CN109161486B - Preparation method of biocontrol trichoderma strain inoculant and application of biocontrol trichoderma strain inoculant in tobacco planting - Google Patents

Preparation method of biocontrol trichoderma strain inoculant and application of biocontrol trichoderma strain inoculant in tobacco planting Download PDF

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CN109161486B
CN109161486B CN201811129770.0A CN201811129770A CN109161486B CN 109161486 B CN109161486 B CN 109161486B CN 201811129770 A CN201811129770 A CN 201811129770A CN 109161486 B CN109161486 B CN 109161486B
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trichoderma
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葛永怡
杨正军
代园凤
陈雪
向婷
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Guizhou University
Guizhou Tobacco Co Ltd Bijie Branch
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract

The invention discloses a preparation method of a biocontrol trichoderma fungicide and application of the biocontrol trichoderma fungicide in tobacco planting. The preparation method of the microbial inoculum comprises the steps of inoculating trichoderma conidium suspension into a solid fermentation culture medium for culture, and collecting conidia seven days later. The collected trichoderma conidia are prepared into a microbial inoculum which is in the form of bacterial powder. When the microbial inoculum is applied during tobacco seedling raising and transplanting, the microbial inoculum has stable bacterium content and good planting effect in the tobacco seedling raising period and the field period. The microbial inoculum of the invention can comprehensively utilize the antibiotic and competition among microbial species to inhibit the black shank and the brown spot of the tobacco. Trichoderma is used as biological pesticide to inhibit plant pathogenic bacteria, and can protect plants, so as to improve the inhibition effect on pathogenic bacteria and solve the problems of chemical pesticide residue and ecological environment.

Description

Preparation method of biocontrol trichoderma strain inoculant and application of biocontrol trichoderma strain inoculant in tobacco planting
Technical Field
The invention belongs to the technical field of biological control of plant diseases, and particularly relates to a preparation method of a biocontrol trichoderma strain microbial inoculum and application of the biocontrol trichoderma strain microbial inoculum in tobacco planting.
Background
For a long time, the prevention and control of plant diseases mainly adopt chemical prevention and control, and are directly applied to natural environments, the environments are places where human beings live, and with the attention of human beings on the environment and the requirement of agricultural sustainable development, people are made to realize that the use of chemical agents can generate larger harm to ecological environment, beneficial organisms and human bodies, and the long-term use of chemical pesticides can cause the increasing drug resistance of pathogenic bacteria and the accumulation of soil pesticide residues, so that the further development of biological prevention and control technology, the research and application of microbial pesticides meet a historical opportunity and technical challenge, and meanwhile, the microbial pesticides can play more and more important roles in the process of promoting the agricultural sustainable development in the future. Therefore, the biological control bacteria are increasingly regarded as a biological pesticide without phytotoxicity, residue and resistance.
At present, trichoderma is widely applied to the fields of biological control and environmental protection. In the agricultural field, more than 50 trichoderma biocontrol bacteria or bacterial manure products are produced in a commercial way internationally, and play an important role in the aspects of preventing plant diseases, repairing organic biological pollution and the like, and some trichoderma can directly promote plant growth due to the fact that the trichoderma can secrete various plant hormones such as indoleacetic acid (IAA), Zeatin (ZA), Gibberellin (GA) and the like, so that the trichoderma has the growth promoting and yield increasing effects of biological fertilizers; meanwhile, part of the trichoderma simultaneously has the functions of dissolving phosphorus, potassium and the like to increase the amount of nutrient substances which can be absorbed by soil, and provides richer nutrition for the growth of plants. As a biopesticide, it has a competitive action, a parasitism action, an antibiotic action, a synergistic antagonism and an induced resistance action, and is widely used for controlling plant diseases, and the disease sources include fungi, bacteria, nematodes, viruses and the like. Biocontrol bacteria are an important part of biological control and are always hot spots of biological control research on plant diseases. With the increasing demand for sustainable agriculture, it is used as a resource-rich antagonistic microorganism and plays a very important role in biological control of plant diseases. The temperature, humidity, illumination, pH, nutrition conditions and the like in the natural environment are complex and changeable, the key of the field planting of the biocontrol bacteria in the natural environment is realized, the relationship between the biocontrol bacteria and the environment is well processed, and the prevention and treatment effect of the biocontrol bacteria is directly influenced.
Disclosure of Invention
In view of the above, the invention provides a preparation method of a biocontrol trichoderma strain microbial inoculum and application of the biocontrol trichoderma strain microbial inoculum in tobacco planting.
In order to solve the technical problem, the invention discloses a preparation method of a biocontrol trichoderma microbial inoculum, which comprises the following steps:
(1) inoculating biocontrol trichoderma strain GF-10 (Trichoderma longibrachiatum GF-10) on a PDA culture medium in a refrigerator stored at 4 ℃, and performing activated culture in a constant-temperature incubator at 28 ℃; washing the GF-10 strain after 5 days of activation culture with 10mL of sterile water to prepare an inoculated spore suspension, counting by a hemocytometer, and keeping the inoculated spore suspension in a refrigerator at 4 ℃ for later use;
(2) adopting a ceramic plate for fermentation, taking bran as a substrate, adding a culture medium, inoculating a trichoderma spore suspension, sealing a preservative film, punching 3-5 small holes on the preservative film by using a bell head, stirring after culturing under the illumination condition, and continuing to culture; after seven days, uncovering the preservative film, sealing the porcelain plate by using two layers of sterilization gauzes, and drying in an incubator; laying 3-6 layers of sterilization gauze in a cover of the pulverizer, pulverizing, obtaining conidium powder on the gauze after pulverizing, collecting the bacterial powder, and storing for later use.
Alternatively, the biocontrol trichoderma strain GF-10 comes from the patent a trichoderma longibrachiatum strain and its use, application No.: 201210521630.4 application date: 2012-12-07, publication No.: CN103122317A, published: 2013-05-29.
Optionally, the culture medium in the step (2) is prepared from the following components in a mass ratio of 1.6 ‰: 10%: 1 per mill: 1.6%: 1% of C6H12O6、(NH4)2SO4、CaCO3And CaSO4(ii) a The thickness of the culture medium is 2-3 cm.
Optionally, the active spores of the trichoderma spore suspension in step (2) are 1 × 107CFU/mL。
Optionally, the active spores of the trichoderma spore suspension in step (2) are 1 × 107CFU/mL。
Optionally, the drying temperature in the step (2) is 30-43 ℃, and the drying time is 12-48 h.
The invention also discloses an application of the biocontrol trichoderma strain preparation in tobacco planting.
Optionally, the application is applied in a seedling raising period and a field period in tobacco planting.
Compared with the prior art, the invention can obtain the following technical effects:
the invention uses bran as solid culture medium, adds MgSO4: glucose: (NH)4)2SO4：CaCO3：CaSO4The spore yield is large, and the large-scale application is facilitated; crushing by a crusher with 3-6 layers of gauze arranged on a crusher cover to obtain the separation of conidia and matrix; simple manufacture, convenient use and stable planting effect.
Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 illustrates the isolation of Trichoderma growth from tobacco tissue according to the invention at various time periods; wherein a represents 72h, b represents 96h, c represents 120h, d represents 144h, e represents 168h, and f represents 192 h.
Detailed Description
The following embodiments are described in detail with reference to the accompanying drawings, so that how to implement the technical features of the present invention to solve the technical problems and achieve the technical effects can be fully understood and implemented.
EXAMPLE 1 preparation of Trichoderma preparation
(1) A biocontrol trichoderma strain GF-10 (Trichoderma longibrachiatum GF-10) (a trichoderma longibrachiatum strain and application thereof, wherein the application number is 201210521630.4 application date: 2012-12-07, the publication number is CN103122317A, the publication number is 2013-05-29) is inoculated on a PDA culture medium in a refrigerator stored at 4 ℃ and is subjected to activation culture in a constant temperature incubator at 28 ℃. Washing the GF-10 strain after 5 days of activation culture with 10mL of sterile water to prepare an inoculated spore suspension, counting by a hemocytometer, and keeping the inoculated spore suspension in a refrigerator at 4 ℃ for later use;
(2) adopts a porcelain plate (25 is multiplied by 30 cm)2) Fermenting with bran as matrix, adding MgSO4：C6H12O6：(NH4)2SO4：CaCO3：CaSO4(1.6 ‰: 10%: 1 ‰: 1.6%: 1%) as culture medium (2-3 cm thick), inoculating 1 × 107CFU/mL trichoderma spore suspension, sealing the preservative film, punching 3-5 small holes on the preservative film by using a bell head, illuminating for 12h/d, stirring after culturing for 47h, and continuing culturing; seven days later, the preservative film is uncovered, the porcelain plate is sealed by two layers of sterilization gauze, and the porcelain plate is dried in an incubator at the temperature of 30-43 ℃ for 12-48 h. Laying 3-6 layers of sterilization gauze in a cover of the pulverizer, pulverizing, obtaining conidium powder on the gauze after pulverizing, collecting the bacterial powder, counting, and storing for later use.
Example 2 field planting of Trichoderma in seedling raising substrate at seedling raising stage
(1) Will be 1 × 109CFU/g trichoderma preparation, respectively applying to special substrate for floating-tray seedling of flue-cured tobacco, mixing uniformly to make the bacteria content in the substrate reach 1 × 107Per gram.
(2) The bacterium-carrying matrix is filled in the hole of the tobacco floating plate, the edge of the floating plate is slightly beaten, the special matrix is fully filled in the hole of the floating plate, and the matrix and the hole of the floating plate are at the same height.
(3) And (3) sowing tobacco seedlings in the floating plate provided with the special matrix for raising the seedlings of the flue-cured tobacco floating plate by using a sowing machine, and ensuring that each hole on the floating plate has one tobacco seed.
(4) And (4) placing the floating disc filled with the tobacco seeds in a water pool to float on the water surface.
(5) The special seedling-raising fertilizer for flue-cured tobacco is applied to a water bed in the middle and later seedling-raising periods.
(6) Samples were collected every ten days and counted for trichoderma (plate count method).
(7) Plate counting method:
preparing a certain Martin type and PDA culture medium; sterilizing; sterilizing the prepared culture medium and the articles to be sterilized, and pouring the culture medium and the articles to be sterilized into a flat plate; separating sample, weighing 10g soil sample, pouring into conical flask containing 90mL sterile water and certain glass beads, shaking for twenty minutes to obtain soil suspension, and diluting to 10% with test tube containing sterile water-4(ii) a Coating, from 10-3The test tube sucks 30uL of separation liquid, adds the separation liquid into a flat plate poured with culture medium, and coats the separation liquid with a coating rod; placing into an incubator for culturing. And counting colonies.
The calculation formula is as follows:
the colonization content (per g) of the bacteria in the matrix is (10000 × number of plate colonies × dilution times)/(amount of soil sample × coating amount).
TABLE 1 colonization of Trichoderma in seedling raising medium
Figure BDA0001813224790000051
Note: the concentration of applied bacteria is the content of bacteria in the matrix; 1-6 respectively show the sampling sequence at 10d intervals over time.
Example 3 field planting of Trichoderma in soil
(1) The method comprises the steps of punching holes with the depth of 20 cm on ridges covered with plastic films by a punching machine during tobacco transplanting, removing tobacco seedlings from a plastic greenhouse, placing the tobacco seedlings into the holes, adding 80-100mL of root fixing water (water, transplanting agent, organic fertilizer, high-efficiency florfenicol, 5L, 1.3886g, 125g, 2.75mL) into the holes, and adding microbial inoculum to ensure that the bacteria content in the root fixing water poured by each plant is 1 x 107Then, the control was applied with root fixing water without bacteria, and covered with soil.
(2) Collecting soil samples every 20 days, and counting the number of trichoderma (plate counting method);
(3) a flat plate counting method, a certain Martin and PDA culture medium is prepared; sterilizing; sterilizing the prepared culture medium and the articles to be sterilized, and pouring the culture medium and the articles to be sterilized into a flat plate; separating a sample; weighing 10g of soil sample, pouring into a conical flask containing 90mL of sterile water and certain glass beads, shaking for twenty minutes to prepare a soil suspension, and diluting to 10% with a test tube containing sterile water-4(ii) a From 10-3The test tube sucks 30uL of separation liquid, adds the separation liquid into a flat plate poured with culture medium, and coats the separation liquid with a coating rod; placing into an incubator for culturing. And counting colonies. The calculation formula is as follows:
the colonization content (number/g) of the bacteria in the matrix is (10000 colonies number dilution times)/(soil sample amount plate adding dilution amount).
TABLE 2 colonization of Trichoderma in seedling substrate in field
Figure BDA0001813224790000061
Note: 1-6 respectively show the sampling sequence at 20d intervals over time.
Example 4 Effect of field Trichoderma on tobacco growth
And observing the growth agronomic characters of the tobacco seedlings every 20 days according to the planting mode of the third embodiment, wherein the experimental results are shown in tables 3 and 4.
TABLE 3 investigation of plant height, leaf number and stem circumference of tobacco plants in experimental and control groups
Figure BDA0001813224790000071
Note: 1-5 show the measurement sequence every 20d over time, respectively.
TABLE 4 investigation of tobacco plant pitch, waist leaf length and waist leaf width of experimental and control groups
Figure BDA0001813224790000072
Note: 1-5 show the measurement sequence every 20d over time, respectively.
From tables 3 and 4, it can be seen that: the application of trichoderma has different influences on the agronomic traits of the tobacco in different investigation periods, and the application of trichoderma has a promoting effect on the pitch, the length of the waist leaves and the width of the waist leaves; the control group with stem girth at the early stage is larger than the experimental group, but the control group at the later stage is larger than the experimental group; in terms of leaf number, the difference between the leaf number of the experimental group and the leaf number of the control group is not large, but the leaf length and the leaf width of the later experimental group are superior to those of the control group; the plant height is higher than the contrast of the strain application; from the overall agronomic characters, the application of the bacteria is beneficial to the growth of the tobacco, and in addition, the tobacco is mainly used as an economic crop for harvesting the tobacco leaves, and the application of the bacteria has a promoting effect on the tobacco leaves, so that the larger yield is obtained; in tables 3 and 4, the reduction in plant height and leaf number at the fifth measurement was due to the topping treatment of tobacco field management.
Example 5 root tissue colonization
(1) According to the planting method of the third embodiment, fresh and mature roots after being applied with the microbial inoculum are washed clean under a tap, soaked in 75% alcohol for 5 minutes, then soaked in diluted 84 disinfectant (diluted according to the ratio of 84 disinfectant: water being 1: 50) for 20 minutes, finally washed with sterile water for four times, and then the water is sucked dry by sterilized filter paper, and cut into small sections with the length of about 0.3cm after the outer skin is removed.
(2) The cut roots were inoculated in Martin's medium with forceps, 3 sections were placed in each dish, and cultured upside down in an incubator at 28 ℃. Observing in different time periods, wherein experimental results are shown in figure 1, white mycelia begin to be generated around root tissues at 72h, colonies can be clearly seen to spread into an oval shape around the root as the center at 96h, white transparent mycelia are arranged at the edge part, and the mycelia are developed; after 120h, the colony diameter around the root tissue is increased, and hyphae are villiform; when the time is 144h, white spore-forming hyphae are formed near the root; after 168h, the colony can be seen to form a dense sporangium ring zone, and the colony is light yellow; after 192 hours, the color of the bacterial colony changes into yellow green, a spore-producing concentric ring taking the root as the center is formed, and the edge of the bacterial colony is irregular; according to the morphological characteristics of the colonies on the plate, the bacteria isolated from the root tissue can be inferred to be GF-10 trichoderma applied in the early stage, which indicates that the bacteria can colonize the root tissue of tobacco.
While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (4)

1. The application of the biocontrol trichoderma fungicide in tobacco planting is characterized in that the preparation method of the fungicide comprises the following steps:
(1) inoculating biocontrol trichoderma strain GF-10 (Trichoderma longibrachiatum GF-10) on a PDA culture medium in a refrigerator stored at 4 ℃, and performing activated culture in a constant-temperature incubator at 28 ℃; washing the GF-10 strain after 5 days of activation culture with 10mL of sterile water to prepare an inoculated spore suspension, counting by a hemocytometer, and keeping the inoculated spore suspension in a refrigerator at 4 ℃ for later use;
(2) adopting a ceramic plate for fermentation, taking bran as a substrate, adding a culture medium, inoculating a trichoderma spore suspension, sealing a preservative film, punching 3-5 small holes on the preservative film by using a bell head, stirring after culturing under the illumination condition, and continuing to culture; after seven days, uncovering the preservative film, sealing the porcelain plate by using two layers of sterilization gauzes, and drying in an incubator; laying 3-6 layers of sterilization gauze in a cover of a pulverizer, pulverizing, obtaining conidium powder on the gauze after pulverizing, collecting the bacterial powder, and storing for later use;
the biocontrol trichoderma GF-10 strain is a trichoderma longibrachiatum strain and application number: 201210521630.4 application date: 2012-12-07, publication No.: CN103122317A, published: 2013-05-29;
the culture medium in the step (2) is prepared from the following components in a mass ratio of 1.6 per mill: 10%: 1 per mill: 1.6%: 1% MgSO4、C6H12O6、(NH4)2SO4、CaCO3And CaSO4(ii) a The thickness of the culture medium is 2-3 cm;
the active spores of the trichoderma spore suspension in the step (2) are 1X 107CFU/mL；
The illumination intensity of the culture in the step (2) is 12h/d, and the culture time is 47 h;
the drying temperature in the step (2) is 30-43 ℃, and the drying time is 12-48 h.
2. The use according to claim 1, characterized in that the use is in the nursery stage and the field stage in tobacco planting.
3. The application of the trichoderma viride strain as claimed in claim 1, wherein the trichoderma biocontrol strain is scattered in a seedling substrate in the seedling stage and is uniformly mixed with the substrate, and the applied active ingredient of the trichoderma biocontrol strain is 1 x 109CFU/g。
4. The use of claim 1, wherein the microbial inoculum is mixed with rooting water during transplanting in a field, and the mixture is applied to tobacco holes and covered with soil.
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WO2015029069A1 (en) * 2013-08-30 2015-03-05 Council Of Scientific & Industrial Research Brachiatin d and process for production thereof
CN106942279A (en) * 2017-05-04 2017-07-14 陕西枫丹百丽生物科技有限公司 A kind of apple tree canker biocontrol agent and its application

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WO2015029069A1 (en) * 2013-08-30 2015-03-05 Council Of Scientific & Industrial Research Brachiatin d and process for production thereof
CN106942279A (en) * 2017-05-04 2017-07-14 陕西枫丹百丽生物科技有限公司 A kind of apple tree canker biocontrol agent and its application

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