CN111019841B - Multifunctional trichoderma reesei and application thereof - Google Patents
Multifunctional trichoderma reesei and application thereof Download PDFInfo
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Abstract
The invention discloses a multifunctional trichoderma reesei and application thereof. The multifunctional Trichoderma reesei is Trichoderma reesei (Trichoderma reesei) H-LS, and the preservation number is CGMCC No. 18108. The invention also discloses application of the trichoderma reesei H-LS in promoting plant growth and/or inhibiting phytopathogen and/or preventing and treating plant diseases caused by phytopathogen infection and/or preventing and treating nematodes and/or improving quality of vegetables and/or forage grass and/or improving soil nutrients. The trichoderma reesei H-LS has the characteristics of high growth speed, large sporulation amount, wide action spectrum, strong stress resistance, capability of realizing mass colonization at plant roots and the like. The invention has great value for preventing and treating related diseases caused by phytopathogen and nematode, and has great application value for promoting the growth of vegetables and forage grass, improving the nutritional quality and improving the physical and chemical properties of soil.
Description
Technical Field
The invention belongs to the technical field of biological control, and particularly relates to a multifunctional trichoderma reesei and application thereof.
Background
At present, the prevention and treatment of plant diseases and nematodes are mainly based on chemical prevention and treatment, and chemical pesticides not only seriously reduce the quality of vegetables and forage grass, pollute the environment and destroy the ecological balance, but also are unsafe to people and livestock. With the emphasis on food safety and ecological safety of people, biological control is one of the important strategies for realizing the sustainable development of modern agriculture. Particularly, the antagonism of the originally existing beneficial microorganisms of plant rhizosphere to target pathogenic microorganisms and insect pests in the nature is utilized to control the insect pests, so that the method has less environmental risk and is an environment-friendly control technology.
Trichoderma is an important biocontrol fungus, and is widely used for preventing and controlling plant diseases and insect pests by the mechanisms of producing various hydrolases and antagonistic substances, and competing nutrition and the like. The commercial registered trichoderma products at home and abroad are applied to the field. However, most of the trichoderma strains in the prior art only have one or a few functions, and the trichoderma strains integrating multiple functions are lacked.
Disclosure of Invention
The first purpose of the invention is to provide a multifunctional trichoderma strain.
The multifunctional Trichoderma strain provided by the invention is Trichoderma reesei (Trichoderma reesei) H-LS, and the preservation number is CGMCC No. 18108. The strain has been preserved in China general microbiological culture Collection center (CGMCC for short, with the address of No. 3 Xilu-Beijing institute of academy of sciences in North Cheng, Chaoyang, Beijing) for 7 months and 15 days in 2019.
The second purpose of the invention is to provide a new application of Trichoderma reesei H-LS CGMCC No. 18108.
The invention provides an application of Trichoderma reesei H-LS CGMCC No.18108 in at least one of (I), (II), (III), (IV), (V), (VI) and (VII): promoting plant growth; (II) preventing and controlling nematodes; (III) inhibition of plant pathogens; (IV) dissolving phosphorus; (V) decomposing potassium; (VI) degrading the cellulose; (VII) producing chitinase.
The invention also provides the application of the Trichoderma reesei H-LS CGMCC No.18108 in the preparation of products; the application of the product is at least one of (I), (II), (III), (IV), (V), (VI) and (VII): promoting plant growth; (II) preventing and controlling nematodes; (III) inhibition of plant pathogens; (IV) dissolving phosphorus; (V) decomposing potassium; (VI) degrading the cellulose; (VII) producing chitinase.
A third object of the invention is to provide a product.
The active ingredient of the product provided by the invention is Trichoderma reesei H-LS CGMCC No.18108 or a bacterial suspension thereof or a culture solution thereof or a fermentation product thereof or a microbial inoculum containing the same; the application of the product is at least one of (I), (II), (III), (IV), (V), (VI) and (VII): promoting plant growth; (II) preventing and controlling nematodes; (III) inhibition of plant pathogens; (IV) dissolving phosphorus; (V) decomposing potassium; (VI) degrading the cellulose; (VII) producing chitinase.
The fourth purpose of the invention is to provide a preparation method of the biological bacterial fertilizer.
The preparation method of the biological bacterial fertilizer provided by the invention comprises the following steps:
1) uniformly mixing a fermentation substrate, a carbon source, a nitrogen source, inorganic salt and water, and sterilizing to obtain a fermentation material;
the fermentation substrate comprises reed grass powder, wheat bran and rice bran;
2) inoculating spore liquid of trichoderma reesei H-LS CGMCC No.18108 into the fermentation material for fermentation culture; obtaining a fermentation product;
3) and air-drying the fermentation product to obtain the biological bacterial fertilizer.
In the above method, in the 1), the carbon source may be at least one of the following substances: glucose, sucrose, molasses;
the nitrogen source may be at least one of the following: peptone, beef extract, yeast extract and soybean cake powder;
the inorganic salt may be at least one of the following: potassium dihydrogen phosphate and dipotassium hydrogen phosphate;
further, the fermentation substrate consists of reed grass powder, wheat bran and rice bran.
The carbon source is glucose;
the nitrogen source is peptone;
the inorganic salt is potassium dihydrogen phosphate.
Furthermore, the mass ratio of the reed grass powder to the wheat bran to the rice bran can be (2-4): 1-3): 1; specifically 3:2: 1.
The mass fraction of the glucose in the fermentation substrate can be 0.5-1.5%; in particular to 1 percent;
the mass fraction of the peptone in the fermentation substrate may be 0.03-0.07%; specifically 0.05%;
the mass fraction of the monopotassium phosphate in the fermentation substrate can be 0.01-0.03%; in particular to 0.02 percent;
the mass fraction of the water in the fermentation substrate may be 30-50%, in particular 40%;
the sterilization condition is specifically high-pressure sterilization at 121 ℃ for 30 min.
In the above method, in the 2), the preparation method of the spore liquid comprises the following steps: culturing Trichoderma reesei H-LS CGMCC No.18108 on a culture medium (the culture medium can be a PDA culture medium) (the culture condition can be 28 ℃ for 5 days), and washing spores (which can be washed by sterilized distilled water) to obtain a spore solution;
further, the concentration of the spore liquid may be 1 × 104-1×108Per mL; specifically 1X 106one/mL.
Inoculating the spore liquid into the fermentation material in a proportion of 2-8% (volume fraction); specifically 5%.
The fermentation culture condition can be (26-30) deg.C for 7-15 days; specifically culturing at 26-30 deg.C for 10 days.
And 3), air-drying until the water content is below 15%, specifically air-drying until the water content is 10%.
The biological bacterial fertilizer prepared by the method also belongs to the protection scope of the invention.
The fifth purpose of the invention is to provide the application of the biological bacterial fertilizer in any one of the following steps:
a. promoting the growth of plants;
b. inhibiting phytopathogens;
c. preventing and treating plant diseases caused by plant pathogenic bacteria infection;
d. preventing and controlling nematodes;
e. improving the quality of vegetables and/or forage grass;
f. improving soil nutrients.
A final object of the invention is to provide a method for promoting plant growth and/or inhibiting phytopathogens and/or controlling plant diseases caused by infestation by phytopathogens and/or controlling nematodes and/or improving the quality of vegetables and/or forage grass and/or improving soil nutrients.
The method for promoting the growth of plants and/or inhibiting phytopathogens and/or controlling plant diseases caused by the infection of the phytopathogens and/or controlling nematodes and/or improving the quality of vegetables and/or forage grass and/or improving soil nutrients, which is provided by the invention, comprises the step of treating the plants with the biological bacterial fertilizer.
In the method, the method for treating the plant can be to mix the biological bacterial manure into a plant seedling raising substrate or plant soil.
In any of the above applications or methods, the plant pathogenic bacteria may be a plant pathogenic fungus; specifically, the microorganism can be Rhizoctonia solani, Fusarium oxysporum, Phytophthora capsici, Pythium melonis, Fusarium triplex, Microcladosporium, anthrax or Alternaria alternata. In a particular embodiment of the invention, said phytopathogenic fungus may in particular be Rhizoctonia solani (Rhizoctonia solani): ACCC 36124, Fusarium oxysporum (Fusarium oxysporum): ACCC 43478, Phytophthora capsici (Phytophthora capsisi): ACCC 37300, Pythium aphanidermatum (Pythium aphanidermatum): ATCC32230, Fusarium oxysporum (Fusarium oxysporum): ACCC 30316, Fusarium tricinctum (Fusarium tricinctum): FDW1, Cladosporium tenuissimum (Cladosporium tenuissimum): CM2, anthrax (Colletotrichum cliviii): PCC1, Alternaria alternata (Alternaria alternata): ATH1-2, Alternaria alternata (Alternaria alternata): ACH 2.
The nematode can be a root-knot nematode. The root-knot nematode is specifically tomato root-knot nematode (melodogyne spp.).
The dissolved phosphorus is the phosphorus which can not be directly utilized by plants (such as lecithin in egg yolk) is converted into the soluble phosphorus which can be utilized by plants.
The potassium-dissolving refers to the conversion of potassium which cannot be utilized by plants (for example, potassium in the form of minerals in potassium feldspar) into soluble potassium which can be utilized by plants.
The degraded cellulose refers to the conversion of cellulose (such as microcrystalline cellulose) that cannot be directly utilized by plants into a carbon source that can be utilized by plants.
The chitinase production means that the trichoderma can degrade the components of cell walls (such as chitin) by secreting important fungal cell wall degrading enzyme-chitinase, and further kill pathogenic fungi in a heavy parasitic process.
The plant growth promotion is embodied in the aspects of increasing the plant height of the plant and/or increasing the stem thickness of the plant and/or increasing the leaf length of the plant and/or increasing the leaf width of the plant and/or increasing the root length of the plant and/or increasing the fresh weight of stems and leaves of the plant and/or increasing the fresh weight of roots of the plant and/or increasing the branch number of the plant and/or increasing the root volume of the plant.
The improvement of the quality of the vegetables and/or the forage grass is realized by improving the content of the feeding nutrient components of the vegetables and/or the forage grass; the feed nutrient content is dry matter and/or crude ash and/or crude protein and/or crude fat and/or crude fiber and/or calcium and/or phosphorus.
The improvement of soil nutrients is characterized by improving the content of available nutrients in soil; the available nutrient is specifically available nitrogen and/or available phosphorus and/or available potassium.
The plant or vegetable or forage may be a dicotyledonous plant or a monocotyledonous plant; the dicotyledons include Solanaceae, Leguminosae, Cucurbitaceae, and Compositae. The Solanaceae plant can be fructus Lycopersici Esculenti, Leguminosae plant can be semen crotonis Laevigatae, Cucurbitaceae plant can be fructus Cucumidis Sativi, and Compositae plant can be herba Cichorii.
The invention provides a trichoderma strain which integrates 7 functions of inhibiting plant pathogenic bacteria, preventing and controlling nematodes, promoting plant growth, dissolving phosphorus, dissolving potassium, degrading cellulose and producing chitinase, so that trichoderma can be successfully colonized in soil, the chitinase is secreted, the field plant pathogenic bacteria and nematodes are inhibited, phosphate, potassium ore and cellulose which are difficult to dissolve in soil are degraded for plant growth, the effect of medicine and fertilizer is achieved, and the maximum economic and ecological benefits are obtained. In addition, the preparation process of the microbial inoculum is simple, is environment-friendly and has wide market prospect.
Deposit description
The strain name is as follows: trichoderma reesei
Latin name: trichoderma reesei
The strain number is as follows: H-LS
The preservation organization: china general microbiological culture Collection center
The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)
Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district
The preservation date is as follows: 7 month and 15 days 2019
Registration number of the preservation center: CGMCC No.18108
Drawings
FIG. 1 is a photograph showing the morphology of colonies after 3 days of incubation at 28 ℃ on PDA solid medium.
FIG. 2 shows the results of example 6, cucumber diseases caused by Rhizoctonia solani.
FIG. 3 shows the results of example 6-cucumber disease caused by Fusarium oxysporum.
FIG. 4 shows the results of example 8-the effect of Trichoderma reesei fungicide applied to industrial tomato seedlings.
FIG. 5 shows the results of example 9, which shows the effect of Trichoderma reesei fungicide applied to industrial seedlings of cucumber.
FIG. 6 shows the results of example 10, which shows the effect of Trichoderma reesei H-LS inoculum in the production of Lacroton lacrosse.
FIG. 7 shows the results of example 11-effect of Trichoderma reesei H-LS inoculum applied in chicory production.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Example 1 obtaining and identification of Trichoderma reesei H-LS
First, acquisition and naming of the Strain H-LS
The strain is derived from healthy cucumber rhizosphere soil in a plot with serious soil-borne diseases (damping-off) in a Beijing Hai lake area, a plurality of multifunctional strains are obtained by screening through antagonistic plant pathogenic bacteria and plant growth promoting characteristics, and the strain with the best comprehensive effect is screened according to the characteristics of bacteriostasis, growth promoting effect, growth speed, spore yield, stress resistance, plant rhizosphere colonization ability and the like, and is named as strain H-LS.
II, identification of strain H-LS
1. Morphological characterization of Strain H-LS
PDA solid medium: potato 200g (boiled and filtered to obtain filtrate), glucose 20g, agar 18g, and distilled water 1000mL, with natural pH.
The morphology picture of the colony of the strain H-LS after being cultured for 3d on the PDA solid medium at 28 ℃ is shown in figure 1, the colony is pure white initially, and old hyphae in the center of the strain H-LS begin to turn into light green after being cultured for 2 d; conidiophores are light green, oval or elliptical, smooth, (3.4-5.1) μm x (2.2-3.0) μm; the middle part of the conidiophores is expanded, and the base width is 1.7-2.6 mu m; maximum width (2.6-3.4) μm; length/maximum width 1.7-3.4.
2. Molecular characterization of Strain H-LS
Extracting genome DNA of the strain H-LS, adopting a universal primer PCR to amplify the ITS zone sequence of the strain H-LS and sequencing. The sequencing result shows that: the coding sequence of ITS zone of strain H-LS is shown in sequence 1 of sequence table.
Third, preservation of Strain H-LS
Based on the results of the morphological identification and ITS sequence analysis in step two, the strain H-LS belongs to Trichoderma reesei (Trichoderma reesei). Trichoderma reesei (Trichoderma reesei) H-LS has been deposited in China general microbiological culture Collection center (CGMCC, address: No. 3 Hosieboldo 1 of Beijing Kogyo Sungzhong, China academy of sciences) in 2019, 7 months and 15 days, and the deposition registration number is CGMCC NO. 18108. Trichoderma reesei (Trichoderma reesei) H-LS is abbreviated as Trichoderma reesei H-LS.
Example 2 Nutrition improving ability of Trichoderma reesei H-LS
Measurement of phosphorus-solubilizing ability (i.e., the ability to convert phosphorus that cannot be directly utilized by plants into soluble phosphorus that can be utilized by plants)
1. Qualitative determination
Monkina organophosphorus solid culture medium: 10.0g of peptone, 3.0g of beef extract, 15.0 g of NaC0, 15g of Agar, 1000mL of water and pH7.0, subpackaging and sterilizing, and adding 3mL of egg yolk solution (egg yolk solution is mixed with fresh egg yolk and 0.9% physiological saline in equal volume) into every 50mL of egg yolk solution when in use.
Inoculating Trichoderma reesei H-LS on Monkina organophosphorus solid culture medium, culturing at 28 deg.C for 7d, and observing its growth condition.
The trichoderma reesei H-LS grows well on the Monkina organic culture medium, can generate a large amount of green spores, and can form an obvious phosphorus dissolving ring, which indicates that the trichoderma reesei H-LS has the capability of decomposing organic phosphorus.
2. Quantitative determination
Menkina organophosphorus liquid medium: 10.0g of peptone, 3.0g of beef extract, 15.0 g of NaC15, 1000mL of water, pH7.0, subpackaging and sterilizing, and adding 3mL of egg yolk solution (egg yolk solution is mixed with fresh egg yolk and 0.9% physiological saline in equal volume) into every 50mL of egg yolk solution when in use, wherein the egg yolk provides difficultly utilized lecithin.
(1) The Trichoderma reesei H-LS is prepared into the strain concentration of 10 by using sterile water6cfu/mL of bacterial suspension.
(2) And (3) sterilizing the bacterial suspension obtained in the step (1) at the high temperature of 121 ℃ for 20 minutes to obtain the inactivated bacterial liquid.
(3) Experimental groups: inoculating 1mL of the bacterial suspension obtained in the step (1) to 50mL of Monkina organophosphorus liquid culture medium, carrying out shaking culture at 28 ℃ and 180rpm for 5d, transferring the whole culture system to a sterile 50mL centrifuge tube, adding 0.5g of phosphorus-free activated carbon, carrying out ultrasonic cell disruption by using a KQ-500DB type numerical control ultrasonic cleaner (the treatment time is 20min, effective phosphorus in cells is released), centrifuging at 4 ℃ and 10000rpm for 15min, and taking a supernatant. The above treatment was carried out using 1mL of the inactivated bacterial suspension obtained in the step (2) in place of the bacterial suspension obtained in the step (1) as a control.
(4) And (4) taking the supernatant obtained in the step (3), and detecting the content of soluble phosphorus by adopting an ammonium molybdate colorimetric method.
P ═ K × V/V1; p is effective phosphorus increment; k, finding the phosphorus content (mg/L) of the color development liquid from the standard curve; v is the volume (mL) of the solution with constant volume during color development; v1 volume of supernatant aspirated (mL) at development.
The phosphorus dissolution amount is the P value of the experimental group-the P value of the control group.
Each set was set to 6 replicates and the results averaged.
The phosphorus dissolution amount is 224.56 +/-15.68 mg/L.
Secondly, measurement of potassium-dissolving ability (i.e. the ability to convert potassium that cannot be utilized by plants into soluble potassium that can be utilized by plants)
1. Qualitative determination
Potassium-dissolving solid culture medium: sucrose 5g, MgS04·7H2O 0.5g、FeCl3 0.005g、Na2HPO42g of potassium feldspar powder (providing potassium which is difficult to utilize), 5g of CaCO3 0.1g、FeCl30.005g, 1000mL of distilled water, 20g of agar, and pH 7.1-7.4.
And (3) streaking and inoculating the Trichoderma reesei H-LS on a potassium-dissolving solid culture medium, and culturing for 5d at 28 ℃. The Trichoderma reesei H-LS grows well on a potassium-dissolving solid culture medium plate, which indicates that the Trichoderma reesei H-LS has potassium-dissolving capacity.
2. Quantitative determination
Potassium-dissolving liquid culture medium: sucrose 5g, MgS04·7H2O 0.5g、FeCl3 0.005g、Na2HPO42g of potassium feldspar powder (providing potassium which is difficult to utilize), 5g of CaCO3 0.1g、FeCl30.005g, 1000mL of distilled water, pH 7.1-7.4.
(1) Preparing Trichoderma reesei H-LS into spore with concentration of 10 by using sterile water6cfu/mL spore suspension.
(2) And (3) sterilizing the spore suspension obtained in the step (1) at the high temperature of 121 ℃ for 20 minutes to obtain the inactivated bacterial liquid.
(3) Experimental groups: inoculating 5mL of the bacterial suspension obtained in the step (2) into 100mL of potassium-dissolving liquid culture medium, culturing at 28 ℃ and 180rpm for 7d, then centrifuging at 4 ℃ and 10000rpm for 10min, and taking supernatant. The above treatment was carried out using 1mL of the inactivated bacterial suspension obtained in the step (2) in place of the bacterial suspension obtained in the step (1) as a control.
(4) And (4) taking the supernatant obtained in the step (3), and measuring the potassium content by using a flame spectrophotometer method.
Potassium-releasing amount-potassium content of experimental group-potassium content of control group.
Each set was set to 6 replicates and the results averaged.
The potassium dissolving amount is 0.87 +/-0.15 mg/L.
Third, determination of cellulose degradation ability
Degrading cellulose culture medium: (NH)4)2SO4 5g、K2HPO4 1g、NaH2PO4 1g、MgS04·7H20.5g of O, 5g of microcrystalline cellulose, 18g of agar and 1000mL of distilled water, and the pH value is 7.1-7.4.
Culturing Trichoderma reesei H-LS on PDA culture medium at 28 deg.C for 5d to obtain bacterial cake with diameter of 5mm, inoculating on culture dish with above degraded cellulose culture medium, and culturing at 28 deg.C for 7 d. Staining with 0.1% Congo red for 0.5h, decolorizing with 1mol/L NaCl solution for 0.5h, and observing whether there is transparent ring.
The results show that: trichoderma reesei H-LS can grow on the cellulose degradation culture medium to generate a large amount of green spores, and transparent circles are generated around colonies, so that the Trichoderma reesei H-LS has the capacity of degrading cellulose.
Example 3 antagonistic Activity of Trichoderma reesei H-LS against phytopathogenic fungi
Determination of chitinase production ability of Trichoderma reesei H-LS
Chitinase-producing medium: (NH)4)2SO4 3g、K2HPO4 1g、NaH2PO4 1g、MgS04·7H20.5g of O, 10g of colloidal chitin, 18g of agar and 1000mL of distilled water, and the pH value is 7.1-7.4.
Culturing Trichoderma reesei H-LS on a PDA solid culture medium at 28 ℃ for 5 days to prepare a bacterial cake with the diameter of 5mm, inoculating the bacterial cake in the center of a chitinase-producing culture medium plate, inverting the bacterial cake in a dark place at 28 ℃ for 7 days, and observing whether a hydrolytic ring exists on the culture medium.
As a result, the Trichoderma reesei H-LS was found to grow on the chitinase production medium, producing a large number of spores, and producing hydrolysis rings around the colonies, indicating that the Trichoderma reesei H-LS has the chitinase production capability.
Trichoderma harzianum can parasitize many other kinds of fungi, and is one of the important reasons why Trichoderma harzianum can be used as a bio-control factor for plant pathogenic bacteria, and Trichoderma harzianum can produce various fungal cell wall degrading enzymes in the process of parasitism, wherein chitinase is the most important. Chitinase is not only an important functional factor for the trichoderma to complete the heavy parasitic pathogenic cells, but also an important elicitor for inducing host plants to generate defense responses.
II, antagonistic ability of Trichoderma reesei H-LS on plant pathogenic fungi
After the pathogenic fungi and the trichoderma reesei H-LS in the table 1 are activated, fungus cakes with the diameter of 5mm are respectively prepared, and the trichoderma reesei H-LS and the pathogenic fungus cakes are respectively transferred to a PDA (personal digital assistant) plate by adopting an opposite method and are separated by 45 mm. And (3) setting the independently inoculated phytopathogen fungus cakes as a control, culturing for 7d at the temperature of 28 ℃, measuring the diameter of the phytopathogen colony and calculating the bacteriostasis rate (%), and repeating for 4 times. The antagonism culture inhibition ratio ═ [ (control colony diameter-treated colony diameter)/control colony diameter ] × 100%.
The results are shown in Table 1. The results show that: the trichoderma reesei H-LS has obvious inhibition effect on 10 tested pathogenic fungi, and meanwhile, the trichoderma reesei H-LS is found to be capable of being parasitized on the pathogenic fungi colonies.
TABLE 1 inhibition of phytopathogenic fungi by Trichoderma reesei H-LS
| Plant diseases | Pathogenic bacteria | Antagonistic culture inhibition (%) |
| Tomato damping off | Rhizoctonia solani ACCC 36124 | 78.93±3.16 |
| Cucumber fusarium wilt | Fusarium oxysporum (Fusarium oxysporum ACCC 43478) | 89.23±1.24 |
| Damping-off of hot pepper | Phytophthora capsici (Phytophthora capsisi ACCC 37300) | 82.94±1.35 |
| Root rot of soybean | Pythium aphanidermatum (Pythium aphanidermatum ATCC 32230) | 76.45±2.17 |
| Blight of cotton | Fusarium oxysporum (Fusarium oxysporum ACCC 30316) | 87.67±2.21 |
| Leaf and leaf spot of Dactylis glomerata | Fusarium triplex (Fusarium tricinctum FDW1) | 85.43±4.21 |
| Leaf spot of alfalfa | Cladosporium tenuissimum CM2) | 62.23±3.19 |
| Anthracnose of hybrid pennisetum alopecuroides | Anthrax bacteria (Colletotrichum cliviii PCC1) | 66.55±2.35 |
| Leaf spot of white clover | Alternaria alternata (Alternaria alternata ATH1-2) | 73.28±1.65 |
| Leaf spot of chicory | Alternaria alternata (Alternaria alternata ACH2) | 78.44±3.24 |
Example 4 determination of nematicidal Activity of Trichoderma reesei H-LS
1. Preparation of Strain fermentation broth
PDB liquid medium: 200g of potatoes (boiled and filtered to obtain filtrate), 20g of glucose and 1000mL of distilled water, and the pH value is natural.
Inoculating Trichoderma reesei H-LS into a 250mL triangular flask, wherein each flask contains 150mL of PDB liquid culture medium, and shaking at 25-28 ℃ and 160rpm for 7d to obtain fermentation liquor. The fermentation liquor and 10 times of dilution liquor thereof are used for respectively treating tomato root-knot nematodes (Meloidogyne spp.) and determining the insecticidal capacity of the Trichoderma reesei H-LS fermentation liquor on the root-knot nematodes.
2. Determination of nematicidal ability
On a sterile cell culture plate, 1mL of fermentation liquor with different concentrations is respectively added into holes, sterile water is used as a control, then 100ul of nematode suspension (100 nematodes) is respectively added into a treatment group and a control group, the treatment group and the control group are placed at room temperature for 24 hours, the death condition of the root-knot nematodes is observed, and the corrected mortality (namely the nematode killing effect) is calculated. Each treatment was repeated 24 times. Corrected mortality (%) - (treated nematode mortality-control nematode mortality)/(1-control nematode mortality) × 100%.
The toxicity effect of the trichoderma reesei H-LS fermentation liquor treated for 24 hours on the root-knot nematode is determined through the experiment, and the result shows that: the trichoderma reesei H-LS fermentation liquor has obvious prevention and control effect on nematodes, the corrected mortality rate of the root-knot nematodes treated by the fermentation liquor is 97%, the prevention and control effect is reduced after the fermentation liquor is diluted by 10 times, and the corrected mortality rate is 74%.
Example 5 preparation of solid fermentation inoculum of Trichoderma reesei H-LS (Fertilizer of Trichoderma reesei H-LS)
The invention takes withered reeds to be treated urgently in autumn and winter in rivers and lakes to prepare grass powder and cheap agricultural and sideline products (wheat bran and rice bran) as fermentation matrixes to ferment and produce the multifunctional Trichoderma reesei H-LS bacterial fertilizer, and the specific preparation method is as follows:
1. preparation of spore liquid
Culturing Trichoderma reesei H-LS in PDA solid culture medium at 28 deg.C for 5d, washing spores with sterilized distilled water to obtain culture medium with concentration of 1 × 106Spore solution/mL for use.
2. Preparation and sterilization of fermented materials
Uniformly mixing reed grass powder (obtained by harvesting withered reeds in winter, drying in the sun and then crushing), wheat bran (a product in the feed veterinary drug market in the west of Rongchang district in Chongqing city) and rice bran (a product in the feed veterinary drug market in the west of Rongchang district in Chongqing city) serving as fermentation substrates according to the mass ratio of 3:2:1, adding 1% of glucose, 0.05% of peptone, 0.01% of dipotassium hydrogen phosphate and 40% of tap water to obtain a fermentation material, sterilizing at 121 ℃ for 30min under high pressure, and cooling for later use.
3. Inoculation and fermentation
Inoculating the spore liquid prepared in the step 1 into the fermentation material prepared in the step 2 according to the ratio of 7 percent (volume fraction), and statically culturing for 10 days at the temperature of 26-30 ℃ to obtain a fermentation product, wherein the spore yield of the fermentation product is up to 6.3 multiplied by 109Per gram.
4. And (3) air-drying the fermentation product obtained in the step (3) until the water content is below 15% (the water content can be 10%), thus obtaining the trichoderma reesei H-LS bacterial fertilizer.
Example 6 prevention of cucumber diseases caused by Rhizoctonia solani and cucumber diseases caused by Fusarium oxysporum by Trichoderma reesei H-LS bacterial fertilizer
Seedling raising substrate: peat, vermiculite and perlite in a volume ratio of 3:1: 1; the turf is a product of Chaomei peat Co., Ltd, Heilongjiang birch and perlite are products of Beijing mineral processing factories in Lingshou county, Hebei province.
Cucumber Zhongnong No. 6: institute of vegetable and flower, academy of agricultural sciences, china.
Rhizoctonia solani (Rhizoctonia solani): ACCC 36124.
Fusarium oxysporum (Fusarium oxysporum): ACCC 43478.
First, the control effect of Trichoderma reesei H-LS bacterial fertilizer to rhizoctonia solani
Experimental groups: and mixing 10g of the trichoderma reesei H-LS bacterial manure prepared in the example 5 and 0.5g of rhizoctonia solani bacterial body per liter of the seedling culture substrate to obtain a culture substrate, sowing seeds of No. 6 midge cucumber according to a conventional method, and performing conventional management. Control group: mixing each liter of seedling culture substrate with 0.5g of rhizoctonia solani thallus to be used as a culture substrate, sowing seeds of No. 6 cucumber Zhongnong according to a conventional method, and performing conventional management. Three replicates were performed, each replicate with 72 plants per group.
The photograph after 15 days of sowing is shown in FIG. 2 (the disease symptoms of the diseased plants are within the circle). The disease symptom of the rhizoctonia solani is that brown disease spots (initially in a water stain shape, rapidly expands, overflows and shrinks to become thin like a line) are generated at the stem base of the seedling, cotyledons are still green without withering, the seedling stems are gradually shrunk until the seedling turns brown and dies because the seedling stems are suddenly laid down from the stem base (or the middle part of the stem) and are attached to a bed surface.
Survival (average of three groups) was counted 30 days after sowing. The results show that: the survival rate of the experimental group was 72%, and the survival rate of the control group was 23%.
Second, the control effect of the Trichoderma reesei H-LS bacterial fertilizer on fusarium oxysporum caused cucumber fusarium wilt
Experimental groups: and mixing 10g of trichoderma reesei H-LS bacterial manure prepared in example 5 and 0.5g of fusarium oxysporum per liter of seedling raising matrix as a culture matrix, sowing seeds of No. 6 midge cucumber according to a conventional method, and performing conventional management. Control group: mixing each liter of seedling substrate with 0.5g of fusarium oxysporum thalli to serve as a culture substrate, sowing seeds of No. 6 cucumber Zhongnong according to a conventional method, and performing conventional management. Three replicates were performed, each replicate with 72 plants per group.
The photograph after 15 days of sowing is shown in FIG. 3 (diseased plants in the circle). The disease symptom of fusarium oxysporum caused wilt is wilting death of the damaged seedling (fusarium oxysporum infects a vascular bundle system of a host plant, destroys a conduction tissue of the plant, and generates toxin to damage crops in the metabolic process of plant growth and development to cause wilting death of the plant).
Survival (average of three groups) was counted 30 days after sowing. The results show that: the survival rate of the experimental group was 68%, and the survival rate of the control group was 27%.
Example 7 control of root knot nematodes in potted tomatoes by Trichoderma reesei H-LS bacterial manure
Seedling raising substrate: peat, vermiculite and perlite in a volume ratio of 3:1: 1; the turf is a product of Chaomei peat Co., Ltd, Heilongjiang birch and perlite are products of Beijing mineral processing factories in Lingshou county, Hebei province.
Tomato medium impurity number 9: institute of vegetable and flower, academy of agricultural sciences, china.
Experimental groups: and mixing each liter of seedling raising substrate with 10g of trichoderma reesei H-LS bacterial manure prepared in the example 5 to serve as a cultivation substrate, sowing seeds of No. 9 hybrid tomato seeds according to a conventional method, and performing conventional management. Control group: a seedling culture medium is adopted as a culture medium, and seeds mixed with No. 9 tomato seeds are sown according to a conventional method and are managed conventionally.
After the tomato seedlings grow for 20 days, 10mL (about 2000 larvae) of root-knot nematode liquid is applied to holes on the periphery of the tomato root system, and tap water is used as a blank control. And after inoculation for 45 days, counting the number of nematodes in rhizosphere soil and tomato roots, and calculating the nematode decline rate in the rhizosphere soil and the tomato roots.
The nematode decline rate (%) in rhizosphere soil was ═ 100% of the number of nematodes in control soil (number of nematodes in control soil-number of nematodes in rhizosphere soil of trichoderma-treated group)/number of nematodes in control soil.
The nematode decline rate (%) of the clubroot was ═ 100% of the number of nematodes in control roots/number of nematodes in trichoderma-treated roots/control roots.
The results show that: 192 nematodes per 100g of rhizosphere soil in the experimental group, 493 nematodes in the control group and 61.05% of nematode reduction rate in the rhizosphere soil after trichoderma treatment; the number of nematodes in each 100g of tomato roots in the experimental group is 143, the number of nematodes in the control group is 627, and the nematode reduction rate in the tomato roots after trichoderma application reaches 77.19%.
Example 8 application of Trichoderma reesei H-LS bacterial manure in tomato industrial seedling cultivation
Seedling raising substrate: peat, vermiculite and perlite in a volume ratio of 3:1: 1; the turf is a product of Chaomei peat Co., Ltd, Heilongjiang birch and perlite are products of Beijing mineral processing factories in Lingshou county, Hebei province.
Tomato medium impurity number 9: institute of vegetable and flower, academy of agricultural sciences, china.
Experimental groups: and mixing each liter of seedling raising substrate with 10g of trichoderma reesei H-LS bacterial manure prepared in the example 5 to serve as a cultivation substrate, sowing seeds of No. 9 hybrid tomato seeds according to a conventional method, and performing conventional management. Control group: a seedling culture medium is adopted as a culture medium, and seeds mixed with No. 9 tomato seeds are sown according to a conventional method and are managed conventionally.
The photograph after 28 days of sowing is shown in FIG. 4. After 30 days of sowing, the plant growth parameters (plant height, stem thickness, fresh weight of stem and leaf, fresh weight of root system, root volume) were measured. Three replicates were performed, each replicate measuring 20 plants per group, and the results averaged.
The results are shown in Table 2. The results show that: compared with a control group, the trichoderma reesei H-LS bacterial manure prepared in the example 5 can obviously improve the plant height, stem thickness, fresh weight of stems and leaves, fresh weight of root systems and root volume of tomato plants.
TABLE 2 measurement results of various parameters of the plants of the experimental group and the control group 30 days after sowing
| Plant height (cm) | Stem diameter (mm) | Fresh weight of stem and leaf (g) | Fresh weight of root system (g) | Root volume (cm)3) | |
| Control group | 12.25±0.51 | 2.65±0.20 | 8.36±0.46 | 1.32±0.23 | 1.27±0.21 |
| Experimental group | 15.32±0.54 | 3.31±0.56 | 12.10±0.42 | 1.76±0.26 | 1.75±0.11 |
After 30 days of sowing, the culture medium of each group is air-dried, and the content of available nitrogen, the content of available phosphorus and the content of available potassium are detected. The method for detecting the content of available nitrogen is an alkaline hydrolysis diffusion method, the method for detecting the content of available phosphorus is a 0.5mol/L sodium bicarbonate leaching-molybdenum-antimony anti-colorimetric method, and the method for detecting the content of available potassium is a 1mol/L ammonium acetate leaching-flame photometry method. The concrete determination steps refer to the method in the literature "Yanjianhong, Wangcheng, Binhanclin. soil agrochemical analysis and environmental monitoring [ M ]. China geodetic Press, 2008.182-184, 282-290 ].
The results are shown in Table 3. The results show that: compared with a control group, the trichoderma reesei H-LS bacterial manure prepared in the example 5 can obviously improve the contents of available nitrogen, available phosphorus and available potassium in a culture medium.
TABLE 3 influence of Trichoderma reesei H-LS bacterial manure on mineral element content in the cultivation substrate
| Available nitrogen (mg/kg) | Available phosphorus (mg/kg) | Effective potassium (mg/kg) | |
| Control group | 73.25±2.13 | 109.99±4.54 | 196.95±9.23 |
| Experimental group | 146.63±36.31 | 271.01±3.98 | 417.29±49.81 |
Example 9 application of Trichoderma reesei H-LS bacterial manure in industrial seedling raising of cucumber
Seedling raising substrate: peat, vermiculite and perlite in a volume ratio of 3:1: 1; the turf is a product of Chaomei peat Co., Ltd, Heilongjiang birch and perlite are products of Beijing mineral processing factories in Lingshou county, Hebei province.
Cucumber Zhongnong No. 6: institute of vegetable and flower, academy of agricultural sciences, china.
Experimental groups: and mixing each liter of seedling raising substrate with 10g of trichoderma reesei H-LS bacterial manure prepared in the example 5 to serve as a cultivation substrate, sowing seeds of No. 6 cucumber Zhongnong according to a conventional method, and performing conventional management. Control group: a seedling culture medium is adopted as a culture medium, and seeds of No. 6 cucumber Zhongnong are sown according to a conventional method and are managed conventionally.
The photograph after 28 days of sowing is shown in FIG. 5. After 30 days of sowing, the plant growth parameters (plant height, stem thickness, fresh weight of stem and leaf, fresh weight of root system, root volume) were measured. Three replicates were performed, each replicate measuring 20 plants per group, and the results averaged.
The results are shown in Table 4. The results show that: compared with a control group, the trichoderma reesei H-LS bacterial fertilizer prepared in the example 5 can obviously improve the plant height, stem thickness, fresh weight of stems and leaves, fresh weight of root systems and root volume of cucumber plants.
Table 4 measurement results of various parameters of cucumber plants of the experimental group and the control group 30 days after sowing
| Plant height (cm) | Stem diameter (mm) | Fresh weight of stem and leaf (g) | Fresh weight of root system (g) | Root volume (cm)3) | |
| Control group | 10.01±0.76 | 2.84±0.36 | 19.14±0.34 | 3.15±0.28 | 3.10±0.24 |
| Experimental group | 15.67±0.68 | 3.73±0.14 | 25.42±0.75 | 4.81±0.37 | 4.70±0.39 |
Example 10 application of Trichoderma reesei H-LS bacterial manure in production of Lacroton lacunaris
The variety of the dolichos lablab L is a product of Chengdu green grass garden species limited company.
The soil to be tested was neutral lime brown purple mud (sandy soil) collected in the field of Rongchang district in Chongqing.
Experimental groups: potted plant test, each pot was filled with 3.5kg of soil, and 10g of the Trichoderma reesei H-LS bacterial manure prepared in example 5 was added. Control group: 3.5kg of soil is filled in each pot, and no bacterial manure is mixed. And sowing the dolichos lablab seeds according to a conventional method and performing conventional management.
The photograph after 60 days of sowing is shown in fig. 6. After sowing for 60 days, the plant growth indexes (plant height, leaf length, leaf width, root length, root fresh weight, stem and leaf fresh weight), the contents of feed nutrients (dry matter, crude protein, crude fat, crude fiber, calcium and phosphorus) and the contents of available soil nutrients (mg/kg) were measured. The method for detecting Dry Matter (DM) is oven drying, and drying at 105 deg.C to constant weight. The method for detecting the Crude Protein (CP) is a semi-micro Kjeldahl method. The method for detecting crude fat (EE) is soxhlet extraction. The method for detecting the Crude Fiber (CF) is an acid-base digestion method. The method for detecting calcium (Ca) is potassium permanganate titration. The method for detecting the phosphorus (P) is a molybdenum-antimony colorimetric-resistance method. The concrete determination steps refer to the literature "Zeliying. feed analysis and feed quality detection technology. 3 rd edition. Beijing: the method of "Nongda Press, China, 2007:1-435.
The results are shown in tables 5 and 6. The results show that: compared with a control group, the trichoderma reesei H-LS bacterial manure prepared in the example 5 can obviously promote the growth of the dolichos lablab L, improve the content of the feeding nutrient components of the dolichos lablab L, is beneficial to the conversion of the refractory nutrients in soil and improves the nutrient condition of the soil.
TABLE 5 measurement of growth and feeding nutritional composition of Croton tiglium plants in experimental and control groups
| Growth index | Experimental group | Control group | Nutrient composition | Experimental group | Control group |
| Plant height (cm) | 104.45±20.10 | 84.2.01±13.22 | Dried substance | 25.93±0.16 | 19.77±0.31 |
| Leaf length (cm) | 10.45±1.14 | 10.40±2.36 | Crude protein | 17.75±0.08 | 13.97±0.02 |
| Leaf width (cm) | 8.2±1.39 | 6.94±2.43 | Crude fat | 3.27±0.02 | 2.87±0.02Cd |
| Root length (cm) | 22.67±3.16 | 12±1.24 | Coarse fiber | 66.85±0.4 | 65.76±0.65Bb |
| Fresh weight of root (g/plant) | 12.45±0.95 | 7.80±1.08 | Calcium carbonate | 11.59±0.05 | 11.2±0.94Ab |
| Fresh weight of stem and leaf (g/plant) | 84.25±8.13 | 64.5±2.12 | Phosphorus (P) | 0.59±0.27 | 0.36±0.08Aa |
TABLE 6 content of available nutrients (mg/kg) in soil of experimental group and control group 60 days after sowing
| Available nitrogen | Available phosphorus | Effective potassium | |
| Control group | 82.6±3.18 | 20.5±1.32 | 105.0±9.23 |
| Experimental group | 102.7±4.71 | 31.7±2.31 | 154.2±7.33 |
Example 11 application of Trichoderma reesei H-LS bacterial manure in chicory production
The chicory is a general product of Bai Lu Cao industry Co.
The soil to be tested was neutral lime brown purple mud (sandy soil) collected in the field of Rongchang district in Chongqing.
Experimental groups: potted plant test, each pot was filled with 3.5kg of soil, and 10g of the Trichoderma reesei H-LS bacterial manure prepared in example 5 was added. Control group: 3.5kg of soil is filled in each pot, and no bacterial manure is mixed. And (4) sowing chicory seeds according to a conventional method and performing conventional management.
The photograph after 60 days of sowing is shown in FIG. 7. Plant growth indicators (plant height, leaf length, leaf width, branch number, fresh root weight, fresh grass weight) and feed nutrient content (dry matter, crude ash, crude protein, crude fat, crude fiber, calcium) were measured 60 days after sowing. The method for detecting Dry Matter (DM) is oven drying, and drying at 105 deg.C to constant weight. The method for detecting the coarse ash is a 550 ℃ burning method. The method for detecting the Crude Protein (CP) is a semi-micro Kjeldahl method. The method for detecting crude fat (EE) is soxhlet extraction. The method for detecting the crude fiber is an acid-base digestion method. The method for detecting calcium (Ca) is potassium permanganate titration. The concrete determination steps refer to the literature "Zeliying. feed analysis and feed quality detection technology. 3 rd edition. Beijing: the method of "Nongda Press, China, 2007:1-435.
The results are shown in Table 7. The results show that: compared with a control group, the trichoderma reesei H-LS bacterial fertilizer prepared in the example 5 can obviously promote the growth of chicory and improve the content of the feeding nutrient components of the chicory.
TABLE 7 influence of Trichoderma inoculum on growth index and feed quality of chicory
| Growth index | Experimental group | Control group | Nutrient composition (%) | Experimental group | Control group |
| Plant height (cm) | 36.18±1.53Aa | 31.83±1.30Bb | Dried substance | 19.51±0.75Aa | 16.89±1.29Ab |
| Leaf length (cm) | 18.13.±1.23Aa | 16.46±0.68Bb | Coarse ash content | 16.99±0.54Aa | 16.13±0.23Aa |
| Leaf width (cm) | 4.91±0.49Aa | 4.15±0.26Ab | Crude protein | 25.03±1.45Aa | 20.07±1.62Bb |
| Number of branches (number) | 8.83±0.87Aa | 6.63±0.98Ab | Crude fat | 6.80±0.16Aa | 5.42±0.52Bb |
| Fresh weight of root (g/plant) | 2.31±2.15Aa | 2.10±2.35Aa | Coarse fiber | 15.43±0.40Aa | 13.66±1.62Bb |
| Fresh weight of grass (g/plant) | 13.12±0.23Aa | 7.50±1.53Bb | Calcium carbonate | 1.56±0.07Aa | 1.44±0.05Ab |
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> university of southwest
<120> multifunctional trichoderma reesei and application thereof
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<170>PatentIn version 3.5
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<213>Artificial Sequence
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Claims (16)
1. Trichoderma reesei (T. reesei) ((T. reesei))Trichoderma reesei) H-LS with the preservation number of CGMCC No. 18108.
2. The use of Trichoderma reesei H-LS as claimed in claim 1 in at least one of (I), (II), (III), (IV), (V), (VI), (VII): promoting plant growth; (II) preventing and controlling nematodes; (III) inhibiting phytopathogenic fungi; (IV) dissolving phosphorus; (V) decomposing potassium; (VI) degrading the cellulose; (VII) producing chitinase.
3. Use of the trichoderma reesei H-LS of claim 1 in the preparation of a product; the application of the product is at least one of (I), (II), (III), (IV), (V), (VI) and (VII): promoting plant growth; (II) preventing and controlling nematodes; (III) inhibiting phytopathogenic fungi; (IV) dissolving phosphorus; (V) decomposing potassium; (VI) degrading the cellulose; (VII) producing chitinase.
4. A product comprising the Trichoderma reesei H-LS or a bacterial suspension thereof or a culture solution thereof or a microbial preparation containing the same as defined in claim 1 as an active ingredient; the application of the product is at least one of (I), (II), (III), (IV), (V), (VI) and (VII): promoting plant growth; (II) preventing and controlling nematodes; (III) inhibiting phytopathogenic fungi; (IV) dissolving phosphorus; (V) decomposing potassium; (VI) degrading the cellulose; (VII) producing chitinase.
5. A preparation method of biological bacterial fertilizer comprises the following steps:
1) uniformly mixing a fermentation substrate, a carbon source, a nitrogen source, inorganic salt and water, and sterilizing to obtain a fermentation material;
the fermentation substrate comprises reed grass powder, wheat bran and rice bran;
2) inoculating the spore solution of trichoderma reesei H-LS of claim 1 into the fermentation material for fermentation culture; obtaining a fermentation product;
3) and air-drying the fermentation product to obtain the biological bacterial fertilizer.
6. The method of claim 5, wherein: in the step 1), the carbon source is glucose;
the nitrogen source is peptone;
the inorganic salt is potassium dihydrogen phosphate.
7. The method of claim 6, wherein: the mass fraction of the glucose in the fermentation substrate is 0.5-1.5%;
the mass fraction of the peptone in the fermentation substrate is 0.03-0.07%;
the mass fraction of the monopotassium phosphate in the fermentation substrate is 0.01-0.03%.
8. The method of claim 5, wherein: in the step 1), the mass ratio of the reed grass powder to the wheat bran to the rice bran is (2-4) to (1-3) to 1.
9. The method of claim 5, wherein: in the step 1), the mass fraction of the water in the fermentation substrate is 30-50%.
10. The method of claim 5, wherein: in the 2), the preparation method of the spore liquid comprises the following steps: culturing the Trichoderma reesei H-LS according to claim 1 on a culture medium, and washing spores to obtain the spore solution.
11. The method of claim 10, wherein: the concentration of the spore liquid is 1 × 104 -1×108Per mL;
the spore liquid is inoculated in the fermentation material in a proportion of 2-8%.
12. The method of claim 5, wherein: in the step 2), the fermentation culture conditions are (26-30) DEG C for 10 days.
13. The method of claim 5, wherein: and 3) air-drying until the water content is below 15%.
14. A biological bacterial fertilizer prepared according to the method of any one of claims 5 to 13.
15. Use of the biological bacterial fertilizer of claim 14 in any one of:
a. promoting the growth of plants;
b. inhibiting plant pathogenic fungi;
c. preventing and treating plant diseases caused by plant pathogenic bacteria infection;
d. preventing and controlling nematodes;
e. improving the quality of vegetables and/or forage grass;
f. improving soil nutrients.
16. A method for promoting plant growth and/or inhibiting phytopathogenic fungi and/or controlling plant diseases caused by infestation of phytopathogens and/or controlling nematodes and/or improving the quality of vegetables and/or forage grass, comprising the step of treating the plants with a biological bacterial fertilizer according to claim 14.
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