Trichoderma fungus y4 and application thereof in promoting rice growth

Abstract

The application discloses a Trichoderma fungus Y4 and application thereof in promoting rice growth, and belongs to the technical field of microorganisms. Trichoderma The classification name of the Trichoderma fungus Y4 is Trichoderma sp. Y4, which is preserved in the China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO: M 2026215. In combination with biological characteristics and molecular identification, the Trichoderma fungus Y4 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 can be increased. The Trichoderma fungus Y4 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 fungus Y4 and its application in promoting rice growth. Background Technology

[0002] Rice ( Oryza sativa Rice (Oryza sativa) is an annual aquatic herbaceous plant belonging to the genus Oryza in the family Poaceae. It boasts extremely high nutritional value and a delicious taste, and is one of the world's three major food crops. In agricultural production, long-term reliance on chemical fertilizers and pesticides not only increases production costs but also leads to a series of problems such as environmental pollution, soil degradation, and pesticide residues, posing a continuous challenge to the healthy and sustainable development of the rice industry. Against this backdrop, developing environmentally friendly and sustainable green agricultural technologies has become an urgent need.

[0003] Microbial fertilizers are biological agents containing live microorganisms that produce specific fertilizer effects during application, thereby increasing plant yield or improving quality. The most important of these microorganisms are plant growth promoters (PGPRs). Plant growth promoters are beneficial microorganisms that live in the soil or are epiphytic on plant roots, promoting nutrient absorption and growth while inhibiting harmful organisms. Studies have shown that PGPRs can affect crops in multiple ways through phosphorus solubilization, nitrogen fixation, and plant hormone production, such as promoting seed germination, improving crop growth and yield, alleviating external stress, and enhancing crop stress resistance. Furthermore, PGPRs can promote the growth of other beneficial microorganisms and inhibit harmful microorganisms. Compared to the application of chemical fertilizers in traditional agriculture, PGPRs not only save on planting costs but also reduce environmental pollution.

[0004] Trichoderma ( Trichoderma Trichoderma harzianum is a widely distributed fungus belonging to the phylum Fungi, subphylum Deuteromycetes, and order Hyphomyales. It has functions in preventing and controlling plant diseases and promoting plant growth. Studies have found that Trichoderma harzianum has good effects on rice seed germination and promoting rice root growth. For example, patent document CN118978982A discloses a strain of Trichoderma harzianum (…). Trichoderma harzianum NFF001, a bacterium, possesses nitrogen-fixing capabilities due to its symbiotic relationship with *Sphingomonas*, providing nitrogen to rice and promoting its growth. Patent document CN118497082A discloses a compound bacterium composed of *Bacillus sicca* 2-3C48 and *Trichoderma harzianum* 1-1322, which has the dual function of promoting the decomposition of straw directly returned to the field and promoting rice growth. Therefore, utilizing *Trichoderma* fungi to act on rice roots to enhance rice's growth-promoting ability or its ability to resist external stress is an effective means of ensuring safe rice production and increasing yields.

[0005] In summary, it is necessary to discover more strains with excellent adaptability, analyze their interaction molecular mechanisms with rice and the rhizosphere microenvironment, 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 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:

[0008] This invention isolated a new Trichoderma species from the roots of cultivated rice. Trichoderma The fungus Y4 (sp.) exhibits the following biological characteristics: rapid growth on PDA medium, forming numerous white aerial hyphae on the surface; colonies are white on the upper surface and yellow on the lower surface; the hyphae are vegetatively branched and septate, and the conidia are round. 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. This strain belongs to the kingdom Fungi, phylum Ascomycota, class Sordariomycetes, order Hypocreales, family Hypocreaceae, and genus Trichoderma, but is a novel species (MLBP > 80). Furthermore, its colony morphology and hyphal-spore structure are significantly different from other species in this genus.

[0009] Therefore, this strain was classified and named Trichoderma sp. Y4 was deposited on January 23, 2026 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2026215.

[0010] Furthermore, the culture conditions for Trichoderma fungus Y4 were as follows: inoculated into potato dextrose agar (PDA) medium and cultured in the dark at 22-25°C.

[0011] This invention has found that inoculating Trichoderma fungus Y4 or its fermentation products into rice growth substrate and co-culturing them can promote rice growth and increase plant biomass.

[0012] Therefore, the present invention provides the application of the aforementioned Trichoderma fungus Y4 in promoting rice growth.

[0013] Furthermore, the indicators for rice growth include at least one of chlorophyll content, leaf nitrogen content, stem and leaf height, root length, stem and leaf fresh weight, and root fresh weight.

[0014] Furthermore, the application includes inoculating the Trichoderma fungus Y4 into a rice planting substrate to promote rice growth.

[0015] As a specific embodiment of the present invention, the application includes: inoculating activated Trichoderma fungus Y4 into sterilized barley grains to obtain solid fermented microbial fertilizer, and then mixing it into rice planting substrate.

[0016] This invention provides a formulation of Trichoderma fungus Y4, which is prepared into a solid fermented microbial fertilizer. The preparation method of the solid fermented microbial fertilizer includes: inoculating activated Trichoderma fungus Y4 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.

[0017] The solid fermented microbial fertilizer is applied during rice seedling raising or cultivation. Specifically, the solid fermented microbial fertilizer is mixed into the seedling substrate, and the germinated rice seeds are sown in the seedling substrate containing Trichoderma Y4 and fermentation products. During the seedling raising process, Trichoderma Y4 and fermentation products promote the growth of rice seedlings.

[0018] Specifically, solid fermented microbial fertilizer is mixed with rice planting substrate at a mass ratio of 1:10.

[0019] As another specific embodiment of the present invention, the application includes: inoculating activated Trichoderma Y4 into PDB medium and culturing it to the exponential phase, and then mixing the bacterial solution into rice planting substrate.

[0020] Specifically, the preparation method of the bacterial solution includes: inoculating strain Y4 into PDB medium and culturing at 25°C and 150 rpm for 3 days.

[0021] The bacterial solution is applied during rice seedling raising or cultivation. Specifically, the bacterial solution is mixed with the rice planting substrate at a volume ratio of 3:7.

[0022] The activation method includes inoculating Trichoderma fungus Y4 into PDA medium and culturing it at 22-25°C for 7 days.

[0023] The beneficial effects of this invention are as follows:

[0024] This invention provides a new Trichoderma genus ( TrichodermaThe fungus Y4, identified as a new species of Trichoderma based on biological characteristics and molecular identification, was used in rice cultivation. Applying 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 Y4 showed a significant increase in chlorophyll content (39.68%), nitrogen content (36.75%), stem and leaf height (23.68%), fresh weight (28.27%), and root fresh weight (7.00%). In hydroponic experiments, rice co-cultured with Y4 showed a significant increase in chlorophyll content (58.07%), nitrogen content (51.66%), stem and leaf height (14.10%), and fresh weight (44.44%). This invention develops Trichoderma fungus Y4 into a bio-fertilizer, providing a new avenue for the application of microorganisms in crop production. Attached Figure Description

[0025] Figure 1 The images show the colony morphology of strain Y4 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.

[0026] Figure 2 Images show the hyphae and spore morphology of strain Y4 under a microscope; where A is a hyphae image and B is a spore image.

[0027] Figure 3 Images of solid-state fermented microbial fertilizer from strain Y4 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.

[0028] Figure 4 This is a phylogenetic tree diagram of the four genes TEF1-SSU-ITS-LSU in strain Y4. 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). The scale bar represents a single base substitution site.

[0029] Figure 5 The results of solid fermentation microbial fertilizer of strain Y4 promoting the growth of potted rice are shown in the figure; where A is the effect of strain Y4 on promoting rice growth, (i) is a picture of the potted plant, and (ii) is a picture of the whole rice plant; BG are statistical bar charts of chlorophyll (SPAD) content, leaf nitrogen content, stem and leaf height, root length, stem and leaf fresh weight and root fresh weight of rice in the control group and the treatment group, respectively (independent samples t test data, ** and **** represent the results of solid fermentation microbial fertilizer promoting the growth of potted rice). P <0.01 and P Significant difference at the 0.0001 level, ns indicates no significant difference.

[0030] Figure 6 The graph shows the results of the Y4 strain's inoculum promoting the growth of hydroponically grown rice. A represents the image of Y4 promoting rice growth, (i) is a hydroponic image, and (ii) is a whole rice plant image. B and C are bar charts representing the chlorophyll (SPAD) content, leaf nitrogen content, stem and leaf height, and stem and leaf fresh weight of the control and treatment groups, respectively (independent samples t-test analysis data, *** and **** represent...). P <0.001 and P (Significant difference at the 0.0001 level). Detailed Implementation

[0031] 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.

[0032] 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.

[0033] Example 1: Isolation and Identification of Microbial Strains

[0034] 1. Strain isolation

[0035] (1) Collect cultivated rice samples from Pinghu, Zhejiang, and rinse the roots of the cultivated rice with tap water to wash away the soil particles and attached substances on the surface.

[0036] (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.

[0037] (3) Cut the disinfected roots into 5 mm long segments;

[0038] (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.

[0039] (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.

[0040] 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.

[0041] A strain was isolated from the root system of common cultivated rice using the above method and named Y4.

[0042] 2. Identification of fungal morphology

[0043] Strain Y4 was inoculated into PDA medium and cultured in the dark at 25°C. The average mycelial growth rate was approximately 2.32-2.53 cm per day. It was then sequentially inoculated into PDB and barley media. The colony morphology and spore characteristics were classified as follows:

[0044] like Figure 1 As shown, strain Y4 grows rapidly on PDA medium, forming a large number of white aerial hyphae on the surface. The colonies are white on the front and yellow on the back.

[0045] like Figure 2 As shown, the mycelium is observed to have vegetative branches and septa under a microscope, and the conidia are round.

[0046] like Figure 3 As shown, strain Y4 produces yellow colonies after 3 days of growth on PDB medium, and turns whitish-brown after 15 days of fermentation on barley medium.

[0047] 3. Molecular identification and phylogenetic analysis

[0048] (1) DNA extraction

[0049] 1) After culturing strain Y4 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.

[0050] The extraction buffer formulation is: 10 mM ethylenediaminetetraacetic acid, 100 mM Tris-hydrochloric acid, and 1 M potassium chloride;

[0051] 2) Place the centrifuge tubes in an MP FastPrep®-24 homogenizer and shake at 65 Hz for 2 min to break the mycelial tissue;

[0052] 3) Centrifuge at 9000 rpm for 10 min, then pipette the supernatant into a new 1.5 mL centrifuge tube;

[0053] 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;

[0054] 5) Add 700 μL of 70% ethanol (v / v) to dissolve impurities, mix gently, and centrifuge at 12000 rpm for 1 min.

[0055] 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.

[0056] (2) PCR amplification of fungal TEF1, SSU rDNA, ITS and LSU rDNA genes

[0057] 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′;

[0058] The primers for sequencing SSU rDNA, ITS, and LSU rDNA are as follows: the upstream primer SSU-F sequence is 5′-GTAGTCATATGCTTGTCTC-3′, and the downstream primer LSU-R sequence is 5′-TCCTGAGGGAAACTTCG-3′.

[0059] 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.

[0060] 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.

[0061] (3) Gene sequencing and sequence analysis

[0062] 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 Y4.

[0063] On the NCBI website, the nucleotide sequences of TEF1 and SSU-ITS-LSU of strain Y4 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 PV157964.1. Trichoderma capillare The similarity of SLO1-1 was 99.54%, and it was similar to strain PV157961.1 (accession number). Trichoderma capillare The KC2-2 sequence showed a 99.54% similarity. The SSU-ITS-LSU three-gene sequence was similar to that of strain MH047196.1. Trichoderma reesei The similarity was 99.56%, and it was similar to the strain with accession number AF510497.1. Trichoderma reesei The similarity to QM6a was 99.56%. Through sequence comparison, strain Y4 belongs to... Trichoderma sp.

[0064] (4) Phylogenetic analysis

[0065] The TEF1 and SSU-ITS-LSU sequences of closely related genera of strain Y4 were searched on NCBI, and a phylogenetic tree of the four genes TEF1-SSU-ITS-LSU of strain Y4 and its closely related genera was constructed. The results showed that the four genes combined to form a single informational site, totaling 7369 bases, including 2676 parsimony informational sites, 6037 variable informational sites, and 742 conserved sites. Using jModelTest 2.1.10, TIM3+G was calculated as the optimal model for both the Bayesian Information Criterion (BIC) and the Akaike Information Criterion (AICc), and a phylogenetic tree based on maximum likelihood (ML) was established. Figure 4 The image shows an ML tree, and from the phylogenetic tree, strain Y4 belongs to... Trichoderma sp., but it is the same as T. capillare , T. reesei , T. hamatum They are not on the same branch, and strain Y4 and their colony morphology and hyphal spore morphology are also very different.

[0066] Based on comprehensive phylogenetic analysis, morphological and biological characteristics, strain Y4 is defined as a new species. Trichoderma pinghuensis sp. nov belongs to the kingdom Fungi, phylum Ascomycota, class Sordariomycetes, order Hypocreales, family Hypocreaceae, and genus Trichoderma.

[0067] Therefore, the strain was named Trichodermasp. Y4 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 2026215, and was confirmed to be viable on January 30, 2026.

[0068] Example 2: Preparation of solid-state fermentation fertilizer by strain Y4

[0069] Preparation of microbial fertilizer: The Y4 strain was cultured in PDA medium for 7 days, and the mycelial cake was collected and 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 per 200 g of barley grains) and fermented at 25℃ for 20 days.

[0070] 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.

[0071] PDB medium: 200 g potato, 5 g glucose, add distilled water to 1000 mL, pH at natural. Autoclave at 121℃ for 15 min.

[0072] Example 3: The promoting effect of Y4 strain solid fermented microbial fertilizer on the growth of potted rice.

[0073] 1. Rice seed germination: 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.

[0074] 2. Potted Planting: The fermented microbial fertilizer from Example 2 was air-dried naturally. The dried microbial fertilizer and rice planting substrate were mixed at a mass ratio of 1:10 and placed into planting pots. The germinated rice seeds were planted into the pots containing the fermented microbial fertilizer (30 seeds and 40 g of fertilizer per pot). Sterile barley seed fertilizer was used as a control group.

[0075] 3. 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.

[0076] 4. Results Analysis: For example... Figure 5As shown, in the pot experiment, the chlorophyll content of rice co-cultured with Y4 increased significantly by 39.68%, the nitrogen content of leaves increased significantly by 36.75%, the stem and leaf height increased significantly by 23.68%, the fresh weight of stems and leaves increased significantly by 28.27%, and the fresh weight of roots increased significantly by 7.00%.

[0077] Example 4: The promoting effect of Y4 bacterial solution on the growth of hydroponic rice

[0078] 1. Strain Y4 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.

[0079] 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 chlorophyll content, leaf nitrogen content, stem and leaf height, and stem and leaf fresh weight of the rice seedlings.

[0080] 3. Results Analysis: For example... Figure 6 As shown, in the hydroponic experiment, when the Y4 bacterial solution content in the culture medium was 30%, the chlorophyll content of rice leaves increased significantly by 58.07%, the nitrogen content of leaves increased significantly by 51.66%, the stem and leaf height increased by 14.10%, and the fresh weight of stems and leaves increased significantly by 44.44%.

[0081] 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 fungus of the genus Trichoderma ( Trichoderma sp.)Y4, characterized in that, The fungus Y4 of the Trichoderma genus is classified as follows: Trichoderma sp. Y4, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M2026215.

2. The Trichoderma fungus Y4 as described in claim 1, characterized in that, The culture conditions for the Trichoderma fungus Y4 were as follows: inoculated in PDA medium and cultured in the dark at 22-25°C.

3. The application of Trichoderma fungus Y4 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, leaf nitrogen content, stem and leaf height, root length, stem and leaf fresh weight, and root fresh weight.

5. The application as described in claim 3, characterized in that, The application includes inoculating the Trichoderma fungus Y4 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 Y4 into sterilized barley grains to obtain solid fermented microbial fertilizer, and then mixing it into rice planting substrate; or inoculating activated Trichoderma Y4 into PDB medium and culturing it to the exponential phase, and then mixing the bacterial solution into rice culture medium.

7. The application as described in claim 6, characterized in that, The solid fermented microbial fertilizer or microbial liquid is applied during rice seedling raising or cultivation.

8. The application as described in claim 6 or 7, characterized in that, The preparation method of the solid fermented microbial fertilizer includes: inoculating activated Trichoderma fungus Y4 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 solid fermented microbial fertilizer.

9. The application as described in claim 6, characterized in that, The activation method includes inoculating Trichoderma fungus Y4 into PDA medium and culturing it at 22-25°C for 7 days.

10. 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.

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