Trichoderma guasei and application thereof
By screening and applying the ferrite-producing Trichoderma Gams fermentation broth, the lack of biological control strains in corn stem rot prevention and control was solved, and significant disease control effects and corn seedling growth promotion were achieved.
Patent Information
- Application Number
- CN202411798273.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-09
- Publication Date
- 2025-03-14
AI Technical Summary
The prior art lacks effective biological control strains to prevent and treat corn stem rot, resulting in corn harvest and economic losses.
Through screening and identification, a ferrite-producing Trichoderma gamsii was found and used to cultivate in fermentation broth and used to soak corn seeds and roots to inhibit the dominant pathogen Fusarium gracilis, the corn stem rot.
Trichoderma Gams significantly inhibited the mycelial growth of Fusarium gracilis, prevented and treated the further expansion of corn stem rot lesions, with an effect of 51.45%, and promoted the growth of corn seedlings.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microorganisms, relates to the separation, identification and application of functional bacteria, and specifically relates to a strain of Trichoderma gemsii and application thereof. Background Art
[0002] Trichoderma is one of the biocontrol microorganisms with application potential in current green agricultural production. Studies have shown that when iron deficiency stress occurs, Trichoderma enhances the plant's absorption and utilization of iron elements involved in cell metabolism by secreting siderophores, thereby promoting plant growth; it can also inhibit the growth of plant rhizosphere pathogens by competing for limited iron elements in the environment.
[0003] Corn stalk rot is a corn disease caused by Fusarium, the dominant pathogen of which is Fusarium graminearum. When the disease is severe, it can cause corn crop failure and huge losses. The extensive use of traditional chemical pesticides and fertilizers has led to the development of drug resistance in plant pathogens. Biological control strains have become the first choice for plant disease and insect pest control in recent years. However, there is still a lack of biological control strains for the prevention and control of corn stalk rot. Therefore, it is of great significance to explore biological control strains for the prevention and control of corn stalk rot. Summary of the invention
[0004] The present invention aims to provide more alternative solutions for the prevention and treatment of corn stalk rot. To achieve this technical goal, the inventors of the present application screened out a siderophore-producing Trichoderma magamsii after a large number of experiments. The Trichoderma magamsii was isolated from the rhizosphere soil of corn plants in a corn field in Longnan City, Gansu Province. The deposit information is as follows:
[0005] Strain name: Trichoderma gemsii
[0006] Latin name: Trichoderma gamsii
[0007] Strain number: Tg-1
[0008] Depository: College of Plant Protection, Gansu Agricultural University
[0009] Address of the depository: No. 1 Yingmen Village, Anning District, Lanzhou City, Gansu Province
[0010] Deposit date: November 17, 2023
[0011] Furthermore, the nucleotide sequence of ITS rDNA of Trichoderma gamsii is shown in SEQ ID NO:1.
[0012] The method for preparing the fermentation liquid of Trichoderma geumens of the present invention is characterized by comprising the following steps: culturing Trichoderma geumens in a fermentation medium at 28° C. and 180 r / min for 7 days.
[0013] Furthermore, the fermentation medium is composed of the following ingredients: 200 g / L potato, 18.34 g / L glucose, 1.88 g / L yeast extract, 0.86 g / L potassium dihydrogen phosphate, 0.63 g / L ferrous sulfate, and 1000 mL distilled water, and is sealed and sterilized at 121° C. for 30 min.
[0014] The invention discloses an application of the Trichoderma gemsii strain in producing a product for preventing and treating corn stalk rot.
[0015] Furthermore, after surface disinfection of corn seeds, 200 mL of Trichoderma gemsii fermentation liquid was added and soaked for 4 hours, and then the treated corn seeds were sown in the farmland.
[0016] The invention discloses an application of the Trichoderma gemsii strain in producing a product for promoting the growth of corn plants.
[0017] Furthermore, the soil matrix used for the growth of corn seedlings was sterilized at 121° C. for 60 minutes and set aside; plump corn seeds were selected for surface disinfection, and then the corn seeds were planted in the sterilized soil matrix; finally, the roots were irrigated with the fermentation liquid of Trichoderma gemsii.
[0018] The invention discloses an application of the Trichoderma gemsii strain in producing a product for inhibiting the expansion of Fusarium graminearum, a dominant pathogen of corn stalk rot.
[0019] Compared with the prior art, the present invention "a strain of Trichoderma gemsii and its application" has the following beneficial effects:
[0020] The invention screens a fungus with the ability to produce siderophore from rhizosphere soil of healthy corn plants, and identifies the fungus as Trichoderma gamsii. The Trichoderma gamsii produces an orange halo on a CAS culture medium and has a good ability to produce siderophore.
[0021] The Trichoderma gamsii provided by the present invention has a growth-promoting effect on corn seedlings. After the corn seedlings are treated by root irrigation with a fermentation solution containing Trichoderma gamsii, the plant height and root length of the corn seedlings in the treatment group are better than those in the control group, the overall growth is better, and the growth-promoting effect is obvious.
[0022] The Trichoderma gamsii provided by the invention has an inhibitory effect on the hyphae growth of Fusarium graminearum, the dominant pathogen of corn stalk rot. When Trichoderma gamsii and Fusarium graminearum are cultured face to face on a culture medium, compared with a culture medium inoculated with only Fusarium graminearum, Trichoderma gamsii can significantly inhibit the hyphae growth of Fusarium graminearum, and has an antagonistic effect on Fusarium graminearum, the dominant pathogen of corn stalk rot.
[0023] The Trichoderma geumii provided by the present invention can prevent and control corn stalk rot caused by Fusarium. Using the Trichoderma geumii fermentation liquid provided by the present invention to soak corn seeds in the field can inhibit the further expansion of corn stalk rot lesions in corn stem internodes, and the prevention and control effect is 51.45%, which is significant.
[0024] In summary, the Trichoderma gamsii provided by the present invention has good siderophore production ability and can promote the growth of corn seedlings; on the other hand, the Trichoderma gamsii can also inhibit the mycelial growth of corn stalk rot pathogens and the further expansion of corn stalk rot lesions, and shows good control effect in preventing and controlling corn stalk rot, which is of great significance for the safe prevention and control of corn stalk rot. At the same time, it is expected to play a role in agricultural production as a safe corn growth promoter. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 This is a picture of the colony growth morphology of Trichoderma gamsii on CAS medium.
[0026] Figure 2 This is a phylogenetic tree of Trichoderma gamsii.
[0027] Figure 3 The growth of corn seedlings in the treatment group and the control group were shown in Figure 2. The treatment group was irrigated with Trichoderma gamsii fermentation liquid; the control group was irrigated with sterile water.
[0028] Figure 4 These are the results of a plate confrontation between Trichoderma gamsii and Fusarium graminearum, the dominant pathogen of corn stalk rot. Figure 4Figure a shows the growth of the colony of Fusarium graminearum inoculated alone in the culture medium; Figure 4 Figure b in the middle shows the colony growth of Trichoderma gamsii and Fusarium graminearum in confrontation culture.
[0029] Figure 5 Shown are the disease conditions of corn stem tissue in the treatment group and the control group. Figure 5 In the figure, "treatment group" means corn seeds treated with Trichoderma geum fermentation liquid, in which corn seeds were soaked with Trichoderma geum fermentation liquid and then inoculated with Fusarium graminearum; "CK" means control group, in which corn seeds were soaked with sterile water and then inoculated with Fusarium graminearum. DETAILED DESCRIPTION
[0030] The technical solution of the present invention is clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0031] Example 1
[0032] This example provides the isolation of Trichoderma gamsii and the identification of its ability to produce siderophores.
[0033] Strain source: The Trichoderma gamsii was isolated from the rhizosphere soil of corn plants.
[0034] PDA culture medium: 200 g / L potato, 20 g / L glucose, 20 g / L agar powder, 1000 mL distilled water, seal and autoclave at 121°C for 30 min, set aside.
[0035] Isolation and purification of strains: Take the rhizosphere soil of maize plants with good growth and no pests and diseases in the maize field (Longnan City, Gansu Province (104°01′-106°35′E, 32°35′-34°32′N)) and bring it back to the laboratory. Take 10 g of soil sample and put it into a triangular flask containing 100 mL of sterile water. Oscillate at 180 r / min and 28°C for 30 min, then let it stand for 10 min. Take 1 mL of the middle layer soil suspension and spread it with sterile water at a concentration of 10. -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7Dilution was performed, and 10 μL of the dilution solution of each concentration was respectively taken and evenly spread on the PDA plate prepared above, and cultured in the dark at 28° C. for 3 days. Single spores were picked for purification culture to obtain purified strains and stored with numbers.
[0036] Strain screening: After activating the above-purified strains, they were inoculated on blue siderophore-producing identification medium (CAS medium) respectively. After culturing at 28°C for 72 hours, the growth of colonies in different culture media was observed. If the strain can grow on CAS medium and an orange halo can be observed around the colony, it means that the strain can secrete siderophores. The larger the orange halo, the stronger the ability of the strain to produce siderophores. The strain with the largest orange halo was selected and numbered as Tg-1. The growth of the Tg-1 strain on CAS medium is shown in the figure. Figure 1 shown. Figure 1 It can be seen that an obvious orange halo appears around the colony of the Tg-1 strain, indicating that the Tg-1 strain has a good ability to produce siderophore.
[0037] Example 2
[0038] This example provides the taxonomic identification of Trichoderma gamsii.
[0039] PD liquid culture medium: 200 g / L potato, 20 g / L glucose, 1000 mL distilled water, seal and autoclave at 121°C for 30 min, set aside.
[0040] The strain was cultured in PD liquid medium at 28°C and 180 r / min for 7 days, and then DNA was extracted for ITSrDNA sequencing. The sequencing results were compared by BLAST on the NCBI website to construct the following: Figure 2 The phylogenetic tree is shown, and the sequence information of the Tg-1 strain is shown in SEQ ID NO: 1. Homology analysis revealed that the Tg-1 strain is Trichoderma gamsii of the genus Trichoderma.
[0041] The Trichoderma gamsii was preserved in the College of Plant Protection, Gansu Agricultural University on November 17, 2023. The strain number is Tg-1, and the classification name is Trichoderma gamsii. The preservation address is: Gansu Agricultural University, No. 1, Yingmen Village, Anning District, Lanzhou City, Gansu Province.
[0042] Example 3
[0043] This example provides a test for the ability of Trichoderma gamsii to promote the growth of corn seedlings.
[0044] To ensure the reliability of the results, three repeated experiments were set up. Each experiment set up a treatment group and a control group: the treatment group was irrigated with the fermentation liquid of Trichoderma gamsii; the control group was only irrigated with sterile water.
[0045] Fermentation medium: 200 g / L potato, 18.34 g / L glucose, 1.88 g / L yeast extract, 0.86 g / L dipotassium hydrogen phosphate, 0.63 g / L ferrous sulfate, 1000 mL distilled water, seal and autoclave at 121°C for 30 min, set aside.
[0046] Preparation of fermentation broth: Culture Trichoderma gemsii in fermentation medium at 28°C and 180 r / min for 7 days and set aside.
[0047] Operation steps: Sterilize the soil matrix for corn seedling growth at 121℃ for 60min and set aside. Select full corn seeds for surface disinfection, and then plant the corn seeds in the sterilized soil matrix, 7 seeds in each treatment group and control group; then irrigate the roots of the treatment group with 20mL Trichoderma gamsii fermentation liquid, and irrigate the roots of the control group with 20mL sterile water. After 30 days, measure the plant height and root length with a vernier caliper.
[0048] Experimental results: The growth data of the treatment group and the control group are shown in Table 1.
[0049] Table 1 Plant growth data of treatment group and control group
[0050]
[0051] Comparing the growth data of corn seedlings in the treatment group and the control group, it can be seen that the root length and plant height data of the treatment group are significantly greater than those of the control group; in addition, the growth of corn in the treatment group and the control group ( Figure 3 ) It can also be seen that the corn seedlings in the treatment group grew better overall, which indicates that Trichoderma gamsii has a significant growth-promoting effect on corn plants.
[0052] Example 4
[0053] This example provides a plate confrontation test of Trichoderma gamsii and Fusarium graminearum.
[0054] PDA culture medium: Weigh 200 g of peeled potatoes, cut into small pieces, boil in distilled water until they can be broken apart, filter through three layers of gauze, add 20 g of glucose and 18 g of agar, stir evenly, and make up to 1000 mL with distilled water. Seal the container and sterilize under high pressure at 121°C for 30 min for later use.
[0055] The test pathogen: Fusarium graminearum, isolated and preserved by the College of Plant Protection, Gansu Agricultural University.
[0056] Experimental method: Using the plate confrontation method, Fusarium graminearum and Trichoderma gamsii with a diameter of 10 mm, cultured for 7 days and of similar mass were inoculated at two points 6 cm apart in the PDA plate culture medium; in the control group, Fusarium graminearum of the same size was inoculated in the center of the plate to observe the growth of the strains.
[0057] Test results: Figure 4 Figure a shows the colony growth of Fusarium graminearum inoculated alone in PDA medium; Figure 4 Figure b in the middle shows the colony growth of Fusarium graminearum and Trichoderma gamsii in confrontation culture. Figure 4 The results showed that Trichodermagamsii had the ability to significantly inhibit the growth of Fusarium graminearum colonies, indicating that it had an antagonistic effect on Fusarium graminearum.
[0058] Example 5
[0059] This example provides the results of a field trial of Trichoderma gamsii.
[0060] PDA culture medium: Weigh 200 g of peeled potatoes, cut into small pieces, boil in distilled water until they can be broken apart, filter through three layers of gauze, add 20 g of glucose and 18 g of agar, stir evenly, and make up to 1000 mL with distilled water. Seal the container and sterilize under high pressure at 121°C for 30 min for later use.
[0061] Corn kernel culture medium: After soaking corn kernels in water for 24 hours, boil them for 45 minutes, put them into a high-temperature resistant plastic bag, tie them tightly with cotton, and sterilize them at 121℃ for 30 minutes for use.
[0062] Fermentation medium: 200.00 g / L potato, 18.34 g / L glucose, 1.88 g / L yeast extract, 0.86 g / L potassium dihydrogen phosphate, 0.63 g / L ferrous sulfate, 1000 mL distilled water, seal and autoclave at 121°C for 30 min, set aside.
[0063] Inoculum preparation: The Fusarium graminearum cultured on PDA medium for 5-7 days was inoculated into sterilized corn kernel culture medium and cultured in a constant temperature box at 25°C. After two weeks, when the mycelium covered the corn kernels, it was used for inoculation test.
[0064] Preparation of fermentation broth: Culture Trichoderma gemsii in fermentation medium at 28°C and 180 r / min for 7 days and set aside.
[0065] Field test: The test field was set up at the experimental station of Gansu Agricultural University, with random block distribution, 3 repetitions, 2m row length, 10 plants per row, 6 rows for each treatment, and protective rows around. After surface disinfection of corn seeds, 200mL Trichoderma gamsii fermentation liquid was added and soaked for 4h. The corn seeds of the control group were soaked in sterile water for the same time. Then the treated corn seeds were sown in the test field, and 20g corn kernel culture medium inoculated with Fusarium graminearum was buried at a distance of 5cm from the corn seeds. Water and fertilizer were routinely managed during the entire growth period. In the late stage of corn milk maturity, the growth of the plant was observed, and the base of the stem was pressed by hand to check the hardness of the stem. The stem was longitudinally cut to observe the color and infection degree of the pith, and then the incidence and disease index of corn stalk rot were calculated according to the survey standard of corn stalk rot in the adult stage. The test results are shown in Table 2.
[0066] Table 2 Field control effect of corn stalk rot
[0067]
[0068] Note:
[0069]
[0070] As shown in Table 2, the Trichoderma gamsii can reduce the occurrence of corn stalk rot. In addition, the expansion of corn stalk rot lesions in the treatment group and the control group ( Figure 5 ) It can also be seen that Trichoderma gamsii can inhibit the further expansion of corn stalk rot lesions in the internodes of corn stems. This shows that Trichoderma gamsii has a preventive and therapeutic effect on corn stalk rot.
[0071] The embodiments described above are only some embodiments of the present invention, not all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but only represents selected embodiments of the present invention. Any other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.
Claims
1. A strain of Trichoderma gemsii, characterized in that Strain name: Trichoderma gemsii; Latin name: Trichoderma gamsii; Strain number: Tg-1; Depository: College of Plant Protection, Gansu Agricultural University; Address of the depository: No. 1, Yingmen Village, Anning District, Lanzhou City, Gansu Province; Date of preservation: November 17, 2023.
2. The Trichoderma gemsii strain according to claim 1, characterized in that The nucleotide sequence of ITSrDNA of the Trichoderma gemsii is shown in SEQ ID NO:
1.
3. The method for preparing the fermentation liquid of Trichoderma geimus according to claim 1, characterized in that: The following steps are involved: The Trichoderma gemsii was cultured in a fermentation medium at 28°C and 180 r / min for 7 days.
4. The method for preparing the fermentation liquid of Trichoderma geimus according to claim 3, wherein The fermentation medium is composed of the following ingredients: 200 g / L potato, 18.34 g / L glucose, 1.88 g / L yeast extract, 0.86 g / L dipotassium hydrogen phosphate, 0.63 g / L ferrous sulfate, and 1000 mL distilled water, and is sealed and sterilized at 121° C. for 30 min.
5. Use of the Trichoderma geimus strain according to any one of claims 1 to 2 in producing a product for preventing and controlling corn stalk rot.
6. The use according to claim 5, characterized in that: After surface disinfection of corn seeds, 200 mL of Trichoderma gemsii fermentation liquid was added and soaked for 4 hours, and then the treated corn seeds were sown in the farmland.
7. Use of the Trichoderma gemsii strain according to any one of claims 1 to 2 in the production of a product for promoting the growth of corn plants.
8. The use according to claim 7, characterized in that: The soil matrix used for the growth of corn seedlings was sterilized at 121°C for 60 minutes and set aside; plump corn seeds were selected for surface disinfection, and then the corn seeds were planted in the sterilized soil matrix; finally, the roots were irrigated with the fermentation liquid of Trichoderma gemsii.
9. Use of the Trichoderma gemsii strain according to any one of claims 1 to 2 in producing a product for inhibiting the expansion of Fusarium graminearum, the dominant pathogen of corn stalk rot.