0065] The preparation of conidia suspension of strain Dz-1118 was the same as in Example 4. The concentration of the obtained conidia suspension was 1×10 8 CFU / mL.

[0066] Preparation of conidia suspension of Fusarium solani was the same as that of strain Dz-1118. The concentration of the conidia suspension was 1×10 7 CFU / mL.

[0067] The plot experiment method was adopted in the fields with serious root rot in the core experimental area of ​​Chengjiang, with a randomized block design.

[0068] Healthy flue-cured tobacco seedlings with 5 to 6 true leaves were transplanted into the field. Two treatments were set up: 1) Dz treatment group: Immediately after transplanting the flue-cured tobacco seedlings, 250 ml of Dz-1118 conidia suspension was irrigated into the roots. Seven days later, 250 ml of Dz-1118 conidia suspension was irrigated into the roots again. 2) Control group (CK): The Dz-1118 conidia suspension was replaced with sterile water, and all other treatments were the same as the Dz treatment group. Each treatment was replicated three times, with a plot area of ​​50 m2. 2 (10 m × 5 m). Tobacco fertilization methods and rates were similar to those for field production. Disease incidence in flue-cured tobacco was measured every 7 days using the same method as in Example 5 until harvest. The incidence rate, disease index, and relative control efficacy on day 28 are shown in Table 2.

[0069] As shown in Table 2, in the field experiment, compared with the CK treatment, the incidence of flue-cured tobacco root rot in the Dz treatment was reduced by 25.1%, and the relative prevention efficiency was up to 51.5%.

[0070] Table 2

[0071] In conclusion, applying Dz-1118 by root irrigation during the tobacco transplanting period can significantly enhance the disease resistance of flue-cured tobacco.

Claims

1. Pseudo-stringy basket fungus ( Talaromyces pseudofuniculosus ) strain Dz-1118, which was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number CGMCC No. 41854.

2. The pseudotrichium strain Dz-1118 according to claim 1 is used to inhibit Fusarium ( Fusarium ) and / or Botrytis cinerea ( Botrytis ) in the application.

3. The use according to claim 2, characterized in that The fusarium is Fusarium solani ( Fusarium solani ), Fusarium oxysporum ( Fusarium oxysporum ), Pseudomonas graminearum ( Fusarium. pseudograminearum ), Fusarium spp. ( Fusarium. verticillioides ) and Fusarium graminearum ( Fusarium graminearum ) at least one of the following.

4. The use according to claim 2, characterized in that The botrytis cinerea is botrytis cinerea ( Botrytis cinerea ).

5. Use of the Talaromyces pseudotrapeziaceus strain Dz-1118 according to claim 1 for preventing and treating at least one of root rot, wilt, stem base rot, ear rot, head blight and gray mold.

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