Trichoderma sp. y3 and application thereof in promoting rice growth

Abstract

The application discloses a trichoderma Y3 and application thereof in promoting rice growth, and belongs to the technical field of microorganisms. Trichoderma The trichoderma Y3 is classified as Trichoderma sp. Y3, is preserved in the China Center for Type Culture Collection, and has a preservation number of CCTCC NO: M 2026214. In combination with biological characteristics and molecular identification, the trichoderma Y3 is identified as a new species of the trichoderma genus. The strain or fermentation product thereof is applied to a rice culture medium, so that the growth of the rice can be effectively promoted, and the plant biomass is increased. The trichoderma Y3 is developed and prepared into a biological bacterial fertilizer, and a new way is provided for the development of the application of microorganisms to crop production.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a Trichoderma Y3 and its application in promoting rice growth. Background Technology

[0002] Rhizosphere growth-promoting fungi, especially Trichoderma ( 木霉属 Fungi, due to their outstanding ability to promote growth, resist disease, and induce systemic resistance, are considered one of the important biological strategies for achieving ecological and high-yield rice cultivation.

[0003] Studies have shown that Trichoderma promotes rice growth and increases yield through multiple mechanisms: First, it enhances nutrient efficiency by secreting organic acids and enzymes to dissolve insoluble nutrients such as phosphorus and potassium in the soil, and enhances the absorption of elements such as nitrogen, iron, and zinc by rice. Second, it regulates root systems by producing hormone analogs (such as IAA) that stimulate root development and expand the absorption area. Third, it acts as a biological control agent, effectively mitigating the damage caused by soil-borne and airborne diseases such as sheath blight, rice blast, and seedling damping-off through competition, hyperparasitism, antibiotic resistance, and the induction of systemic resistance in rice. Fourth, it enhances stress resistance by helping rice alleviate abiotic stresses such as drought and salinity. These combined effects provide an effective way to reduce the use of chemical fertilizers and pesticides and improve rice yield and quality.

[0004] For example, patent document CN 121203832 A discloses a microbial inoculant, Trichoderma harzianum ( 木霉属 哈茨木霉 ) and Trichoderma fusiforme ( 短密木霉 The combination of Tb12 and other fungi showed significant control effects against Fusarium wilt; *Pseudomonas pulmonarius* (…) 粉红粘帚霉 NF-06 and Trichoderma acicularis ( 木霉属 棘孢木霉 When used in combination, the effect of killing nematodes is obvious; Trichoderma harzianum ( 哈茨木霉 ) and Trichoderma acicularis ( 棘孢木霉 The combination of these two fungi promotes rice growth. Patent document CN121086908A discloses white-rot fungi (…). 黄孢原毛平革菌 NDM3-2 and Trichoderma spiralis ( 螺旋木霉 Applying the HJR7 combination to soil containing straw and shiitake mushroom residue can improve the fertility of newly cultivated land and has a significant advantage in promoting rice growth.

[0005] As an important aspect of sustainable agriculture, the microbial fertilizer industry needs to screen strains with excellent adaptability, deeply analyze their interaction molecular mechanisms with rice and the rhizosphere microenvironment, and then develop efficient compound microbial fertilizers and optimize application techniques to promote the standardized and large-scale application of Trichoderma biopharmaceuticals in green rice production. Summary of the Invention

[0006] The purpose of this invention is to provide a microorganism that promotes rice growth and to develop it into a bio-fertilizer for safe and efficient rice production.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention isolated a new Trichoderma species from the roots of cultivated rice. 木霉属 The fungus Y3 (sp.) exhibits the following biological characteristics: rapid growth on PDA medium, initially producing abundant aerial hyphae on the medium surface, followed by the formation of numerous green spores and slimeballs on the colony surface; the hyphae are vegetatively branched and septate, and the conidia are elliptical. The TEF1 and SSU-ITS-LSU sequences of this strain are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. A phylogenetic tree of the four genes TEF1-SSU-ITS-LSU for this strain and its closely related species was constructed, identifying the strain as belonging to the kingdom Fungi, phylum Ascomycota, class Sordariomycetes, order Hypocreales, family Hypocreaceae, and genus Trichoderma. 木霉属 (sp.), but it is an independent branch (MLBP>80), and its colony morphology and hyphal spore structure are significantly different from other species in the genus, thus identifying this strain as a new species.

[0008] Therefore, this strain was classified and named 木霉属 sp.Y3 was deposited on January 23, 2026 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2026214.

[0009] Furthermore, 木霉属 The culture conditions for sp. Y3 (Trichoderma Y3) are as follows: inoculated into potato dextrose agar (PDA) medium and cultured in the dark at 22-25°C.

[0010] This invention has found that inoculating Trichoderma Y3 or its fermentation products into rice growth substrate and co-culturing them can promote rice growth and increase rice plant biomass.

[0011] Therefore, the present invention provides the application of the aforementioned Trichoderma Y3 in promoting rice growth.

[0012] Furthermore, the indicators for rice growth include at least one of chlorophyll content, nitrogen content, stem and leaf height, root length, aboveground fresh weight, and underground fresh weight.

[0013] Furthermore, the application includes inoculating the Trichoderma Y3 into a rice planting substrate to promote rice growth.

[0014] As a specific embodiment of the present invention, the application includes: inoculating activated Trichoderma Y3 into sterilized barley grains to obtain solid fermented microbial fertilizer, and then mixing it into rice planting substrate.

[0015] Preferably, the solid fermented microbial fertilizer and the rice planting substrate are mixed at a mass ratio of 1:10.

[0016] As another specific embodiment of the present invention, the application includes: inoculating activated Trichoderma Y3 into PDB medium and culturing it to the exponential phase, and then mixing the bacterial solution into rice planting substrate.

[0017] Preferably, the bacterial solution is mixed with the rice planting substrate at a volume ratio of 3:7.

[0018] This invention provides a formulation of Trichoderma Y3, which is prepared into a solid fermented microbial fertilizer. The preparation method of the solid fermented microbial fertilizer includes: inoculating activated Trichoderma Y3 into PDB medium and dark-culturing it at 22-25℃ and 100-150 rpm until the exponential phase; then adding 150 mL of mycelial suspension per 200 g of barley grains and dark-culturing at 22-25℃ until the mycelium covers the barley grains, thereby obtaining the solid fermented microbial fertilizer.

[0019] The activation method includes: inoculating Trichoderma Y3 into PDA medium and culturing it at 22-25℃ for 7 days.

[0020] The present invention also provides the application of the aforementioned Trichoderma Y3 solid fermentation fertilizer in promoting rice growth.

[0021] Furthermore, the solid fermented microbial fertilizer is applied during rice seedling raising or cultivation.

[0022] Specifically, the solid fermented microbial fertilizer can be applied by mixing the solid fermented microbial fertilizer into the seedling substrate, and then sowing the germinated rice seeds in the seedling substrate containing Trichoderma Y3. During the seedling raising process, Trichoderma Y3 promotes the growth of rice seedlings.

[0023] The beneficial effects of this invention are as follows: This invention provides a new Trichoderma genus ( 木霉属Based on biological characteristics and molecular identification, strain Y3 of *Trichoderma* was identified as a new species of the genus *Trichoderma*. Application of this strain or its fermentation products to rice culture media effectively promoted rice growth and increased plant biomass. In pot experiments, rice co-cultured with *Trichoderma* Y3 showed a significant increase in leaf chlorophyll content (40.51%), leaf nitrogen content (37.41%), stem and leaf height (19.14%), stem and leaf fresh weight (53.71%), and root fresh weight (39.04%). In hydroponic experiments, rice co-cultured with *Trichoderma* Y3 showed a 41.56% increase in stem and leaf height, a 59.03% increase in stem and leaf fresh weight, and a 47.40% increase in root fresh weight. These findings indicate that strain Y3 and its fermentation products can promote rice growth and significantly increase rice biomass, and can be used to prepare bio-fertilizers, providing a new avenue for the application of microorganisms in crop production. Attached Figure Description

[0024] Figure 1 The images show the colony morphology of strain Y3 after 7 days of growth on PDA medium; where A is the front view of the colony and B is the back view of the colony.

[0025] Figure 2 Images show the hyphae and spore morphology of strain Y3 under a microscope; where A is a hyphae image and B is a spore image.

[0026] Figure 3 Images of solid-state fermented microbial fertilizer from strain Y3 are shown below. Image A shows the strain grown on PDB medium at 25°C and 150 rpm for 3 days. Image B shows the frontal growth of strain A after 15 days of fermentation on barley medium. Image C shows the side growth of strain A after 15 days of fermentation on barley medium.

[0027] Figure 4 This is a phylogenetic tree diagram of the four genes TEF1-SSU-ITS-LSU in strain Y3. The tree is composed of TEF1, SSU, ITS, and LSU sequence datasets. The topology of the tree is the result of the ML method. The values ​​on the branches are ML expansion values ​​(MLBP), and the scale bar represents 0.5 base substitution sites.

[0028] Figure 5 The results of solid fermentation microbial fertilizer from strain Y3 promoting the growth of potted rice are shown in the figure. A represents the effect of strain Y3 on rice growth, (i) is a picture of the potted plant, and (ii) is a picture of the whole rice plant. BG are bar charts showing the chlorophyll (SPAD) content, leaf nitrogen content, root length, stem and leaf height, stem and leaf fresh weight, and root fresh weight of the control and treatment groups, respectively (independent samples t-test analysis data, ** and **** represent...). P <0.01 and PSignificant difference at the 0.0001 level, ns indicates no significant difference.

[0029] Figure 6 This image shows the results of the Y3 strain's inoculum promoting hydroponic rice growth. A represents a hydroponic image; B represents a whole rice plant image; and C and D are bar charts showing the stem and leaf height, root length, stem and leaf fresh weight, and root fresh weight of the control and treatment groups, respectively (data analyzed using independent samples t-test, **** indicates...). P Significant difference at the 0.0001 level, ns indicates no significant difference. Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0032] Example 1: Isolation and Identification of Microbial Strains 1. Strain isolation (1) Collect cultivated rice samples from Zhejiang Province, rinse the roots of cultivated rice with tap water to wash away soil particles and attached substances on the surface; (2) Select healthy root tissues for surface disinfection. Disinfect with 75% alcohol for 2 min, then disinfect with 1% sodium hypochlorite for 4-5 min, and finally rinse with sterile distilled water 4-5 times to remove residual sodium hypochlorite solution. (3) Cut the disinfected roots into 5 mm long segments; (4) Pour 15 mL of sterilized PDA medium (with chloramphenicol added to the medium to reach 50 mg / L to inhibit bacterial growth) into 7 cm culture dishes after cooling to 50-60℃; after the medium has cooled and solidified, use tweezers to transfer the cut root segments into the medium for incubation. (5) Invert the petri dish and incubate it in a constant temperature incubator at 25℃. After 3-7 days of incubation, pick the hyphae of a single colony and place them on a new PDA plate.

[0033] PDA medium: 200 g potato, 20 g glucose, 15 g agar powder, add distilled water to 1000 mL, pH at rest. Autoclave at 121℃ for 15 min.

[0034] A strain was isolated and named Y3 through the above isolation and purification steps.

[0035] 2. Identification of fungal strain morphology Strain Y3 was inoculated in PDA medium and cultured in the dark at 25°C. The average mycelial growth rate was approximately 2.65-2.85 cm per day. Colony morphology and spore characteristics were classified on PDA, PDB, and barley media.

[0036] like Figure 1 As shown, strain Y3 grows rapidly on PDA medium. In the early stage, a large number of white aerial mycelia are formed, and in the later stage, a large number of green spores are formed on the surface of the colony. The front of the colony is green and the back is white.

[0037] like Figure 2 As shown, the hyphae are vegetatively branched and septate, and the conidia are elliptical, as observed under a microscope.

[0038] like Figure 3 As shown, strain Y3 colonies turned white after 3 days of growth on PDB medium; the colonies also turned white after 15 days of fermentation on barley medium.

[0039] 3. Molecular identification and phylogenetic analysis (1) DNA extraction 1) After culturing strain Y3 on a PDA plate at 25°C for 10 days, scrape the mycelium from the plate with a toothpick and put it into a sterilized 1.5 mL centrifuge tube containing 500 μL of extraction buffer and an appropriate amount of quartz sand. The extraction buffer formulation is: 10 mM ethylenediaminetetraacetic acid, 100 mM Tris-hydrochloric acid, and 1 M potassium chloride; 2) Place the centrifuge tubes in an MP FastPrep®-24 homogenizer and shake at 65 Hz for 2 min to break the mycelial tissue; 3) Centrifuge at 9000 rpm for 10 min, then pipette the supernatant into a new 1.5 mL centrifuge tube; 4) Add an equal volume of isopropanol to the supernatant, invert the mixture several times, centrifuge at 12000 rpm for 10 min, discard the supernatant, and retain the precipitate; 5) Add 700 μL of 70% ethanol (v / v) to dissolve the impurities, mix gently, and centrifuge at 12000 rpm for 1 min. 6) Discard the supernatant, invert the centrifuge tube onto absorbent paper to evaporate excess ethanol. The white precipitate is the genomic DNA to be extracted. Add 50 μL of sterile water and dissolve at room temperature for 15 min. After dissolution, store the genomic DNA in a -20°C freezer.

[0040] (2) PCR amplification of fungal TEF1, SSU rDNA, ITS and LSU rDNA genes TEF1 primers: The upstream primer EF1 sequence is: 5′-ATGGGTAAGGA(A / G)GACAAGAC-3′, and the downstream primer EF2 sequence is GGA(G / A)GTACCAGT(G / C)ATCATGTT-3′; SSU rDNA, ITS and LSU rDNA three-gene sequencing primers: upstream primer SSU-F sequence: 5′-GTAGTCATATGCTTGTCTC-3′, downstream primer LSU-R sequence: 5′-TCCTGAGGGAAACTTCG-3′; PCR amplification was performed in a 30 μL reaction system containing: 1.5 μL each of forward and reverse primers, 15 μL of Green TaqMix, 1.5 μL of template DNA, and 10.5 μL of ddH2O.

[0041] PCR amplification was performed on a Langqi MG96G PCR instrument. Reaction conditions: 95℃ pre-denaturation for 3 min, followed by 35 cycles of: 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min; and a final extension at 72℃ for 10 min.

[0042] (3) Gene sequencing and sequence analysis The target DNA fragment was sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing. After rigorous verification, the sequencing results yielded the DNA fragment sequences shown in SEQ ID NO.1 and SEQ ID NO.2, namely the TEF1 and SSU-ITS-LSU gene sequences of strain Y3.

[0043] On the NCBI website, the nucleotide sequences of TEF1 and SSU-ITS-LSU of strain Y3 were analyzed using BLAST and compared with homologous or similar nucleotide sequences in the GenBank database. BLAST comparison revealed that the TEF1 sequence was similar to that of strain with accession number MN307404.1. 钩状木霉 The similarity of SFC20180510-M09 was 99.30%, and it was similar to strain OR779500.1. 木霉属 The similarity of sp. T30064 was 97.24%. The SSU-ITS-LSU three-gene sequence was similar to that of strain ON399101.1. 绿色木霉 The similarity of T2C1.4 was 97.78%, and it was similar to strain PP386584.1 (accession number). 木霉属 The similarity of sp. 47S was 97.75%. Through sequence comparison, strain Y3 belongs to... 木霉属 sp.

[0044] (4) Phylogenetic analysis The TEF1 and SSU-ITS-LSU sequences of closely related genera of strain Y3 were searched on NCBI, and a phylogenetic tree of the four genes TEF1-SSU-ITS-LSU combined with those of strain Y3 and its closely related genera was constructed. The results showed that the four genes combined information loci totaled 7260 bases, including 2111 parsimony information loci, 3898 variable information loci, and 1891 conserved loci. Using jModel Test 2.1.10, the optimal model for the Bayesian Information Criterion (BIC) and Akaike Information Criterion (AICc) was determined to be TIM3, and a phylogenetic tree based on maximum likelihood (ML) was constructed. The ML phylogenetic tree was constructed using PAUP. Figure 4 The image shows an ML tree. From the phylogenetic tree, strain Y3 belongs to... 木霉属 sp., but it is the same as 钩状木霉 Not in the same branch and strain Y3 and 钩状木霉 The colony morphology and hyphae spore morphology also differ greatly.

[0045] Based on comprehensive phylogenetic analysis, morphological and biological characteristics, strain Y3 is defined as a new species. 木霉属 稻 sp. nov belongs to the kingdom Fungi, phylum Ascomycota, class Sordariomycetes, order Hypocreales, family Hypocreaceae, and genus Trichoderma sp.

[0046] Therefore, the strain was named 木霉属 sp. Y3 was deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on January 23, 2026, with accession number CCTCC NO: M 2026214, and was confirmed to be viable on January 30, 2026.

[0047] Example 2: The promoting effect of Y3 strain solid fermented microbial fertilizer on the growth of potted rice. 1. Germination of rice (CO39) seeds: Wash rice seeds in sequence with clean water, 1% NaClO (soak for 10 min), and sterile water. Place the washed seeds in a glass basin containing moist absorbent paper and germinate in the dark at 37℃ for 2 days.

[0048] 2. Preparation of microbial fertilizer: The Y3 strain was cultured in PDA medium for 7 days, and the mycelial cake was placed in 150 mL PDB medium and cultured at 25℃ and 150 rpm for 3 days. Then, the mycelial suspension was poured into barley grains that had been autoclaved (121℃ for 30 min) (150 mL of mycelial solution for every 200 g of barley grains) and fermented at 25℃ for 20 days.

[0049] 3. Potted Planting: Allow the fermented microbial fertilizer to air dry naturally. Mix the dried microbial fertilizer with rice planting substrate at a mass ratio of 1:10 and fill the pots with the mixture. Plant the germinated rice seeds (30 seeds and 40 g of fertilizer per pot) into the pots containing the fermented microbial fertilizer. Use sterile barley seed fertilizer as a control group.

[0050] 4. Phenotypic analysis of rice plants: After culturing rice for 15 days, the chlorophyll and nitrogen content of rice leaves were measured using a SPAD-502 (Japan) chlorophyll content meter. The rice seedlings were pulled out of the soil substrate by the roots, washed repeatedly with clean water, and the water on the rice plants was dried with absorbent paper. The height of the rice stems and leaves, the length of the roots, and the fresh weight of the stems, leaves and roots were measured.

[0051] 5. Results Analysis: For example... Figure 5 As shown, in the pot experiment, the chlorophyll content of rice co-cultured with Y3 increased significantly by 40.51%, the nitrogen content of leaves increased significantly by 37.41%, the stem and leaf height increased significantly by 19.14%, the fresh weight of stems and leaves increased significantly by 53.71%, and the fresh weight of roots increased significantly by 39.04%.

[0052] Example 3: The promoting effect of Y3 bacterial solution on the growth of hydroponic rice 1. Strain Y3 was inoculated into 150 mL PDB medium / 250 mL Erlenmeyer flask and cultured at 25℃ and 150 rpm for 3 days. A mixture of 30 mL bacterial culture and 70 mL pure water was used as the treatment group, and pure water was used as the control.

[0053] 2. Sprinkle a small amount of perlite on the surface of the prepared culture solution (to prevent rice seeds from sinking to the bottom), transfer the germinated rice seeds into culture bottles (15 seeds per bottle), and culture for 15 days (add pure water once during the culture process). After that, take photos to record and measure the stem and leaf height, root length, fresh weight of stems and leaves, and fresh weight of roots of the rice seedlings.

[0054] 3. Results Analysis: For example... Figure 6 As shown, in the hydroponic experiment, when the content of Y3 bacterial solution in the culture medium was 30%, the stem and leaf height of rice increased by 41.56%, the fresh weight of stems and leaves increased significantly by 59.03%, and the fresh weight of roots increased significantly by 47.40%.

[0055] The above description is merely a specific embodiment of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent modifications or substitutions made based on the essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A type of Trichoderma ( Trichoderma sp.)Y3, characterized in that, Trichoderma Y3 is classified as Trichoderma sp. Y3, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 2026214.

2. The Trichoderma Y3 as described in claim 1, characterized in that, The culture conditions for Trichoderma Y3 are as follows: inoculated into PDA medium and cultured in the dark at 22-25℃.

3. The application of Trichoderma Y3 as described in claim 1 or 2 in promoting rice growth.

4. The application as described in claim 3, characterized in that, The indicators for rice growth include at least one of the following: chlorophyll content, nitrogen content, stem and leaf height, root length, aboveground fresh weight, and underground fresh weight.

5. The application as described in claim 3, characterized in that, The application includes inoculating the Trichoderma Y3 into a rice planting substrate to promote rice growth.

6. The application as described in claim 5, characterized in that, The applications include: inoculating activated Trichoderma Y3 into sterilized barley grains to obtain solid fermented microbial fertilizer, and then mixing it into rice planting substrate; or inoculating activated Trichoderma Y3 into PDB medium and culturing it to the exponential phase, and then mixing the bacterial solution into rice planting substrate.

7. The application as described in claim 6, characterized in that, Solid fermented microbial fertilizer is mixed with rice planting substrate at a mass ratio of 1:10; microbial liquid is mixed with rice planting substrate at a volume ratio of 3:

7.

8. A Trichoderma Y3 solid fermentation fertilizer, characterized in that, The method for preparing the solid fermented microbial fertilizer includes: inoculating the activated Trichoderma Y3 as described in claim 1 into PDB medium and dark-culturing it at 22-25℃ and 100-150 rpm until the exponential phase; then adding 150 mL of mycelial suspension per 200 g of barley grains and dark-culturing it at 22-25℃ until the mycelium covers the barley grains to obtain the solid fermented microbial fertilizer.

9. The application of Trichoderma Y3 solid fermentation fertilizer as described in claim 8 in promoting rice growth.

10. The application as described in claim 9, characterized in that, The solid fermented microbial fertilizer is applied during rice seedling raising or cultivation.

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