Trichoderma longibrachiatum and application thereof

Abstract

The invention discloses Trichoderma longibrachiatum and application thereof in biological control, the strain is Trichoderma longibrachiatum Tr-8, the Trichoderma longibrachiatum Tr-8 is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.41721, the preservation date is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC No.41721, and the preservation number is CGMCC No.41721. And the telephone number is 010 to 64807355. Experiments prove that the biological control effect is remarkably improved by compounding a straw substrate which is preferentially degraded and bacillus subtilis thalli in the culture process, the relative control effect of original bacteria on tobacco bacterial wilt reaches 57.14%, and the relative control effect of the original bacteria on tobacco bacterial wilt reaches 71.43% after the substrate is compounded; by reasonably optimizing the substrate, the biological control effect on plant pathogenic microorganisms can be obviously enhanced.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a long-branched Trichoderma and application thereof in biological control. Background Art

[0002] With the adjustment of the planting industry structure and the expansion of facility agriculture, the quality of agricultural soil has gradually deteriorated in recent years. At present, the prevention and control of plant diseases still mainly rely on chemical pesticides, but this has led to soil salinization, compaction and imbalance of soil microbial communities, which in turn has aggravated the problems of soil-borne diseases and plant continuous cropping obstacles. In order to effectively prevent and control plant diseases and reduce the use of chemical pesticides, more and more countries and regions have begun to pay attention to biological control technology. This technology uses beneficial microorganisms and their products to contain harmful organisms in crops. As a green, economical and sustainable solution, it has been a research hotspot in recent years. As an important biocontrol microorganism, Trichoderma has the biocontrol characteristic of inhibiting the growth of pathogenic microorganisms by secreting a variety of antibiotics. However, Trichoderma still faces challenges such as unstable effects during field application. Therefore, the integrative omics approach is used to deeply explore the substrate preference of Trichoderma, which is of great practical significance for improving its survival rate, antibacterial effect and overall biocontrol performance in the field.

[0003] The long-branched Trichoderma Tr-8 is a strain of long-branched Trichoderma that has both biomass degradation and antibacterial capabilities. The results of genome analysis showed that it has abundant cellulose degradation enzymes and diverse secondary metabolite encoding genes, and can be regarded as a potential biocontrol strain for efficient utilization of waste biomass and simultaneous production of a large number of antibacterial substances. Summary of the invention

[0004] The present invention provides a strain having both biomass degradation and antibacterial capabilities, the strain being Trichoderma longibrachiatum Tr-8, which is deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.41721 and a deposit date of December 16, 2024. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Tel: 010-64807355.

[0005] Herein, Trichoderma longibrachiatum Tr-8 may also be referred to as "Tr-8", "T. longibrachiatum-8" or "Trichoderma longibrachiatum Tr-8".

[0006] On the other hand, the present invention also provides a fermentation method of the above-mentioned strain, which comprises the step of fermenting the strain using a culture medium.

[0007] In one embodiment, the carbon source of the culture medium is selected from one or any combination of wheat straw, wheat bran, sodium carboxymethyl cellulose, xylan, starch, and colloidal chitin.

[0008] In one embodiment, the nitrogen source of the culture medium is selected from one or any combination of soybean meal, yeast extract, corn meal, keratin, collagen, and gelatin.

[0009] In one embodiment, the culture medium comprises Mandels nutrient salt solution.

[0010] In one embodiment, the fermentation temperature is 25-40°C, preferably, 30°C.

[0011] In one embodiment, the fermentation time is 1-10 days, preferably, 4-6 days.

[0012] In one embodiment, the strain is fermented by co-culturing with other microorganisms; in one embodiment, it is co-cultured with Bacillus subtilis.

[0013] Preferably, the co-cultivation comprises the following steps: culturing Bacillus subtilis to an OD of 600 After that, Bacillus subtilis and Tr-8 are co-inoculated into the culture medium and co-cultured for a period of time to obtain a fermentation liquid.

[0014] In one embodiment, the fermentation method further comprises the step of obtaining a cell-free fermentation broth.

[0015] On the other hand, the present invention also provides the use of the above-mentioned strain or its fermentation liquid in antagonizing plant pathogens.

[0016] Preferably, the plant pathogenic bacteria is selected from one or any several of Pseudomonas solanacearm, Ralstoniasolanacearum and Streptomyces ipomoeae.

[0017] On the other hand, the present invention also provides the use of the above-mentioned strain or its fermentation liquid in preparing plant biocontrol agents.

[0018] In one embodiment, the biocontrol agent is used to control plant bacterial wilt.

[0019] In one embodiment, the bacterial wilt is caused by Ralstonia solanacearum.

[0020] In one embodiment, the biocontrol agent is used to control diseases caused by plant pathogens; preferably, the plant pathogens are selected from one or any combination of Pseudomonas solanacearm, Ralstonia solanacearum and Streptomycesipomoeae.

[0021] On the other hand, the present invention also provides a plant biocontrol agent, which comprises the above-mentioned strain or its fermentation liquid. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0023] Figure 1 .Physiological and chemical characteristics of Trichoderma longifolia Tr-8 under different carbon sources; Among them, A. biomass; B. protein content; C. endoglucanase activity; D. xylanase activity.

[0024] Figure 2 .Physiological and chemical characteristics of Tr-8 under different nitrogen sources; Among them, A. biomass; B. protein content; C. pH; D. casein enzyme activity.

[0025] Figure 3 .Detection of extracellular functional enzymes of Tr-8 after 4 days of growth on various carbon and nitrogen sources using LC-MS / MS. A. Heat map comparison of selected CAZymes detected in the extracellular fermentation broth during growth on various carbon sources; B. Heat map comparison of selected proteases detected in the extracellular fermentation broth during growth on various nitrogen sources.

[0026] Figure 4 In vitro antagonism test. The effects of Tr-8 sterile fermentation filtrate on the inhibition of pathogens under different single substrates and composite substrates were determined through in vitro experiments (1. wheat straw 2. wheat bran 3. corn gluten meal 4. soybean meal).

[0027] Figure 5 .Antagonistic analysis of biological control experiment of bacterial wilt in potted plants. A. Antagonistic experiment between Tr8 and pathogens in tobacco potted plants. B. Plant wet weight. C. Disease index and relative control effect of different treatment groups. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with specific examples, but the present invention is not limited by the examples. The materials, reagents, instruments and methods used in the following examples, unless otherwise specified, are conventional materials, reagents, instruments and methods in the art and can be obtained through commercial channels.

[0029] Example 1. Methods and Materials

[0030] The long-branch Trichoderma used in this embodiment is a long-branch Trichoderma with both biomass degradation and antibacterial ability screened by the inventor. The strain is Trichoderma longibrachiatum Tr-8, which is deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration Committee, with a deposit number of CGMCC No.41721, and a deposit date of December 16, 2024. The address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Tel: 010-64807355.

[0031] (1) Strain culture

[0032] 200 μL of spores (10 6 CFU / ml) was inoculated into 100ml seed culture medium (glucose potato water culture medium) at 30°C and cultured at 200rpm for 2 days. One percent (W / V) of different carbon sources and nitrogen sources were added to the Mandels nutrient solution. In the carbon source experiment, wheat straw, wheat bran, sodium carboxymethyl cellulose, xylan, starch, and colloidal chitin were added to the nutrient solution separately. For the nitrogen source experiment, the culture medium was supplemented with soy flour, yeast extract, corn flour, keratin, collagen, and gelatin. The seed solution was inoculated at a ratio of three percent (V / V) and cultured at 30°C with shaking at 200rpm for 4 days, and samples were collected every day. Eight layers of gauze were used to separate the fungal biomass from the supernatant and centrifuged at 8000rpm for 15 minutes at 4°C. The supernatant was stored at 4°C and used for further experiments.

[0033] Composite culture (also called co-culture): Inoculate Bacillus subtilis in LB medium at 37°C and shake at 200 rpm for about 12-24 hours until OD 600Reach 0.8-1.2. Inoculate B.subtilis and Tr-8 in equal proportions (3 ml each) into 100 ml Mandels nutrient solution containing 1% (W / V) straw, use a 300 ml conical flask and then co-cultivate at 30°C and 200 rpm for 4 days, and collect samples every day. Use eight layers of gauze to separate the bacteria and the supernatant, and centrifuge at 8000 rpm for 15 minutes at 4°C. The supernatant is stored at 4°C and used for further experiments.

[0034] (2) Determination of dry cell weight and extracellular protein content

[0035] The dry weight of fungal mycelium was used to characterize the total biomass. The mycelium was filtered through gauze and dried together at 50°C until constant weight. The protein concentration in the filtered culture supernatant was determined using the Bradford method: 150 μL of protein sample and 150 μL of Coomassie Brilliant Blue G-250 dye were reacted at room temperature for 10 minutes. Each sample was tested in triplicate, and all mixtures were measured at 595 nm using a microplate spectrophotometer (Tecan, Morrisville, NC, USA). Bovine serum albumin (0.1 mg / mL) was used to obtain a standard curve.

[0036] (3) Protease activity assay

[0037] Casein was used as a substrate and casein degradation activity was determined by the Folin phenol method. After diluting the supernatant with 50mM Tris-HCl (pH8.0), 100mL of enzyme solution was added to 2% (w / v) casein and incubated at 40°C for 10 minutes, followed by the addition of 200μL 0.4M trichloroacetic acid (TCA, Sigma-Aldrich, St.Louis, MO, USA) to terminate the reaction. After centrifugation at 10,000 × g for 10 minutes at 4°C, 100μL of supernatant was mixed with 500μL 0.4M Na2CO3 and 100μL Folin phenol and incubated at 40°C for 20 minutes. The absorbance was then measured at 660nm. In the control experiment, TCA was added before incubation. The unit of enzyme activity is defined as the amount of enzyme required to hydrolyze casein and produce 1μmol of tyrosine per minute at the same temperature.

[0038] (4) Determination of endoglucanase activity

[0039] The reducing sugar concentration and endoglucanase and xylanase activities were measured using the dinitrosalicylic acid method. 1% xylan (w / v) or 1% sodium carboxymethylcellulose (CMC, Sangon Biotech, Shanghai, China) (w / v) in sodium hydrogen phosphate / citric acid buffer (pH 6.0) was used as a substrate to measure xylanase or endoglucanase activity. The filtered culture supernatant (400 μL) was mixed with 600 μL 1% xylan, reacted at 30°C for 30 min, and the xylanase activity was determined; the filtered culture supernatant (400 μL) was mixed with 600 μL 1% CMC and reacted at 30°C for 30 minutes to measure the endoglucanase activity. Then 800 μL DNS was added to each sample and boiled for 10 minutes. The mixture was made up to 10 mL, and the absorbance at 550 nm was measured using a UV spectrophotometer (Puyuan Instruments, Ltd., Shanghai, China).A standard curve was prepared using 1 mg / mL xylose or glucose.

[0040] (5) Liquid chromatography tandem mass spectrometry (LC–MS / MS) analysis of extracellular functional enzymes

[0041] The samples were concentrated by ultrafiltration (3-kDa molecular weight cutoff membrane; Sigma-Aldrich, St. Louis, MO, USA) and precipitated using acetone containing 10% (w / v) trichloroacetic acid (TCA) and 0.1% (w / v) dithiothreitol (DTT, Sigma-Aldrich). The mixture was incubated on ice overnight and then harvested by centrifugation at 8000×g for 30 min at 4°C. After removing the supernatant, the pellet was washed three times with 80% ice-cold acetone to remove residual TCA. Finally, the precipitated protein was dried and dissolved in ultrapure water.

[0042] After the protein concentration was determined by the Bradford method, 25 μg of protein solution was incubated with 50 μL of denaturation buffer (0.5 M Tris-HCl, 2.75 mM EDTA, 6 M guanidine hydrochloride, Sigma-Aldrich) and 30 μL of 1 M DTT at 37 ° C for 2 hours to reduce disulfide bonds. Then, 50 μL of 1 M iodoacetamide (IA, Sigma-Aldrich) was added for cysteine ​​alkylation in the dark at room temperature for 1 hour. Each sample was then transferred to a Microcon YM-10 centrifuge tube (3-kDa membrane; Millipore, Burlington, MA, USA) and washed three times with 360 μL of 25 mM NH4HCO3 (Sigma-Aldrich) by centrifugation at 14,000 × g for 15 minutes at 4 ° C. The washed protein was digested by incubation with trypsin (Sigma-Aldrich) at 37 ° C at a ratio of 1:25 (w / w, trypsin: protein) overnight. After desalting by C18 ZipTip (Millipore, Burlington, MA, USA), the peptides were dissolved in 10 μL of 0.1% (v / v) trifluoroacetic acid (TFA, Sigma-Aldrich).

[0043] The separated proteins were analyzed by a nanosystem (Thermo Fisher, Waltham, Massachusetts, USA) coupled to a 1,000,000 FWHM high-resolution Nano Orbitrap Fusion Lumos Tribrid mass spectrometer system (Thermo Scientific, USA). Separation was performed using a nanoViper C18 silica column (Acclaim PepMap RSLC, 75 μm×25 cm, 2 μm, Thermo, USA) with a mobile phase system consisting of solvent A (0.1% [v / v] formic acid) and solvent B (80% [v / v] acetonitrile containing 0.1% [v / v] formic acid) at a flow rate of 300 nL / min. The following gradient program was used: 0–4 min, solvent B increased from 2% to 10%; 4–44 min, solvent B increased from 10% to 28%; 44–54 min, solvent B increased from 28% to 38%; 54–57 min, solvent B increased from 38% to 100%; 57–60 min, solvent B remained at 100%. The eluted peptides were sprayed into the mass spectrometer through a nanospray ionization source. The Nano Orbitrap Fusion Lumos Tribrid mass spectrometer was operated in data-dependent acquisition mode, using Xcalibur 4.2.47 software (Thermo Scientific, USA), and the interval between main scans was set to 3 s. Full scan MS spectra (from 350 to 1800 m / z) were detected in the Orbitrap at a resolution of 60,000 (at 400 m / z). Precursor ions with intensities greater than 5.0e4 in the quadrupole were selected for MS / MS fragmentation analysis with a normalized collision energy of 30% and a resolution of 15,000 in the Orbitrap detector. Dynamic exclusion was used within 50 seconds to avoid repeated selection of peptides. Three replicates were performed on wheat straw, wheat bran, CMC, colloidal chitosan, soybean meal, yeast extract, and corn meal on the fourth day for each sample.

[0044] (6) Database search

[0045] Data search was performed using Proteome Discover software version 1.4 (Thermo Fisher Scientific) in combination with the SEQUEST search engine. The reference database of T. longibrachiatum was downloaded from UniProt (http: / / www.uniprot.org). The parameters for the MS / MS search were set as follows: (1) Trypsin was used to digest the protein, and two missed cleavages were allowed; (2) The precursor mass tolerance was set to 10 ppm and the fragment mass tolerance was 0.8 Da; (3) Oxidation of methionine was selected as a dynamic modification, and carbamidomethylation of cysteine ​​residues was selected as a fixed modification. Only peptides containing at least six amino acid residues with 95% confidence (q≤0.05) were included in the results, and the false positive discovery rate was set to 1%. The relative abundance of proteins was characterized by peptide spectrum matching (PSM). Previous studies have shown that there is a linear correlation between PSM values ​​and protein abundance. The relative content of each protein is the ratio of the PSM value of the protein to the PSM value of the total protein. Subsequent processing was performed using Matlab R2014a, and the hierarchical clustering and heat map generation were performed using the Euclidean distance metric and the average linkage method.

[0046] (7) Antimicrobial activity analysis of strain confrontation culture and sterile fermentation filtrate in vitro

[0047] The plant pathogenic bacteria Pseudomonas solanacearm was provided by Professor Sun Caiyun of Shandong University, and Ralstonia solanacearum (CGMCC NO.1.12711) and Streptomyces ipomoeae (CGMCC NO.4.1381) were obtained from the China Microbiological Culture Collection (CGMCC, Beijing, China). The plant pathogenic bacteria were inoculated in LB medium at 37°C, shaken at 200 rpm for about 12-24 hours until the OD600 reached 0.8-1.2, and then streaked on LB agar medium at 37°C for 24 hours. Plant pathogenic fungi were inoculated with bacterial cakes on bran agar plates and cultured at 30°C for 10 days.

[0048] The potential of Trichoderma isolates to fight plant pathogens was evaluated by confrontation culture. Plant pathogens and activated strains of strain Tr-8 were placed on bran agar medium with d=90 mm (45 mm between each other). Plant pathogens were cultured alone as a control, and three replicates were set for each group. After all plates were cultured in an incubator at 30°C for 10 days, the confrontation growth state of the strain plates was observed.

[0049] The antimicrobial activity of the supernatant of strain Tr-8 cultured on different substrates was analyzed. The supernatant was filtered once with a 0.45μm-pore-sized filter, and then filtered three times with a 0.22-μm-pore-sized sterile filter under sterile conditions. All pathogens were then tested by diffusion method using an Oxford cup (inner diameter 6.0mm). When the indicator bacteria are pathogenic bacteria, 1% of the indicator strain with OD600=1.2 was injected into 12ml LB agar medium at 45°C, shaken evenly to pour the plate, and 200μL of the treated supernatant was dropped into the Oxford cup, cultured at 37°C for 24h, and finally the size of the inhibition zone was observed.

[0050] (8) Biocontrol experiment on tobacco bacterial wilt in potted plants

[0051] The tobacco bacterial wilt caused by Ralstonia solanacearum was used as the research object to study the actual biocontrol effect under different fermentation substrates. The tobacco seeds were sterilized in 5% (v / v) hypochlorous acid solution for 10 minutes, then rinsed three times with sterile deionized water, and germinated in a 28°C culture dish on sterile gauze. After germination, they were carefully transplanted into pots filled with sterilized peat soil, with 2 plants transplanted in each pot, and 4 pots in each treatment group, and cultured in a culture room at 25°C, 14 hours of light and 10 hours of darkness. After 30 days of seed germination, the two groups were treated with biocontrol bacteria root irrigation. One group was irrigated with 150 ml of fermentation liquid with straw as the carbon source per pot, and the other group was irrigated with 150 ml of fermentation liquid with a composite substrate per pot. The other two groups were irrigated with sterile water, and the biocontrol bacteria were irrigated again after 7 days. After 7 days, the pathogen root irrigation inoculation treatment was carried out, 50 ml per plant, and the control group was only irrigated with sterile water. The inoculated plants were further cultured in a culture room at 25°C and 75% humidity. The disease index and relative control effect were observed after 15 days.

[0052] Example 2, experimental results

[0053] (1) Physiological and biochemical assays of Tr8 under different carbon and nitrogen substrates

[0054] like Figure 1 As shown, under different carbon source substrates (wheat straw, wheat bran, sodium carboxymethyl cellulose, xylan, starch or colloidal chitin), the biomass of Tr8 increased rapidly on the first day, the protein content tended to stabilize on the second day, and the activity of lignocellulase reached the highest peak on the second day; the extracellular enzyme system was efficiently induced by cellulose substrates.

[0055] like Figure 2As shown, under different nitrogen source substrates (soybean flour, yeast extract, corn flour, keratin, collagen or gelatin), the biomass of Tr8 increased on the first day, the protein content stabilized on the second day, the casein activity reached a peak on the third day, and the secretion was significantly delayed.

[0056] like Figure 3 As shown in the figure, the proteome showed that wheat straw as a carbon source induced more extracellular carbohydrate active enzymes in Tr8, and there were many types of cellulose degrading enzymes and they were efficiently expressed, while only 3 S8 family and 2 A1 family proteases were expressed in small amounts. Protein degradation mode: The serine proteases of the S8 family decomposed the complete globulin, which was then degraded into polypeptide fragments by the A1 family proteases, but the other metalloproteinases were not available for cooperative degradation, so the protein degradation ability was relatively weak. This result preliminarily clarified that cellulose substrates such as wheat straw are the preferred substrates of T. longibrachiatum-8, which are conducive to bacterial growth and induce a large number of extracellular degrading enzymes, which is a key influencing factor for its efficient colonization in natural habitats.

[0057] (2) Antimicrobial activity analysis of the Tr8 strain in vitro by confrontation culture and sterile fermentation filtrate

[0058] like Figure 4 As shown in the figure, Tr8 has direct antagonism against three plant pathogens (Pseudomonas solanacearm, Ralstoniasolanacearum and Streptomyces ipomoeae). The cell-free filtrates under different fermentation substrates also showed obvious inhibition zones, and the complex culture with Bacillus subtilis ( Figure 4 Composte substrate) can improve the antibacterial ability of the fermentation supernatant.

[0059] (3) Antagonistic analysis of the biocontrol experiment of tobacco bacterial wilt in potted plants

[0060] like Figure 5 As shown in the figure, the leaves of tobacco plants irrigated with Tr8 and the composite culture substrate of Tr8 and Bacillus subtilis were thicker and greener than those of the healthy control group, and the plant wet weight was 2 and 3 times that of the healthy control group, respectively, showing its growth-promoting ability. The pathogen-treated group showed obvious leaf wilting, and its plant wet weight was lower than that of the healthy control group; Tr8 had a certain biocontrol effect on tobacco bacterial wilt, reaching 57.14%; the composite culture ( Figure 5 The disease index of Tr-8+Complex substrates (Tr-8+Cs) in the experiment decreased, and the relative prevention and treatment effect was as high as 71.43%.

[0061] Although the present invention has been disclosed as above in the form of preferred embodiments, it is not intended to limit the present invention. Without departing from the spirit and scope of the present invention, those skilled in the art can make various changes and modifications on this basis. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain having both biomass degradation and antibacterial capabilities, the strain being Trichoderma longibrachiatum Tr-8, which is deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No. 41721 and a deposit date of December 16, 2024. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Tel: 010-64807355.

2. A method for fermenting the strain according to claim 1, comprising the step of fermenting the strain using a culture medium.

3. The method according to claim 2, characterized in that The carbon source of the culture medium is selected from one or any combination of wheat straw, wheat bran, sodium carboxymethyl cellulose, xylan, starch and colloidal chitin.

4. The method according to claim 2, characterized in that: The nitrogen source of the culture medium is selected from one or any combination of soybean meal, yeast extract, corn meal, keratin, collagen, and gelatin.

5. The method according to claim 2, characterized in that: The strain is fermented by co-culturing with other microorganisms.

6. The method according to claim 2, characterized in that The method further comprises the step of obtaining a cell-free fermentation broth.

7. Use of the strain according to claim 1 or the fermentation liquid obtained by any one of the methods of claims 2 to 6 in antagonizing plant pathogens.

8. Use of the strain according to claim 1 or the fermentation liquid obtained by any one of the methods of claims 2 to 6 in preparing a plant biocontrol agent.

9. A plant biocontrol agent, comprising the strain according to claim 1 or the fermentation liquid obtained by any one of the methods of claims 2-6.

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