CN114431255B - Application of trichoderma asperellum-like and plant growth promoting microbial agent - Google Patents
Application of trichoderma asperellum-like and plant growth promoting microbial agent Download PDFInfo
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- CN114431255B CN114431255B CN202111267622.7A CN202111267622A CN114431255B CN 114431255 B CN114431255 B CN 114431255B CN 202111267622 A CN202111267622 A CN 202111267622A CN 114431255 B CN114431255 B CN 114431255B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
- A01N63/38—Trichoderma
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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Abstract
The invention relates to the technical field of microorganisms, in particular to application of trichoderma asperellum and a plant growth promoting microbial agent. Application of trichoderma asperellum in promoting plant growth; and the strain is utilized to prepare the microbial agent. The invention provides the preparation of the trichoderma asperellum-like microbial agent and the application of the trichoderma asperellum-like microbial agent in promoting plant growth.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to application of trichoderma asperellum and a plant growth promoting microbial agent.
Background
Trichoderma (Trichoderma spp.) is a filamentous fungus that is widely found in nature, and was first reported and described by Peroson at the end of the 18 th century. Trichoderma is belonging to the Ascomycota phylum, ascomycota, the Phlebomycetes (Sordariomycetes), the Hypocrea order, the Hypocreaceae family (Hypofaceeae), more than 250 Trichoderma species have been reported. The trichoderma has important economic value, is widely applied to various aspects of agriculture, and particularly has very broad application prospect in the aspects of biological control antagonism and plant growth promoting bacteria.
Wheat is an important source of human grains, is one of main grain crops next to rice, and has great influence on human life. Wheat is also the main ration crop in China, and in 2019, the sowing area and the total yield of wheat in China respectively account for 20.4% and 20.1% of the total sowing area and the total yield of grain crops. Therefore, the method for preventing and controlling the wheat diseases and insect pests and promoting the wheat yield and income increase has very important significance for guaranteeing the national economy development.
Disclosure of Invention
The invention aims to provide an application of trichoderma asperellum-like and a plant growth promoting microbial agent.
In order to achieve the above purpose, the invention adopts the technical scheme that:
application of trichoderma asperellum, application of trichoderma asperellum in promoting plant growth; wherein, the trichoderma asperellum is with a preservation number of CGMCC No.19914, a preservation date of 2020, 6 and 9 days, a preservation unit of China general microbiological culture Collection center, and an address: the classification of North Chen Xili No.1, 3 in the Chaoyang area of Beijing is named Trichoderma asperellum (Trichoderma asperelloides).
A plant growth-promoting microbial agent contains the trichoderma asperellum.
The number of viable bacteria of the trichoderma asperellum in the microbial inoculum is 10 8 -10 10 CFU/g。
The microbial inoculum is obtained by adding activated trichoderma asperellum into a solid culture medium for fermentation culture.
The fermentation culture conditions are 5-40deg.C, preferably 20-30deg.C, for 3-40 days, preferably 5-15 days.
The solid culture medium is agricultural waste and/or agricultural product processing byproducts; or a solid medium containing agricultural waste and/or agricultural product processing byproducts.
When the solid culture medium is agricultural waste and/or agricultural product processing byproducts, mixing the agricultural waste and/or agricultural product processing byproducts with water to prepare the culture medium to be completely wet;
when the solid culture medium is a solid culture medium containing agricultural wastes and/or agricultural product processing byproducts, the agricultural wastes and/or agricultural product processing byproducts are mixed with water to prepare the culture medium which is completely wet, and then nutrient substances such as glucose, sucrose, peptone, yeast powder and the like can be added into the culture medium.
The agricultural waste is one or more of chaff, crop straw (such as crop straw of corn, soybean, wheat and the like), peanut shell, corn husk and the like; the agricultural product processing by-product is one or more of wheat bran, bean dreg, rice bran, pericarp, fruit dreg, vegetable dreg, cake dreg and the like.
The solid culture medium is prepared by adding water to one or more of the agricultural wastes and/or agricultural product processing byproducts, and the compounding proportion is correspondingly adjusted according to specific requirements, wherein one or more of the agricultural wastes and/or agricultural product processing byproducts can be mixed according to any proportion, only the energy required by the growth of microorganisms is provided, and the amount of other elements in the compounding can be added according to the requirements; the components of the whole culture medium are aimed at providing carbon source, nitrogen source and trace elements for the growth of microorganism.
The microbial inoculum can be powder, suspension or granule.
Use of a microbial agent for promoting plant growth.
The plant is wheat.
The invention has the advantages that:
trichoderma asperellum belongs to trichoderma, is a beneficial bacterial strain, can colonize on the root surface of a plant, and promotes the increase of plant root number; the culture medium for preparing the microbial inoculum is agricultural waste or agricultural product processing byproducts, the material is low in price and easy to obtain, the problem of processing the waste/byproducts is solved, the microbial inoculum is very environment-friendly, and the development of green and environment-friendly agriculture is facilitated.
The specific embodiment is as follows:
the following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
The strain adopted by the invention is trichoderma which is separated from marine pinecone, and the strain is identified as trichoderma asperellum (Trichoderma asperelloides). The strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation date of 2020, 6 and 9 days and a preservation number of CGMCC No.19914.
The microbial agent is obtained by culturing and culturing the trichoderma reesei, the amount of thalli contained in the microbial agent can be changed within a large range, for example, the number of viable bacteria of trichoderma asperellum with the preservation number of CGMCC No.19914 is 10 in each gram of the microbial agent 8 -10 10 CFU; preferably, the number of viable bacteria of trichoderma asperellum with the preservation number of CGMCC No.19914 is 10 in each gram of microbial agent 9 -10 10 CFU. During the culturing, the concentration of viable bacteria can be obtained by conventional methods, such as a blood cell count method or an OD value observation method.
The culture medium can be various culture mediums capable of being used for culturing trichoderma asperellum, for example, the culture medium is one or more of agricultural wastes such as chaff, crop straws (such as crop straws of corn, soybean, wheat and the like), peanut shells, corn husks and the like; the agricultural product processing by-product is one or more mixed solid culture mediums of wheat bran, bean dregs, rice bran, pericarp, fruit dregs, vegetable dregs, cake dregs and the like.
Wherein, the above culture mediums can be sterilized according to conventional sterilization method, for example, at 115-125deg.C under 1.5-2 standard atmospheric pressure for 10-30 min.
Further, the preparation method of the microbial agent can comprise the following steps: the trichoderma asperellum is inoculated on potato dextrose agar solid medium (PDA), cultured for 5-7 days in a 25-28 ℃ incubator, and then the bacteria-containing potato dextrose agar solid medium (PDA) is cut into small pieces and added into a conical flask filled with the solid medium. After large-scale fermentation culture for 3-40 days at 5-40 ℃, taking out the culture medium with trichoderma asperellum, airing, and grinding into powder by a grinding machine to prepare microbial powder.
The trichoderma asperellum or the microbial agent is applied to promoting plant growth.
Optionally, the plant is wheat.
Further, the trichoderma asperellum or the microbial agent containing the trichoderma asperellum is diluted by 200 to 25000 times by sterile water and used for seed soaking for 1 to 12 hours and then is sown.
The present invention will be described in further detail with reference to the following examples, but the scope of the present invention is not limited to the following examples.
Example 1
Trichoderma asperellum was inoculated into potato dextrose agar solid medium (PDA), cultured in an incubator at 25℃for 5 days, and then the aforementioned bacteria-containing potato dextrose agar solid medium (PDA) was cut into small pieces and added to a conical flask containing the solid medium. After large-scale fermentation culture at 25 ℃ for 6 days, taking out the bacteria with the solid culture medium, airing, and grinding into powder by a powder grinding machine to prepare microbial powder. The viable count of Trichoderma asperellum in the obtained microbial powder is 5×10 9 CFU/g, the microbial powder is the microbial agent of the embodiment.
The solid culture medium is a mixture of chaff and wheat bran according to a mass ratio of 2:1.
Example 2
The trichoderma asperellum-like agent is used for examining the effect of promoting the growth of wheat.
In this example, wheat of the variety Jimai 22 was used as an experimental crop. Selecting 600 wheat seeds with full and complete grains, soaking the wheat seeds in 75% alcohol for 1 min for seed disinfection, washing the wheat seeds with sterile water for 5-6 times, and scalding the wheat seeds with sterile water at 50-60 ℃ for 10 min. 0.1g of trichoderma asperellum-like agent in the above example 1 was weighed, 100mL of sterile water was added to dilute 1000 times to obtain a microbial inoculum dilution, wheat seeds were soaked in the prepared microbial inoculum dilution as an experimental group, and wheat seeds were soaked in an equal amount of sterile water as a control group for 1 hour. Glass petri dishes (1500 mm in diameter) were placed on an ultra-clean bench, 2 pieces of filter paper which had been sterilized and sufficiently moistened in advance were placed in each petri dish, and seeds were placed on the filter paper with a single layer with the front side facing upward with forceps, and 100 seeds were placed in each petri dish. Three groups of experimental and control groups were repeated. The petri dish was covered and placed in a room with the temperature maintained at 26 ℃. 10 wheat seedlings were randomly extracted from each of the experimental group and the control group by the day 7 of cultivation, the root number, root length and stem length of the wheat seedlings were recorded, and the average value thereof was recorded (see table 1).
Table 1 Trichoderma asperellum preparation diluted 1000 times for 1h seed soaking, number of seedlings, root length and stem length of wheat seeds on seventh day
Example 3
In this example, wheat of the variety Jimai 22 was used as an experimental crop. Selecting 600 wheat seeds with full and complete grains, soaking the wheat seeds in 75% alcohol for 1 min for seed disinfection, washing the wheat seeds with sterile water for 5-6 times, and scalding the wheat seeds with sterile water at 50-60 ℃ for 10 min. 0.02g of trichoderma asperellum-like agent in the above example 1 was weighed, 100mL of sterile water was added to dilute 5000 times to obtain a microbial agent diluent, wheat seeds were soaked in the prepared microbial agent diluent as an experimental group, and wheat seeds were soaked in an equal amount of sterile water as a control group for 3 hours. Glass petri dishes (1500 mm in diameter) were placed on an ultra-clean bench, 2 pieces of filter paper which had been sterilized and sufficiently moistened in advance were placed in each petri dish, and seeds were placed on the filter paper with a single layer with the front side facing upward with forceps, and 100 seeds were placed in each petri dish. Three groups of experimental and control groups were repeated. The petri dish was covered and placed in a room with the temperature maintained at 26 ℃. 10 wheat seedlings were randomly extracted from each of the experimental group and the control group by the day 7 of cultivation, the root number, root length and stem length of the wheat seedlings were recorded, and the average value thereof was recorded (see table 2).
Table 2. Acidocella-like trichoderma asperellum agent diluted 5000 times for 3 hours of seed soaking, the number of seedlings, root length and stem length at the seventh day of wheat seed germination
Example 4
In this example, wheat of the variety Jimai 22 was used as an experimental crop. Selecting 600 wheat seeds with full and complete grains, soaking the wheat seeds in 75% alcohol for 1 min for seed disinfection, washing the wheat seeds with sterile water for 5-6 times, and scalding the wheat seeds with sterile water at 50-60 ℃ for 10 min. 0.1g of trichoderma asperellum-like agent in the above example 1 was weighed, 100mL of sterile salt (sodium chloride) water with a concentration of 3%o was added for dilution 1000 times to obtain a microbial agent diluent, wheat seeds were soaked in the prepared microbial agent diluent as an experimental group, and 3%o of sterile salt water was used for soaking the wheat seeds as a control group for 1 hour. Glass petri dishes (1500 mm in diameter) were placed on an ultra-clean bench, 2 pieces of filter paper which had been sterilized and sufficiently moistened in advance were placed in each petri dish, and seeds were placed on the filter paper with a single layer with the front side facing upward with forceps, and 100 seeds were placed in each petri dish. Three groups of experimental and control groups were repeated. The petri dish was covered and placed in a room with the temperature maintained at 26 ℃. 10 wheat seedlings were randomly extracted from each of the experimental group and the control group by the day 7 of cultivation, the root number, root length and stem length of the wheat seedlings were recorded, and the average value thereof was recorded (see table 3).
Table 3 Trichoderma asperellum-like agent diluted 1000 times 3%
Example 5
In this example, wheat of the variety Jimai 22 was used as an experimental crop. Selecting 600 wheat seeds with full and complete grains, soaking the wheat seeds in 75% alcohol for 1 min for seed disinfection, washing the wheat seeds with sterile water for 5-6 times, and scalding the wheat seeds with sterile water at 50-60 ℃ for 10 min. 0.02g of trichoderma asperellum-like agent in the above example 1 was weighed, 100mL of 10%o sterile saline (sodium chloride) was added for dilution 5000 times to obtain a microbial agent diluent, wheat seeds were soaked in the prepared microbial agent diluent as an experimental group, 10%o sterile saline equivalent was used for soaking the wheat seeds as a control group, and the soaking time was 3 hours. Glass petri dishes (1500 mm in diameter) were placed on an ultra-clean bench, 2 pieces of filter paper which had been sterilized and sufficiently moistened in advance were placed in each petri dish, and seeds were placed on the filter paper with a single layer with the front side facing upward with forceps, and 100 seeds were placed in each petri dish. Three groups of experimental and control groups were repeated. The petri dish was covered and placed in a room with the temperature maintained at 26 ℃. 10 wheat seedlings were randomly extracted from each of the experimental group and the control group by the day 7 of cultivation, the root number, root length and stem length of the wheat seedlings were recorded, and the average value thereof was recorded (see table 4).
Table 4. Trichoderma asperellum-like agent diluted 5000 times 10%
As is clear from the data in tables 1 to 4, in both fresh water and brine, the microbial agent containing Trichoderma asperellum with the preservation number of CGMCC No.19914 in example 1 has the effect of promoting the growth of wheat, and the microbial agent containing Trichoderma asperellum with the preservation number of CGMCC No.19914 in example 1 has the effect of promoting the rooting of wheat, so that the root number of wheat is increased, compared with the control group.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner without contradiction. The various possible combinations of the invention are not described in detail in order to avoid unnecessary repetition.
Furthermore, any combination of the various embodiments of the present invention is possible, as long as it does not depart from the gist of the present invention, which is also regarded as the content of the present invention.
Claims (9)
1. An application of trichoderma asperellum, which is characterized in that: application of trichoderma asperellum in promoting rooting of wheat; wherein, the trichoderma asperellum is with a preservation number of CGMCC No.19914, a preservation date of 2020, 6 and 9 days, a preservation unit of China general microbiological culture Collection center, and an address: the classification name of the trichoderma asperellum is Trichoderma asperellum (Trichoderma asperellum) of North Chen Xili No.1, 3 in the Chaoyang area of Beijing cityTrichoderma asperelloides)。
2. The application of a plant growth promoting microbial agent in promoting wheat rooting is characterized in that: the microbial inoculum comprises trichoderma asperellum-like according to claim 1.
3. Use according to claim 2, characterized in that: the number of viable bacteria of the trichoderma asperellum in the microbial inoculum is 10 8 -10 10 CFU/g。
4. Use according to claim 2 or 3, characterized in that: the microbial inoculum is obtained by adding activated trichoderma asperellum into a solid culture medium for fermentation culture.
5. The use according to claim 4, wherein: the fermentation culture condition is 5-40 ℃, and the culture is carried out for 3-40 days.
6. The use according to claim 4, wherein: the solid culture medium is agricultural waste and/or agricultural product processing byproducts.
7. The use according to claim 4, wherein: the solid medium contains agricultural waste and/or agricultural product processing byproducts.
8. Use according to claim 6 or 7, characterized in that: the agricultural waste is one or more of crop straw, peanut shell or corn husk; the agricultural product processing by-product is one or more of wheat bran, bean dreg, rice bran, pericarp, fruit dreg, vegetable dreg or cake dreg.
9. Use according to claim 2, characterized in that: the microbial inoculum is powder, suspension or granule.
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| US8716001B2 (en) * | 2009-02-06 | 2014-05-06 | Cornell University | Trichoderma strains that induce resistance to plant diseases and/or increase plant growth |
| EP2478090B1 (en) * | 2009-09-17 | 2016-09-07 | University Of Saskatchewan | Method for increasing plant growth using the fungus trichoderma harzianum |
| RU2607785C2 (en) * | 2015-06-03 | 2017-01-10 | Филоненко Владимир Александрович | Biological product for stimulating the growth of plants and their protection against phytopathogens on the basis of strains of trichoderma, strains of trichoderma for the production thereof (variants), method for producing a biological product on the basis of such strains |
| RU2634415C1 (en) * | 2016-08-02 | 2017-10-26 | Общество с ограниченной ответственностью "Органик парк" | Fungus strain trichoderma asperellum for obtaining biological preparation of complex action for crop growing |
| CN108575994B (en) * | 2018-03-13 | 2020-09-15 | 上海交通大学 | Preparation and application of growth-promoting trichoderma and bacillus composite wettable powder |
| CN111394261B (en) * | 2020-05-07 | 2021-11-09 | 南京农业大学 | Saline-alkali-tolerant trichoderma asperellum and application thereof |
| CN112625923B (en) * | 2021-01-18 | 2022-05-31 | 中国科学院烟台海岸带研究所 | Trichoderma asperellum and application thereof |
| CN112746024A (en) * | 2021-01-19 | 2021-05-04 | 南京农业大学 | Trichoderma composite microbial agent and bio-organic fertilizer prepared from same |
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