Trichoderma harzianum of africa with herbicidal ability and its application
Patent Information
- Application Number
- CN202611364333.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-09-04
- Publication Date
- 2026-10-13
AI Technical Summary
[0018]在本发明的实施例中，对四种杂草牛筋草、马唐、狗尾草和稗草的防治能力测试中病情指数结果分别为83.8，70.3，75.3和76.6，对田间常见的四种杂草均具有较强的防治能力。
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Figure CN122898368A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to a herbicide Trichoderma harzianum and its applications. Background Technology
[0002] Biological control is a method of controlling target diseases using biocontrol microorganisms, including biocontrol fungi and biocontrol bacteria. Compared to chemical control, biological control has the advantages of being environmentally friendly, safe, and sustainable in agriculture. Weeds seriously harm crop growth and cause significant economic losses. Currently, chemical control is the commonly used method for weed control in the field, but long-term use of chemical herbicides can cause environmental pollution and lead to herbicide resistance in weeds. As a green and sustainable control method, biological control plays an important role in the field of weed control.
[0003] Goosegrass (Eleusine indica (L.) Gaertn.), crabgrass (Digitaria sanguinalis (L.) Scop.), barnyard grass (Echinochloa crus-galli (L.) P. Beauv.), and foxtail grass (Setaria viridis (L.) P. Beauv.) are common field weeds, and there are reports of using biocontrol microorganisms to control them. Common fungi that control weeds mainly include those in the genera *Fusarium*, *Alternaria*, *Botrytis*, *Stemona*, *Anthracis*, and *Hydroceta*. Common bacteria that control weeds mainly include those in the genera *Pseudomonas*, *Xanthomonas*, and *Flavobacterium*.
[0004] Live herbicides are a relatively new subcategory of biopesticides, currently in the early stages of industrialization and promotion, with a market share far lower than that of chemical herbicides. Globally, more than ten fungal herbicides have been registered, with representative products including Devine (a palm fungus formulation) and Collego (a specific formulation of Colletotrichum gloeosporioides) from the United States. These products achieve over 90% control efficacy against specific target weeds, but their market penetration is slower than that of chemical herbicides, and their current overall market share is still less than 5%. Most marketed products are highly specific formulations, capable of controlling only one or a few target weeds, making it difficult to meet the comprehensive control needs of complex weed communities in farmland.
[0005] To address the industry pain points caused by the long-term and extensive use of chemical herbicides, such as weed resistance, pesticide residues, and damage to farmland ecosystems, and to ensure crop yield safety, reduce field control costs, and maintain the micro-ecological balance of farmland, there is an urgent need to develop microbial strains that can replace chemical herbicides for green weed control in agriculture. These strains have significant value in many aspects, including ecological security, industrial development, and scientific research innovation. Summary of the Invention
[0006] The purpose of this invention is to overcome the problems of herbicide resistance in weeds, pesticide residues, and ecological damage in existing technologies.
[0007] To address this, the present invention provides a herbicide-resistant Trichoderma afroharzianum strain C39, classified and named Trichoderma afroharzianum, which was deposited on July 29, 2026 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCCNo:68749.
[0008] In an embodiment of the present invention, colony morphology observation of C39 showed that strain C39 could produce a fluffy white mycelial layer in potato dextrose medium, and annular yellowish-green spore rings could be observed. Through genomic DNA extraction and ITS sequence PCR amplification of C39, the ITS sequence of strain C39 was searched using NCBI Blast (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), revealing that the ITS sequence of strain C39 had 100% similarity to *Trichoderma afroharzianum*.
[0009] The present invention also provides a culture of the aforementioned Trichoderma harzianum.
[0010] According to an embodiment of the present invention, the culture is prepared by inoculating the *Trichoderma harzianum* into potato dextrose medium and culturing it. Specifically, the *Trichoderma harzianum* is inoculated into potato dextrose medium and cultured at 23-33°C for 5-10 days; preferably, the *Trichoderma harzianum* is inoculated into potato dextrose medium and cultured at 28°C for 7 days.
[0011] The present invention also provides a composition comprising the aforementioned Trichoderma harzianum and / or the aforementioned culture.
[0012] On the other hand, the present invention also provides the use of the *Trichoderma harzianum*, the culture, or the composition, wherein the use includes at least one of the following:
[0013] (1) Application in weed control;
[0014] (2) Application in the preparation of weed control products.
[0015] Preferably, the weeds are selected from: goosegrass, crabgrass, foxtail grass, and barnyard grass.
[0016] Furthermore, the present invention provides a method for controlling weeds, wherein the method involves applying the *Trichoderma harzianum*, the culture, or the composition to the weed plants or soil. Preferably, the weeds are selected from: goosegrass, crabgrass, foxtail grass, and barnyard grass.
[0017] Specifically, the method involves preparing the spores of *Trichoderma harzianum* into a suspension, which is then sprayed onto the leaves of weeds, the surface of plants, or by drip irrigation, broadcasting, or mixing into the soil. According to an embodiment of the invention, the method collects the spores from the culture.
[0018] In the embodiments of the present invention, the disease index results of the control ability tests on four weeds—goosegrass, crabgrass, foxtail grass, and barnyard grass—were 83.8, 70.3, 75.3, and 76.6, respectively, indicating that the disease has a strong control ability against the four common weeds in the field.
[0019] The beneficial effects obtained by the present invention through the above technical solution include at least the following:
[0020] Previous reports have shown that *Trichoderma harzianum* inhibits the germination of *Eleusine indica* seeds, but its herbicidal activity against mature *Eleusine indica* plants has not been statistically analyzed. *Trichoderma viride*, a fungus belonging to the genus *Trichoderma*, has an inhibitory effect on the germination of *Setaria viridis* seeds. The *Trichoderma harzianum* C39 provided by this invention exhibits herbicidal activity against *Eleusine indica*, *Barnyardgrass*, *Digitaria sanguinalis*, and *Setaria viridis*, which has not been reported with other *Trichoderma* species. This provides a strain resource for developing biological control technologies for weeds, improves the effectiveness of environmental management and agricultural ecological environment applications, and has the advantages of being green, environmentally friendly, and sustainable in agriculture. Simultaneously, it improves the biological pesticide product system, enriches the variety of green pesticide products, and its potential application value also includes: the discovery of novel herbicidal lead compounds in the secondary metabolites of weed-controlling fungi, providing a new resource direction for the creation of new pesticides and contributing to the overall technological advancement of the agricultural microbial industry. Attached Figure Description
[0021] Figure 1 This is a photograph of the colony morphology of strain C39 after culturing at 28°C for 7 days on potato dextrose medium.
[0022] Figure 2 A phylogenetic tree of C39 strains was constructed to select different Trichoderma species with high ITS sequence similarity to strain C39.
[0023] Figure 3 Photos showing the weed control effect of C39 strain on goosegrass, crabgrass, foxtail grass and barnyard grass after 10 days. CK refers to the negative control, where no C39 strain was sprayed and only water was sprayed.
[0024] Strain preservation information:
[0025] The C39 strain of this invention was deposited on July 29, 2026, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC NO:68749 and classification name Trichoderma afroharzianum. Detailed Implementation
[0026] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.
[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0029] Unless otherwise specified, all reagents and materials used in the following examples were purchased from reputable chemical or biological reagent / material suppliers, and all reagents were of analytical grade.
[0030] The basic components of the culture medium used in the examples are as follows:
[0031] Potato glucose medium: 4g potato extract powder, 20g glucose, 15g agar, and distilled water to a final volume of 1L.
[0032] Example 1: Isolation of strain C39
[0033] Diseased *Eleusine indica* plants were collected, and affected areas were selected and disinfected before being placed on potato dextrose agar. The potato dextrose agar containing the diseased *Eleusine indica* parts was placed in a constant temperature incubator at 28°C. After 7 days of incubation, eight fungal strains with different colony morphologies, textures, and colors were selected from the agar. Colonies exhibiting a yellow-green upper surface and a yellow lower surface were selected and named C39. C39 colonies were transferred to potato dextrose agar and purified using streak plating. The purified plates were placed in a constant temperature incubator and incubated at 28°C. After single colonies grew on the plates, streak plating was repeated for purification. After three generations, pure C39 cultures were obtained. C39 strains were stored at 4°C for subsequent research and at -80°C for long-term preservation.
[0034] Example 2: Identification of strain C39
[0035] The taxonomic position of strain C39 was identified using morphological (colony morphology) and molecular biological (phylogenetic analysis) methods.
[0036] 1. Morphological identification
[0037] Potato glucose medium inoculated with strain C39 was placed in a 28°C incubator. After 7 days, the colony morphology of strain C39 was observed. Figure 1 As shown in the figure. Observation revealed that strain C39 can produce a fluffy white mycelial layer, and annular yellowish-green spore rings can be observed.
[0038] 2. Molecular biological identification
[0039] (1) Extraction of genomic DNA from strain C39
[0040] Potato glucose medium inoculated with strain C39 was placed in a 28°C incubator, and the bacterial cells were scraped off after 7 days. Genomic DNA of strain C39 was extracted using a fungal genomic DNA extraction kit.
[0041] (2) Amplification of ITS sequence of strain C39
[0042] PCR amplification of the ITS sequence of strain C39 was performed using universal ITS amplification primers with C39 genomic DNA as a template.
[0043] The 25µL PCR amplification system includes 12.5µL of DNA polymerase mixture (containing DNA polymerase, Mg...) 2+ The sample contained 4 types of dNTPs, PCR amplification buffer, etc., 1 µL each of universal amplification primers (upstream and downstream), 2 µL of LC39 genomic DNA, and 8.5 µL of ddH2O.
[0044] The PCR amplification program for the ITS sequence of strain C39 was as follows: pre-denaturation at 94℃ for 3 min, followed by 30 cycles of denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, and extension at 72℃ for 1 min. A final extension at 72℃ for 10 min was performed. The PCR amplification products were detected by agarose gel electrophoresis, and sequencing was performed after confirming the correct bands.
[0045] (3) Phylogenetic analysis
[0046] The ITS sequence of strain C39 was compared using the NCBI Blast search program (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The comparison results showed that the ITS sequence of strain C39 was 100% similar to that of *Trichoderma afroharzianum*. To further clarify the taxonomic position of strain C39, a phylogenetic tree of strain C39 was constructed for phylogenetic analysis. A phylogenetic tree was constructed using the ITS sequence of strain C39 and other *Trichoderma* species with high sequence similarity, such as... Figure 2 As shown. Phylogenetic analysis revealed that strain C39 is most closely related to *T. afroharzianum*.
[0047] Based on the morphological and molecular biological characteristics of strain C39, it was identified as *Trichoderma harzianum*. Strain C39 was deposited at the Guangdong Provincial Microbial Culture Collection Center, accession number: GDMCC No:68749.
[0048] Example 3: Control ability of strain C39 against four weeds
[0049] The herbicidal ability of strain C39 against four common field weeds—goosegrass, crabgrass, foxtail, and barnyard grass—was tested. Weed seeds were soaked in a 1000 mg / L gibberellin solution for 24 hours to break dormancy, then rinsed with water and evenly scattered in petri dishes lined with three layers of filter paper. Once the coleoptiles emerged, the seeds were transplanted into 8.5 cm × 8.5 cm square pots containing a mixture of seedling substrate and organic fertilizer (3:1). The square pots were placed in a greenhouse with a light intensity of L / D = 12 h / 12 h, a temperature of (28±5)℃, a relative humidity of (60±20)%, and a 200 μmol / L chlorine dioxide concentration. -2 s -1 Temperature in darkness: (20±4)℃, relative humidity: (50±20)%.
[0050] Potato glucose medium inoculated with strain C39 was placed in a 28°C incubator, and spores were collected after 7 days. The collected C39 spores were prepared into a spore suspension and sprayed on four weed species, with water spraying serving as a control. Both the control and treatment species were kept in a moist environment for 10 days, and the disease incidence was investigated and the disease index was calculated. The disease index was calculated using the formula: [Σ(number of diseased leaves × corresponding disease severity value) / (total number of investigated leaves × highest disease severity value)] × 100.
[0051] Weed disease is assessed using a 4-level grading system: Level 0: healthy plants with no visible lesions; Level 1: lesion area less than 25%; Level 2: lesion area 26-50%; Level 3: lesion area 51-75%; Level 4: lesion area exceeding 75% of leaf area.
[0052] Weed control ability tests revealed that, Figure 3 As shown, compared with the negative control CK (no C39 spore suspension), the disease severity of *Eleusine indica* was severe after spraying with *C39 spore suspension*. After spraying with C39, disease symptoms gradually appeared over time. When the disease severity was investigated at 10 days, level 4 disease was found to be the most common. The disease index of *Eleusine indica* after spraying with C39 was 83.8.
[0053] Compared with the negative control (CK) that was not sprayed with C39 strain spore suspension, 10 days after inoculation with C39 strain spore suspension, the disease indices of the three weeds—barnyard grass, foxtail grass, and barnyard grass—were slightly lower than those of goosegrass. The disease index for barnyard grass was 70.3, for foxtail grass it was 75.3, and for barnyard grass it was 76.6.
Claims
1. A type of Trichoderma afroharzianum, characterized in that, The accession number is: GDMCCNo:68749.
2. The culture of Trichoderma harzianum as described in claim 1.
3. The culture as described in claim 2, characterized in that, The culture was prepared by inoculating the Trichoderma harzianum in potato glucose medium and culturing it.
4. The culture as described in claim 3, characterized in that, The culture is prepared by inoculating the African Trichoderma harzianum into potato dextrose medium and culturing it at 23-33°C for 5-10 days.
5. A composition, characterized in that, The composition contains the African Trichoderma harzianum as described in claim 1 and / or the culture as described in any one of claims 2 to 4.
6. The use of *Trichoderma harzianum* as described in claim 1, the culture as described in any one of claims 2 to 4, or the composition as described in claim 5, characterized in that, The application includes at least one of the following: (1) Application in weed control; (2) Application in the preparation of weed control products; The weeds were selected from: goosegrass, crabgrass, foxtail grass, and barnyard grass.
7. A method for controlling weeds, characterized in that, Apply the Trichoderma harzianum of claim 1, the culture of any one of claims 2 to 4, or the composition of claim 5 to weed plants or soil; The weeds were selected from: goosegrass, crabgrass, foxtail grass, and barnyard grass.
8. The method as described in claim 7, characterized in that, The spores of Trichoderma harzianum as described in claim 1 are prepared into a suspension, and then sprayed on the leaves of weeds, on the surface of plants, or drip-irrigated, broadcast, or mixed into the soil.
9. The method as described in claim 8, characterized in that, The spores are collected from the culture according to any one of claims 2 to 4.