CN114836329B - Trichoderma harzianum HB40609 and application thereof - Google Patents
Trichoderma harzianum HB40609 and application thereof Download PDFInfo
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- CN114836329B CN114836329B CN202210545934.8A CN202210545934A CN114836329B CN 114836329 B CN114836329 B CN 114836329B CN 202210545934 A CN202210545934 A CN 202210545934A CN 114836329 B CN114836329 B CN 114836329B
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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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- 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
- C12N3/00—Spore forming or isolating processes
Abstract
The Trichoderma harzianum (Trichoderma harzianum) HB40609 provided by the invention is preserved in the China general microbiological culture Collection center of the culture Collection of microorganisms at 3 months and 7 days in 2022, and the preservation number is CGMCC No.40142; the invention provides application of Trichoderma harzianum (Trichoderma harzianum) HB40609 in preventing and treating tomato leaf blight, preparing a biocontrol preparation for preventing and treating diseases caused by fusarium oxysporum and preparing a preparation for preventing and treating tomato leaf blight; the Trichoderma harzianum (Trichoderma harzianum) HB40609 provided by the invention can effectively inhibit fusarium oxysporum; the treated tomato seedlings show a more remarkable disease-resistant effect on fusarium oxysporum; meanwhile, after measuring chlorophyll fluorescence related parameters and hormone content in leaves, the Trichoderma harzianum (Trichoderma harzianum) HB40609 strain can protect a photosynthetic system II in tomato seedlings and can induce the change of the hormone content of plants so as to improve disease resistance.
Description
Technical Field
The invention relates to the technical field of microorganisms and biofertilizers, and particularly relates to trichoderma harzianum HB40609 and application thereof.
Background
Fusarium oxysporum (Fusarium oxysporum) is a pathogenic bacterium causing tomato blight, has a wide spread range and is difficult to eradicate. At present, a large amount of chemical agents are mainly used in production to kill pathogenic bacteria, but the negative effects of pesticide residue of agricultural products and harm to the ecological environment are brought, and meanwhile, the drug resistance of the pathogenic bacteria is improved. Therefore, the search for a method for quickly and effectively biologically controlling tomato blight has been receiving increasing attention.
Trichoderma is a fungus widely found in nature. Trichoderma spp belongs to Deuteromycotina, class Hyphomycetes, order Moniliales, family Moniliaceae. Trichoderma is widely existed in soil, rhizosphere, leaf periphery, seed and other ecological environments, and is also an excellent biocontrol bacterium. Due to the wide adaptability, broad spectrum and multi-mechanism of trichoderma, the trichoderma is always the key object of biological control research of plant diseases, but no trichoderma capable of obviously resisting tomato blight and improving tomato yield is applied at present.
Disclosure of Invention
The purpose of the invention is as follows: the Trichoderma harzianum has an inhibiting effect on fusarium oxysporum, can protect a photosynthetic system II in tomato seedlings, and improves the disease resistance of plants.
The technical scheme is as follows: the Trichoderma harzianum (Trichoderma harzianum) strain is preserved in the China general microbiological culture Collection center at 3, 7 and 2022 days, and the addresses are as follows: xilu No. 1 Hospital No. 3, beijing, chaoyang, north; the preservation number is CGMCC No.40142.
The invention provides application of Trichoderma harzianum (Trichoderma harzianum) HB40609 in preventing and treating tomato leaf blight.
The invention provides application of Trichoderma harzianum (Trichoderma harzianum) HB40609 in protecting a tomato plant photosynthetic system.
The invention provides application of Trichoderma harzianum (Trichoderma harzianum) HB40609 in preparing a biocontrol microbial inoculum for preventing and treating diseases caused by fusarium oxysporum.
The invention provides application of Trichoderma harzianum (Trichoderma harzianum) HB40609 in preparing a biocontrol microbial inoculum for preventing and treating tomato leaf blight.
The invention provides a biocontrol microbial inoculum which comprises Trichoderma harzianum HB40609.
The invention provides a biocontrol microbial inoculum, which is a conidium eluent.
The invention provides a method for preventing and treating tomato leaf blight, which comprises the step of dipping root in conidium eluent before transplanting tomato seedlings.
Further, the viable bacteria concentration of the conidia eluate was 1x10 7 One per ml.
The invention provides a method for preparing a biocontrol microbial inoculum, the preparation method of conidium eluent comprises the following steps,
step 1, inoculating Trichoderma harzianum (Trichoderma harzianum) HB40609 strain on a PDA plate, and culturing conditions are as follows: culturing for 3d under the illumination condition at the temperature of 25-28 ℃ to obtain a single colony;
step 2, taking out mycelium blocks from the edge of the single colony, inoculating the mycelium blocks to a PDA (personal digital Assistant) plate for culture for 7d, wherein the culture conditions are as follows: irradiating at 25 deg.C to obtain conidium;
Has the advantages that: compared with the prior art, the invention has the advantages that: (1) The invention provides a Trichoderma harzianum (Trichoderma harzianum) HB40609 strain with biocontrol effect; (2) The Trichoderma harzianum HB40609 provided by the invention can effectively inhibit Fusarium oxysporum; tomato seedlings treated by mixed spore liquid of Trichoderma harzianum (Trichoderma harzianum) HB40609 strain and fusarium oxysporum show a more remarkable disease-resistant effect on fusarium oxysporum; (3) Meanwhile, after measuring chlorophyll fluorescence related parameters and hormone content in leaves, the Trichoderma harzianum (Trichoderma harzianum) HB40609 strain can protect a photosynthetic system II in tomato seedlings and can induce the change of the hormone content of plants so as to improve disease resistance; (4) The Trichoderma harzianum HB40609 provided by the invention can regulate the change of resistance hormone in a plant body, induce the defense reaction of tomato seedlings, improve the disease resistance and promote the robust growth of the plant.
Drawings
FIG. 1 shows the inhibition effect of Trichoderma harzianum HB40609 strain on Fusarium oxysporum hypha growth after co-culture with Fusarium oxysporum on PDA medium for 5 days;
FIG. 2 shows the phenotypic differences of tomato plants after the tomato seedlings are root-soaked with different spore eluents;
FIG. 3 shows the disease index of plants after the tomato seedlings are soaked in the root by different spore eluents;
FIG. 4 shows the plant morbidity after the tomato seedlings are root-soaked with different spore eluents;
FIG. 5 shows the chlorophyll fluorescence content difference of each treated tomato leaf after root soaking.
FIG. 6 shows the activity of defensive enzymes and H in tomato plants treated after root soaking 2 O 2 The content is changed;
in the figure, control represents a clear water treatment, F.oxy represents a treatment with Fusarium oxysporum, 609 and F.oxy represents a mixed treatment with Trichoderma harzianum strain HB40609 and Fusarium oxysporum.
Detailed Description
The technical solution of the present invention is described in detail below with reference to the accompanying drawings, but the scope of the present invention is not limited to the embodiments.
Example 1 isolation, purification, identification and preservation of Trichoderma harzianum HB40609
1. Isolation and purification of Trichoderma harzianum HB40609
Taking soil containing trichoderma back from the field, naturally drying the collected soil sample, grinding, and sieving. Weighing 10g of soil sample, placing the soil sample in a flask containing 90mL of sterile water, fully oscillating the soil sample by using a vortex instrument, sucking 10mL of suspension liquid to dilute the suspension liquid in 90mL of sterile water, sucking 10mL of suspension liquid from the diluted suspension liquid to dilute the suspension liquid in 90mL of sterile water, and so on until the volume fraction is 10 -3 、10 -4 、10 -5 . Sucking 0.1mL suspension from the soil suspension with each content by using a micro-pipette, dispersing and dripping the suspension on a Ma Dingshi-Bengal red plate, uniformly coating the suspension by using a sterilized triangular glass rod, standing for 20min, and then placing the suspension in a constant-temperature incubator at 25 DEG CThe medium was cultured in an inverted state and immediately purified after colony formation.
Trichoderma harzianum (Trichoderma harzianum) HB40609 strain is inoculated on a PDA plate for culture, the temperature of the culture is preferably 25 ℃, and the time is preferably 3d, so as to obtain a single colony. The composition of the PDA plate is not particularly limited in the present invention, and a plate medium which is conventional in the art may be preferably used.
The formulations of the PDA plates used in the examples included: 200g of potato, 20g of glucose and 10g of agar powder, and distilled water is supplemented to 1000ml. In the preparation of the PDA plate, this example was performed by slicing cleaned peeled potatoes, boiling in water for about half an hour, filtering off the potatoes with gauze, adding glucose, adding agar powder to water, stirring well, adding to the above solution, adding water to a constant volume, stirring to dissolve, packaging, and sterilizing.
The strain preserved in short term is preserved in a PDA slant test tube in a refrigerator at 4 ℃; the strain preserved for a long time is placed in a refrigerator at minus 80 ℃.
2. Identification of Trichoderma harzianum HB40609
Culturing Trichoderma in PDA plate for 3 days, punching the cake with a punch with diameter of 0.5cm, placing on PDA plate, culturing at 25 deg.C under illumination for 5 days, and observing colony shape and color.
The branching condition of the conidiophores peduncles of the trichoderma and the morphology of the conidiophores are observed under a microscope, white aerial hyphae are formed at the beginning of trichoderma colonies, are circular and quickly expand to the periphery, yellow green spores are gradually generated from the center of the colonies, and the whole colonies are completely changed into green along with the expansion of the hyphae to the periphery. The width of the trichoderma hyphae is about 1-2.8 microns, the main branches are thicker, and the secondary branches are slightly thinner and are distributed on two sides of the main branches in pairs. The bacterial colony generates round yellow-green conidia, the conidia peduncles are vertically and symmetrically diverged, the conidia are single or clustered, and the conidia placed at the temperature of 24-30 ℃ are easy to germinate. Species identification was performed in combination with culture traits and with reference to the trichoderma population classification system of Rifai and Bissett.
3. Deposit of Trichoderma harzianum HB40609
Trichoderma harzianum (Trichoderma harzianum) HB40609, which was deposited at the China general microbiological culture Collection center on 3, 7/2022, and the addresses are: xilu No. 1 Hospital No. 3, beijing, chaoyang, north; the preservation number is CGMCC No.40142.
Example 2 preparation of Trichoderma harzianum HB40609 conidium eluate
Step 1, inoculating Trichoderma harzianum (Trichoderma harzianum) HB40609 strain on a PDA plate, and culturing conditions are as follows: culturing for 3d under the condition of illumination at the temperature of 25-28 ℃ to obtain a single colony;
step 2, taking out mycelium blocks from the edge of the single colony, inoculating the mycelium blocks to a PDA (personal digital Assistant) plate for culture for 7d, wherein the culture conditions are as follows: obtaining conidia at 25 ℃;
Example 3 plate challenge experiment of Trichoderma harzianum (Trichoderma harzianum) HB40609
Respectively activating the preserved Trichoderma harzianum (Trichoderma harzianum) HB40609 strain and Fusarium oxysporum on a PDA flat plate, punching a hole on a PDA culture medium flat plate by using a puncher in an ultra-clean workbench to obtain a bacterial cake with the size of 5 x 5mm, transferring the two bacteria to a new PDA flat plate, and culturing for 7 days at room temperature.
As shown in FIG. 1, trichoderma harzianum (Trichoderma harzianum) HB40609 strain can occupy more growth space and nutrition of the plate in the same time and obviously inhibit the growth of Fusarium oxysporum hyphae.
Example 4 Trichoderma harzianum HB40609 controls tomato leaf blight
1. Experimental materials
Preparing fusarium oxysporum spore eluent: inoculating fusarium oxysporum on a PDA culture medium, and culturing at 25 ℃ for 3d to obtain fusarium oxysporum colonies; inoculating the marginal hyphae of the fusarium oxysporum to a PDA culture dish for culture for 7d; adding sterile water into the culture dish, scraping and washing conidia on the surface of the bacterial colony, mixing uniformly to obtain fusarium oxysporum conidia eluent, adjustingIts concentration is 1x10 7 One per ml.
Trichoderma harzianum (Trichoderma harzianum) HB40609 conidium eluate obtained in example 2, and counted under a microscope using a hemocytometer, the conidium eluate and conidium suspension concentration were adjusted to 1X10 7 One per ml.
2. Experimental methods
Taking out tomato seedlings growing for 3 weeks from the plug tray, washing the roots with clear water, respectively soaking in clear water and Trichoderma harzianum HB40609 strain spore eluent (1 x 10) 7 Seed/ml), fusarium oxysporum spore eluent (1 x 10) 7 Per ml), trichoderma harzianum (Trichoderma harzianum) HB40609 strain and Fusarium oxysporum spore mixed eluent (1 x 10) 7 Seed/ml), soaking the root for 30min, planting in planting square boxes, planting 1 plant in each planting square box, and keeping water and fertilizer normal. The experiment is repeated three times, the pH value of the matrix in the square box is adjusted to be about 7, and the environmental temperature is about 20 ℃. Each group treated 24 strains. After treatment, the tomatoes showed phenotypic differences at the seedling stage, as shown in fig. 2.
As can be seen from FIG. 2, the tomato seedlings in the mixed spore solution treatment group and the single Trichoderma harzianum HB40609 treatment group grew strongly, and the disease resistance of the plants was remarkably enhanced.
Dividing the tomato plants into 4 grades according to disease states:
level 0: the plant grows robustly, leaves are naturally unfolded, green and bright, and the stem is thick;
stage 1: standing, wherein the leaf etiolated wilting area is less than 50% of the total leaf area, and the stem base is browned;
and 2, stage: the leaf yellowing wilting area of the plant is more than 50 percent of the total leaf area, the top leaves are yellow and shriveled, and the stem is withered and yellow
Crimping;
and 3, stage: the plant is short and withered, the leaves are withered, yellow, curled and shriveled, only the growing point survives, and the stem is wilted;
4, level: the plants wither to death.
Calculating disease index according to disease grade: disease index (%) = (number of diseased plant at each stage × corresponding disease stage)/(total number of diseased plant × highest disease stage) × 100%. The disease index is used as the inhibition effect of the effluent of the conidium of the Trichoderma harzianum HB40609 strain on the effluent of Fusarium oxysporum spores.
Calculating the morbidity (judging the tomato plant with the blight disease at the level of 2-4): incidence (%) = number of diseased plants treated/total number of diseased plants treated × 100%. The disease incidence is used as the control effect of the eluent of the conidium of the Trichoderma harzianum HB40609 strain on the tomato blight caused by Fusarium oxysporum.
The result is shown in figure 4, after the tomato seedlings are treated by the fusarium oxysporum spore eluent, the incidence rate of plants is more than 80%, and the incidence rate of the tomato seedlings soaked in the mixed spore liquid of trichoderma harzianum HB40609 and fusarium oxysporum is about 15%, which shows that the trichoderma harzianum HB40609 can obviously inhibit the activity of fusarium oxysporum, prevent pathogenic bacteria from invading the tomatoes, reduce the incidence rate of tomato seedling blight and have obvious biocontrol effect on tomato blight.
Example 5
Taking out tomato seedlings growing for 3 weeks from the plug tray, washing the roots with clear water, and respectively soaking in clear water and Trichoderma harzianum HB40609 strain spore eluent (1 x 10) 7 Seed/ml), fusarium oxysporum spore eluent (1 x 10) 7 One/ml), mixed eluent of trichoderma harzianum HB40609 strain and fusarium oxysporum spore (1 x 10) 7 One/ml) for 15-20 min, planting in a plug tray, and treating 24 plants in each group.
Sampling after phenotype difference of the four treatments, picking the leaves at the same part of the tomato, carrying out dark treatment for 15min, and measuring chlorophyll fluorescence related parameters Fv/Fm, ETR = [ (Fm ' -F) Fm ' ]xPFD, qP = (Fm ' -Fs)/Fv ' and NPQ = (Fm-Fm ')/Fm ' = Fm/Fm ' -1 in the samples by using a fluorometer PAM series.
Fv/Fm reflects the potential maximum photosynthetic capacity (i.e., photosynthetic efficiency) of a plant;
ETR is the apparent photosynthetic electron transfer rate;
qP is fluorescence quenching caused by photosynthesis, and reflects the level of photosynthetic activity;
NPQ reflects the ability of a plant to dissipate excess light energy as heat, i.e., photoprotective ability.
The results are shown in fig. 5, trichoderma inhibits the activity of fusarium oxysporum, prevents tomato seedlings from being infected with diseases, protects the plant photosynthetic system II from being damaged, and improves the leaf photosynthetic efficiency.
Example 6
Taking out tomato seedlings growing for 3 weeks from the plug tray, washing the roots with clear water, and respectively soaking in clear water and Trichoderma harzianum HB40609 strain spore eluent (1 x 10) 7 Seed/ml), fusarium oxysporum spore eluent (1X 10) 7 Per ml), mixed eluent (1 x 10) of Trichoderma harzianum HB40609 strain and Fusarium oxysporum spore 7 One/ml) for 15-20 min, planting in a plug tray, and treating 24 plants in each group.
Sampling after phenotype difference occurs in the four treatments, picking the leaves at the same part of the tomato and determining H in the samples of each treatment group 2 O 2 Content, CAT, POD and SOD enzyme activity.
The results are shown in FIG. 6, where Trichoderma improved the activity of defensive enzymes in tomato plants while significantly reducing H compared to Fusarium oxysporum treated groups 2 O 2 Content, and improves disease resistance of tomato seedlings.
As noted above, while the present invention has been shown and described with reference to certain preferred embodiments, it is not to be construed as limited to the invention itself. Various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (9)
1. Trichoderma harzianum (Trichoderma harzianum) HB40609, and the preservation number of the strain is CGMCC No.40142.
2. The use of Trichoderma harzianum (Trichoderma harzianum) HB40609 as claimed in claim 1 for the control of tomato leaf blight.
3. Use of Trichoderma harzianum (Trichoderma harzianum) HB40609 according to claim 1 for protecting the photosynthetic system of tomato plants.
4. Use of Trichoderma harzianum (Trichoderma harzianum) HB40609 as claimed in claim 1 for the preparation of a biocontrol agent for the control of diseases caused by fusarium oxysporum.
5. The use of Trichoderma harzianum (Trichoderma harzianum) HB40609 as claimed in claim 1 for the preparation of a biocontrol agent for the control of tomato leaf blight.
6. The biocontrol microbial inoculum is characterized in that: comprising Trichoderma harzianum (Trichoderma harzianum) HB40609 as claimed in claim 1.
7. The biocontrol microbial inoculum of claim 6 wherein: the type of the microbial inoculum is conidium eluent.
8. A method for preventing and treating tomato leaf blight is characterized by comprising the following steps: dipping the conidium eluate of claim 7 into roots before transplanting the young tomato seedlings.
9. The method for controlling tomato leaf blight according to claim 8, wherein: the viable bacteria concentration of the conidium eluent is 1x10 7 Each/ml.
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| IL69368A (en) * | 1983-07-28 | 1990-03-19 | Yissum Res Dev Co | Fungicidal compositions containing trichoderma harzianum t-35 active against fusarium and method for using them |
| AT397811B (en) * | 1991-11-05 | 1994-07-25 | Lignocell Holz Biotech | STEMS OF TRICHODERMA MUSHROOM, FUNGICIDE MADE THEREOF, AND METHOD FOR THE USE THEREOF |
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| CN103146586B (en) * | 2013-03-11 | 2014-07-09 | 赵斌 | Trichoderma harzianum strain for controlling plant fungus diseases and application thereof |
| CN103952320B (en) * | 2014-04-28 | 2016-04-13 | 唐山金土生物有机肥有限公司 | One strain trichoderma harziarum and the application in the reaping hook blight of prevention and control lawn thereof |
| MA41534B2 (en) * | 2017-11-24 | 2019-11-29 | Univ Ibn Tofail | Production, formulation and recycling of a biofungicide and biostimulant product based on trichoderma asperellum |
| CN109182137B (en) * | 2018-09-13 | 2020-03-31 | 北京市农林科学院 | Disease-preventing growth-promoting trichoderma africanum and application thereof |
| CN110272833B (en) * | 2019-08-05 | 2021-07-27 | 海南大学 | Trichoderma harzianum HL119 and application thereof |
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