Trichoderma longibrachiatum and application thereof

Abstract

The application discloses a Trichoderma longibrachiatum strain and application thereof in biological control, wherein the strain is Trichoderma longibrachiatum Tr-8, is preserved in the China General Microbiological Culture Collection Center (CGMCC), has a preservation number of CGMCC No. 41721, a preservation date of December 16, 2024, and an address of No. 3, Xili, Beichen West Road, Chaoyang District, Beijing, China, and a telephone number of 010-64807355 of the Institute of Microbiology, Chinese Academy of Sciences. Experiments prove that during the cultivation process, the straw substrate and bacillus subtilis bacteria bodies are compounded to preferentially degrade, and the biological control effect is significantly improved. The relative control effect of the original bacteria on tobacco bacterial wilt is 57.14%, and after the substrate is compounded, the relative control effect is as high as 71.43%. Reasonable optimization of the substrate can significantly enhance the biological control effect of the bacteria on plant pathogenic microorganisms.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a long-branched Trichoderma and its application in biological control. Background Technology

[0002] With the adjustment of planting structure and the expansion of facility agriculture, agricultural soil quality has gradually deteriorated in recent years. Currently, plant disease control still mainly relies on chemical pesticides, which leads to soil salinization, compaction, and imbalance of soil microbial communities, further exacerbating soil-borne diseases and continuous cropping obstacles. To effectively control plant diseases and reduce the use of chemical pesticides, more and more countries and regions are focusing on biological control technologies. This technology, which uses beneficial microorganisms and their products to control harmful organisms in crops, is a green, economical, and sustainable solution and has become a research hotspot in recent years. Trichoderma, as an important biocontrol microorganism, inhibits the growth of pathogenic microorganisms by secreting various antibiotics. However, Trichoderma still faces challenges such as unstable efficacy in field applications. Therefore, using integrative omics methods to deeply explore the substrate preferences of Trichoderma is of significant practical importance for improving its survival rate, antibacterial effect, and overall biocontrol performance in the field.

[0003] Trichoderma longicornis Tr-8 is a Trichoderma longicornis strain screened by the inventors that possesses both biomass degradation and antibacterial capabilities. Genomic analysis results show that it has abundant cellulose-degrading enzymes and diverse genes encoding secondary metabolites, and can be regarded as a potential biocontrol strain for the efficient utilization of waste biomass while simultaneously producing a large amount of antibacterial substances. Summary of the Invention

[0004] This invention provides a strain with both biomass degradation and antibacterial capabilities. The strain is *Trichoderma longibrachiatum* Tr-8, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41721, deposited on December 16, 2024. The address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, China. The telephone number is 010-64807355.

[0005] In this article, Trichoderma longibrachiatum Tr-8 can also be referred to simply as "Tr-8", "T. longibrachiatum-8" or "T. longibrachiatum Tr-8".

[0006] On the other hand, the present invention also provides a fermentation method for the above-mentioned strain, the method comprising 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 flour, yeast extract, corn flour, keratin, collagen, and gelatin.

[0009] In one embodiment, the culture medium comprises a Mandels nutrient 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 another embodiment, it is co-cultured with Bacillus subtilis.

[0013] Preferably, the co-culture includes the following steps: culturing Bacillus subtilis to OD... 600 The concentration was set to 0.5-2.0 (preferably 0.8-1.2); then, Bacillus subtilis and Tr-8 were co-inoculated into the culture medium and co-cultured for a period of time to obtain the fermentation broth.

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

[0015] On the other hand, the present invention also provides the application of the above-mentioned strains or their fermentation broth in antagonizing plant pathogens.

[0016] Preferably, the plant pathogen is selected from one or more of Pseudomonas solanacearm, Ralstonia solanacearum, and Streptomyces ipomoeae.

[0017] On the other hand, the present invention also provides the use of the above-mentioned strains or their fermentation broth in the preparation of plant biocontrol agents.

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

[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 more of Pseudomonas solanacearm, Ralstonia solanacearum, and Streptomyces ipomoeae.

[0021] On the other hand, the present invention also provides a plant biocontrol reagent, the reagent comprising the above-mentioned strain or its fermentation broth. Attached Figure Description

[0022] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this 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 Extracellular functional enzymes of Tr-8 were detected using LC-MS / MS after 4 days of growth on various carbon and nitrogen sources. A. Comparison of heatmaps of selected CAZymes detected in the extracellular fermentation broth during growth on various carbon sources; B. Comparison of heatmaps of selected proteases detected in the extracellular fermentation broth during growth on various nitrogen sources.

[0026] Figure 4 In vitro antagonism assay. The effects of different single and complex substrates on the inhibition of pathogens by Tr-8 aseptic fermentation filtrate 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 a biocontrol experiment on bacterial wilt in potted plants. A. Antagonistic experiment between Tr8 and the pathogen in potted tobacco. B. Plant wet weight. C. Disease index and relative control effect of different treatment groups. Detailed Implementation

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

[0029] Example 1, Method and Materials

[0030] The *Trichoderma longibrachiatum* strain used in this embodiment is a *Trichoderma longibrachiatum* strain screened by the inventors that possesses both biomass degradation and antibacterial capabilities. The strain is *Trichoderma longibrachiatum* Tr-8, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41721, deposited on December 16, 2024. The address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, telephone: 010-64807355.

[0031] (1) Straw culture

[0032] 200 μL of spores of *Trichoderma longibrachiatum* Tr-8 (hereinafter also referred to as "Tr-8" or "T. longibrachiatum-8") were added. 6 The fungal biomass (CFU / ml) was inoculated into 100 ml of seed culture medium (glucose-potato water medium) and cultured at 200 rpm for 2 days at 30°C. One percent (w / v) of different carbon and nitrogen sources were added to the Mandels nutrient solution. For 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 medium was supplemented with soybean flour, yeast extract, corn flour, keratin, collagen, and gelatin. The seed culture was inoculated at a ratio of three percent (v / v) and cultured at 30°C with shaking at 200 rpm for 4 days, with samples collected daily. The fungal biomass and supernatant were separated using eight layers of gauze and centrifuged at 8000 rpm for 15 minutes at 4°C. The supernatant was stored at 4°C for further experiments.

[0033] Co-culture (also known as combined culture): Bacillus subtilis is inoculated into LB medium at 37°C and shaken at 200 rpm for about 12-24 hours until OD is reached. 600The concentration was adjusted to 0.8-1.2. *B. subtilis* and Tr-8 were inoculated in equal proportions (3 ml each) into 100 ml of Mandels nutrient solution containing 1% (w / v) straw in a 300 ml Erlenmeyer flask and co-cultured at 30°C and 200 rpm for 4 days, collecting samples daily. The bacterial cells and supernatant were separated using eight layers of gauze and centrifuged at 8000 rpm for 15 minutes at 4°C. The supernatant was stored at 4°C for further experiments.

[0034] (2) Determination of bacterial dry 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. 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 three times, and all mixtures were measured at 595 nm using a microplate spectrophotometer (Tecan, Morrisville, NC, USA). A standard curve was obtained using bovine serum albumin (0.1 mg / mL).

[0036] (3) Protease activity assay

[0037] Casein was used as a substrate, and its hydrolytic activity was determined by the Folin-Ciocalteu method. After diluting the supernatant with 50 mM Tris-HCl (pH 8.0), 100 mL of enzyme solution was added to 2% (w / v) casein and incubated at 40 °C for 10 min. The reaction was then terminated by adding 200 μL of 0.4 M trichloroacetic acid (TCA, Sigma-Aldrich, St. Louis, MO, USA). After centrifugation at 10,000 × g for 10 min at 4 °C, 100 μL of the supernatant was mixed with 500 μL of 0.4 M Na₂CO₃ and 100 μL of Folin-Ciocalteu, and incubated at 40 °C for 20 min. The absorbance was then measured at 660 nm. In the control experiment, TCA was added before incubation. Enzyme activity is defined as the amount of enzyme required to hydrolyze casein and produce 1 μmol of tyrosine per minute at the same temperature.

[0038] (4) Endoglucanase activity assay

[0039] Reducing sugar concentration and endoglucanase and xylanase activities were measured using the dinitrosalicylic acid method. Xylanase or endoglucanase activities were measured using 1% xylan (w / v) or 1% sodium carboxymethyl cellulose (CMC, Sangon Biotech, Shanghai, China) (w / v) in sodium hydrogen phosphate / citrate buffer (pH 6.0) as substrates. Xylanase activity was measured by mixing 400 μL of filtered culture supernatant with 600 μL of 1% xylan and reacting at 30 °C for 30 min. Endoglucanase activity was measured by mixing 400 μL of filtered culture supernatant with 600 μL of 1% CMC and reacting at 30 °C for 30 min. Then, 800 μL of DNS was added to each sample and the mixture was boiled for 10 min. The mixture was brought to a final volume of 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) Analysis of extracellular functional enzyme systems by liquid chromatography-tandem mass spectrometry (LC-MS / MS)

[0041] The sample was concentrated by ultrafiltration (3-kDa molecular weight cutoff membrane; Sigma-Aldrich, St. Louis, Missouri, 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 precipitate 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 protein concentration was determined using the Bradford method, 25 μg of protein solution was incubated with 50 μL of denaturing 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 h to reduce disulfide bonds. Then, cysteine ​​alkylation was performed in the dark at room temperature with 50 μL of 1 M iodoacetamide (IA, Sigma-Aldrich) for 1 h. Each sample was then transferred to a Microcon YM-10 centrifuge tube (3-kDa membrane; Millipore, Burlington, MD, USA) and washed three times at 4 °C by centrifugation at 14,000 × g for 15 min with 360 μL of 25 mM NH4HCO3 (Sigma-Aldrich). The washed protein was digested overnight at 37 °C with trypsin (Sigma-Aldrich) at a 1:25 (w / w, trypsin:protein) ratio. After desalting with C18 ZipTip (Millipore, Burlington, Massachusetts, USA), the peptides were dissolved in 10 μL of 0.1% (v / v) trifluoroacetic acid (TFA, Sigma-Aldrich).

[0043] The isolated proteins were analyzed using a nanosystem coupled to a 1,000,000 FWHM high-resolution Nano Orbitrap Fusion Lumos Tribrid mass spectrometer system (Thermo Scientific, USA) on a Thermo Fisher (Waltham, MA, USA) device. Separation was performed using a nanoViper C18 silica column (Acclaim PepMap RSLC, 75 μm × 25 cm, 2 μm, Thermo, USA) with a mobile phase 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 via 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), with a 3-second time interval between master scans. 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 an intensity greater than 5.0e4 in the quadrupole were selected for MS / MS fragmentation analysis at a normalized collision energy of 30% and a resolution of 15,000 in an Orbitrap detector. Dynamic exclusion was used within 50 seconds to avoid duplicate peptide selection. Each sample was subjected to three replicate experiments on day four on wheat straw, wheat bran, CMC, colloidal chitosan, soybean flour, yeast extract, and corn flour.

[0044] (6) Database search

[0045] Data searches were performed using Proteome Discover software version 1.4 (Thermo Fisher Scientific) in conjunction with the SEQUEST search engine. The reference database for *T. longibrachiatum* was downloaded from UniProt (http: / / www.uniprot.org). The parameters for the MS / MS search were set as follows: (1) the protein was digested with trypsin, allowing for two missed digestions; (2) the precursor mass tolerance was set to 10 ppm and the fragment mass tolerance to 0.8 Da; (3) methionine oxidation was selected as the dynamic modification, and carbamoyl methylation of cysteine ​​residues was selected as the fixed modification. Peptides containing at least six amino acid residues with a 95% confidence level (q≤0.05) were included in the results, with a false positive rate of 1%. The relative abundance of proteins was characterized by peptide profiling (PSM). Previous studies have shown a linear correlation between PSM values ​​and protein abundance. The relative abundance of each protein was the ratio of its PSM value to the total PSM value of the protein. Subsequent processing was performed using Matlab R2014a, with hierarchical clustering and heatmap generation employing Euclidean distance metric and average connectivity method.

[0046] (7) Analysis of antibacterial activity of strain confrontation culture and aseptic fermentation filtrate

[0047] The plant pathogenic bacteria *Pseudomonas solanacearm* were provided by Professor Caiyun Sun of Shandong University, while *Ralstonia solanacearum* (CGMCC NO. 1.12711) and *Streptomyces ipomoeae* (CGMCC NO. 4.1381) were obtained from the China Microbial Culture Collection Center (CGMCC, Beijing, China). All plant pathogenic bacteria were inoculated into LB agar at 37°C and shaken at 200 rpm for approximately 12-24 hours until the OD600 reached 0.8-1.2, followed by streaking on LB agar at 37°C for 24 hours. The plant pathogenic fungi were inoculated into mycelial cakes on bran agar plates and incubated statically at 30°C for 10 days.

[0048] The potential of Trichoderma isolates against plant pathogens was evaluated using confrontation culture. Mycelial discs of the plant pathogen and an activated strain of Tr-8 were placed on bran agar plates with a d = 90 mm diameter (45 mm apart). Separate cultures of the plant pathogen served as controls, with three replicates for each group. After incubating all plates at 30°C for 10 days, the growth status of the strains on the confrontation plates was observed.

[0049] The antimicrobial activity of the supernatant from strain Tr-8 cultured on different substrates was analyzed. The supernatant was filtered once through a 0.45 μm pore-sized filter, and then three times under aseptic conditions through a 0.22 μm pore-sized sterile filter. Subsequently, all pathogens were tested using the Oxford cup (6.0 mm inner diameter) diffusion method. When the indicator bacterium was a pathogen, 1% of the indicator strain with OD600 = 1.2 was injected into 12 ml LB agar medium at 45 °C, shaken thoroughly, and poured into plates. 200 μL of the treated supernatant was added dropwise to an Oxford cup, and the plates were incubated at 37 °C for 24 h. Finally, the size of the inhibition zone was observed.

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

[0051] This study investigated the biocontrol effects of different fermentation substrates on tobacco bacterial wilt caused by *Ralstonia solanacearum*. Tobacco seeds were sterilized in a 5% (v / v) hypochlorous acid solution for 10 min, rinsed three times with sterile deionized water, and germinated in sterile gauze-covered petri dishes at 28°C. After germination, two seedlings were carefully transplanted into pots filled with sterilized peat moss, with four pots per treatment group. The plants were cultured in a 25°C incubator under 14 hours of light and 10 hours of darkness. Thirty days after germination, two groups underwent root drenching with biocontrol agents. One group received 150 ml of fermentation broth using straw as a carbon source per pot, while the other group received 150 ml of fermentation broth containing a compound substrate per pot. The other two groups were watered with sterile water. Seven days later, a second root drenching with biocontrol agents was performed. Seven days later, pathogen inoculation was performed, with 50 ml of the pathogen per plant. The control group received only sterile water. The inoculated plants were cultured in a culture room at 25°C and 75% humidity. Disease index and relative control effect were observed after 15 days.

[0052] Example 2, Experimental Results

[0053] (1) Physiological and biochemical determination 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 day 1, the protein content tended to stabilize on day 2, and the lignocellulase activity reached its peak on day 2; the extracellular enzyme system was highly 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 day 1, the protein content tended to stabilize on day 2, the caseinase activity reached its peak on day 3, and the secretion was significantly delayed.

[0056] like Figure 3 As shown in the proteomic analysis, wheat straw, as a carbon source, induces a greater number of extracellular carbohydrate-active enzymes in Tr8, with a diverse and highly efficient expression of cellulose-degrading enzymes. However, only three S8 family proteases and two A1 family proteases are expressed in small amounts. The protein degradation pattern is as follows: S8 family serine proteases break down intact globulins, which are then degraded into polypeptide fragments by A1 family proteases. However, the lack of cooperation from other metalloproteinases results in relatively weak protein degradation capacity. These results preliminarily clarify that cellulose substrates such as wheat straw are preferred substrates for *T. longibrachiatum*-8. These substrates promote cell growth and induce a large number of extracellular degrading enzymes, which is a key factor influencing its efficient colonization in its natural habitat.

[0057] (2) Analysis of antibacterial activity of strain Tr8 in filtrate after confrontation culture and aseptic fermentation

[0058] like Figure 4 As shown, Tr8 exhibits direct antagonistic activity against three plant pathogens (Pseudomonas solanacearm, Ralstonia solanacearum, and Streptomyces ipomoeae). Cell-free filtrates from different fermentation substrates also showed significant inhibition zones, and its activity was further enhanced when cultured in combination with Bacillus subtilis. Figure 4 The use of a composite substrate can enhance the antibacterial properties of the fermentation supernatant.

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

[0060] like Figure 5 As shown, tobacco plants treated with Tr8 root irrigation and those treated with a mixture of Tr8 and Bacillus subtilis showed thicker, darker green leaves than the healthy control group, with wet weights two and three times higher, respectively, demonstrating its growth-promoting ability. The pathogen-treated groups showed significant leaf wilting, with lower wet weights than the healthy control group; Tr8 showed a certain biocontrol effect against tobacco bacterial wilt, reaching 57.14%; the mixture culture (… Figure 5 The disease index of Tr-8+ Complex substrates (Tr-8+Cs) decreased, with a relative prevention and control effect of up to 71.43%.

[0061] Although the present invention has been disclosed above with reference to 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 modifications and variations on this basis. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A bacterial strain possessing both biomass degradation and antibacterial capabilities, wherein the strain is *Trichoderma longifolia* (…). Trichoderma longibrachiatum Tr-8 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41721.

2. A method for fermenting the strain of claim 1, the method comprising the step of fermenting the strain using a culture medium, wherein the carbon source of the culture medium is selected from wheat straw.

3. The method according to claim 2, characterized in that, The nitrogen source of the culture medium is selected from one or more of soybean flour, yeast extract, corn flour, keratin, collagen, and gelatin.

4. The method according to claim 2, characterized in that, The strain was fermented by co-culturing with Bacillus subtilis.

5. The method according to claim 2, characterized in that, The method also includes the step of obtaining cell-free fermentation broth.

6. The application of the strain according to claim 1 or the fermentation broth obtained by any one of claims 2-5 in antagonizing plant pathogens, wherein the pathogens are selected from... Ralstonia solanacearum and Streptomyces ipomoeae Any one of them.

7. Use of the strain of claim 1 or the fermentation broth obtained by any of the methods of claims 2-5 in the preparation of plant biocontrol agents, wherein the biocontrol agents are used to control bacterial wilt of plants.

8. A plant biocontrol agent, the agent comprising the strain of claim 1 or the fermentation broth obtained by any of the methods of claims 2-5.

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