CN117305135A - Trichoderma pseudokoningii T0027 and application thereof in preventing and treating soft rot of kiwi fruits - Google Patents
Trichoderma pseudokoningii T0027 and application thereof in preventing and treating soft rot of kiwi fruits Download PDFInfo
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- CN117305135A CN117305135A CN202311418881.4A CN202311418881A CN117305135A CN 117305135 A CN117305135 A CN 117305135A CN 202311418881 A CN202311418881 A CN 202311418881A CN 117305135 A CN117305135 A CN 117305135A
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- Prior art keywords
- trichoderma
- trichoderma pseudokoningii
- soft rot
- pseudokoningii
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
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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
- C12N1/145—Fungal isolates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G17/00—Cultivation of hops, vines, fruit trees, or like trees
- A01G17/005—Cultivation methods
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
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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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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
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- C12N1/14—Fungi; Culture media therefor
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/885—Trichoderma
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention discloses trichoderma pseudokoningii T0027 and application thereof in preventing and treating soft rot of kiwi fruits, belonging to the technical field of biological prevention and treatment, wherein the trichoderma pseudokoningii T0027 is preserved to the China general microbiological culture Collection center (China general microbiological culture Collection center) on 7-month 17 of 2023, and the preservation number is as follows: CGMCC No.40722. The experiment of the invention shows that the effect of the trichoderma pseudokoningii T0027 on antagonizing the viticola reaches 83.55%, the antagonism effect of the fermentation liquor on pathogenic bacteria reaches 78.01%, and the antibacterial effect is good, so that the invention can be used for preventing and treating kiwi fruit soft rot caused by the viticola; the biocontrol experiment proves that the biocontrol effect of the strain and the fermentation liquor thereof on the soft rot of the kiwi fruit reaches 73.56 percent, and the invention provides a safe and effective new medicament for the biological control of the soft rot of the kiwi fruit.
Description
Technical Field
The invention relates to the technical field of biological control, in particular to trichoderma pseudokoningii T0027 and application thereof in controlling soft rot of kiwi fruits.
Background
Kiwi fruit (Actinidia chinensis Planch), native to China, has very high nutritional value and is called "fruit king". The vitamin C content is higher than that of most fruits, and it is reported that 100-200mg per 100g fresh fruit. In addition, the kiwi fruits have certain treatment effects on scurvy and diabetes due to the fact that the kiwi fruits contain a large amount of nutrient substances required by human bodies, and are widely accepted in daily life as a natural health-care food.
The easy rot of the picked kiwi fruits is a difficult problem which restricts the quality development of the industry and is not overcome, and the diseases are mainly found in the mature and storage period of the fruits. As the main yield area of the kiwi fruits has the characteristics of abundant precipitation and moist climate, bacteria are easy to breed so as to induce various plant diseases, and the incidence rate of soft rot of the kiwi fruits in the main yield area in China reaches 20 percent according to incomplete statistics. The infected fruit is mainly in a dark green water stain shape halo at the junction of the disease health, and the disease can lead to the decay of the whole fruit and finally lose the edible value.
In order to reduce the damage degree of soft rot and improve the quality of kiwi fruits, synthetic bactericides are increasingly widely used, however, long-term use of chemical bactericides not only causes drug resistance of pathogenic bacteria of the kiwi fruits, but also causes residues of fruit pesticides, threatens human health and pollutes the environment. In recent years, people gradually pursue green pollution-free organic foods, and are worried about products with labels of pesticide, food, fruits and vegetables, so the aim of searching efficient, pollution-free and low-residue pesticide substitutes is to be urgently solved. Trichoderma as an environment-friendly biocontrol bacterium has the advantages of high efficiency, safety, environment friendliness, broad antibacterial spectrum and the like, and is regarded as one of excellent substitutes for chemical bactericides. However, no report on the prevention and treatment of actinidia arguta soft rot by trichoderma pseudokoningii (Trichoderma ko ningiopsis) has been found so far.
Disclosure of Invention
The invention aims to provide trichoderma pseudokoningii T0027 and application thereof in preventing and treating soft rot of kiwi fruits, so as to solve the problems in the prior art, and the trichoderma pseudokoningii T0027 can obviously inhibit pathogenic bacteria, can be used for preventing and treating soft rot of kiwi fruits caused by the plasmodium, and provides a safe and effective new medicament for biological prevention and treatment of soft rot of kiwi fruits.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides trichoderma pseudokoningii (Trichoderma koningiopsis) T0027 which is preserved in 2023, 7 and 17 days to China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation address of Beijing, chaoyang, north Chen Xidelu No. 1, 3 and a preservation number: CGMCC No.40722.
The invention also provides a microbial inoculum comprising the strain, fermentation liquor or metabolite of trichoderma pseudokoningii T0027.
Further, the trichoderma pseudokoningii T0027 is cultured in the following manner:
the trichoderma pseudokoningii T0027 is inoculated into a Chlamydia medium which takes starch, maltose and lactose as carbon sources and beef extract as nitrogen sources for culture.
Further, the culturing conditions are as follows: the pH is 5-7, and the culture temperature is 15-25 ℃.
The invention also provides an application of the trichoderma pseudokoningii T0027 in preparation of antibacterial agents.
Further, the antimicrobial agent comprises an plasmodium viticola antimicrobial agent.
The invention also provides a method for preventing and treating actinidia soft rot, which comprises the step of applying the trichoderma pseudokoningii T0027 or the microbial inoculum on the actinidia.
The invention also provides application of the trichoderma pseudokoningii T0027 or the microbial inoculum in preparing biocontrol drugs for preventing and treating kiwi soft rot.
Further, the trichoderma pseudokoningii T0027 can exert the biocontrol effect of preventing and treating the soft rot of the kiwi fruits by inhibiting the plasmopara viticola.
The invention discloses the following technical effects:
according to the invention, trichoderma pseudokoningii (Trichoderma koningiopsis) T0027 is obtained by separating and screening saprophyticus, and a plate counter experiment shows that the trichoderma pseudokoningii strain has an effect of 83.55% on antagonizing the plasmopara viticola, and the antagonizing effect of the fermentation liquor on pathogenic bacteria reaches 78.01%, so that the saprophyticus has a good antibacterial effect, and can be used for preventing and treating actinidia arguta soft rot caused by the plasmopara viticola. The biocontrol experiment proves that the biocontrol effect of the strain and the fermentation liquor thereof on the soft rot of the kiwi fruit reaches 73.56 percent, and the invention provides a safe and effective new medicament for the biological control of the soft rot of the kiwi fruit.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing the re-mailing structure of Trichoderma reesei hyphae to Phellinus linteus (C, scale = 10 μm) under a microscope as a culture image (A-B) of Trichoderma reesei T0027 against Phellinus linteus plates;
FIG. 2 is a photograph of colony morphology, conidiophore and conidium of Trichoderma T0027; a: colony morphology; B. c: conidiophores and bottle stems; d: conidium; scale = 10 μm;
FIG. 3 is a diagram of the ML phylogenetic tree of Trichoderma pseudokoningii T0027;
FIG. 4 shows the inhibitory effect of different concentrations of sterile fermentation filtrate of Trichoderma pseudokoningii T0027 on Botrytis cinerea; a:20% of a base; b:25%; c:30%; d:35%; e:40%; f:0% of the total weight of the composition;
FIG. 5 is a biological profile of Trichoderma pseudokoningii strain T0027; a: different temperature conditions; b: different pH conditions; c: different nitrogen sources; d: different carbon sources;
FIG. 6 shows the control effect of the sterile fermentation filtrate of Trichoderma pseudokoningii T0027 on soft rot of macaque; CK: inoculating only the pathogenic bacteria of the viticola; t0027: after 24 hours inoculation of pathogenic bacteria, the mixture is soaked in trichoderma fermentation broth for 20 minutes.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
EXAMPLE 1 isolation, identification and preservation of Trichoderma strains
1 isolation and purification of Trichoderma strain
Sapwood samples were collected in a six-disc water city national name lake park and rinsed clean. Then scraping the part with a blade to be about 1cm thick, putting the part into 75% alcohol to soak for 30s, and putting into 5% sodium hypochlorite to soak for 60s. After washing 5 times with sterile water, the residual water was sucked dry with sterile filter paper. Finally inoculating on double antibody PDA culture medium, culturing at 25 deg.C for 2-3d. The above self-cleaning operations are all performed under aseptic conditions.
After hypha grows out of the flat plate, transferring the flat plate to a new PDA culture medium for 2d, transferring the flat plate to the new PDA culture medium for 2-3 times of purification, and placing the flat plate in a refrigerator at 4 ℃ for preservation for later use, and then using the flat plate in an antagonistic bacteria screening experiment.
2 screening for antagonistic bacteria
Test pathogenic bacteria:
the pathogenic bacteria of the kiwi fruit soft rot disease are Puccinia viticola (Botryosphaeria dothidea), which is provided by the microbiology laboratory of the university of Sedum, biological sciences and technology.
The pre-purified trichoderma strain and the tested pathogenic bacteria are inoculated to a PDA culture medium flat plate for activation, and two opposite screening is carried out (trichoderma and kiwi soft rot pathogenic bacteria are respectively inoculated on two sides of the flat plate and are 3cm apart), so that the trichoderma strain T0027 with good biocontrol effect is obtained.
Inhibition ratio (%) = (radius of control group pathogenic bacteria colony-radius of treatment group pathogenic bacteria colony)/radius of control group pathogenic bacteria colony x 100%.
The antibacterial effect is shown in figure 1, and the plate opposite result shows that the antibacterial effect of the trichoderma strain T0027 on the plasmopara viticola reaches 83.55%, and a large amount of trichoderma hyphae are observed to wind and re-attach on the plasmopara viticola hyphae through the plug-in cultivation, so that pathogenic bacteria growth space is rapidly contented and digested, and the plasmopara viticola hyphae are inhibited from growing. The trichoderma T0027 has strong re-mailing capability to the viticola.
3 identification of strains
3.1 morphological characterization observations
The screened strain T0027 is inoculated on a PDA plate, cultured at 25 ℃ until bacterial colony is sporulated, a temporary loading piece is manufactured by taking a lactic acid phenol cotton blue dye solution as a carrier, and a Mingmei microscopic imaging system is used for shooting morphological characteristics of the strain and measuring the size. The morphological characteristics of the strain are shown in figure 2, the strain can grow to be full of a plate in a PDA culture medium for 72 hours, no obvious ring lines appear, the initial period of hypha is white, light green fine particles are generated from the middle to a powdery spore stack after about 5 days of culture, and the strain is gradually green and covers the plate (A in figure 2); the conidiophore directly produces bottle peduncles or produces secondary branches, the secondary branches are multiple pairs, the top ends of the secondary branches are in vortex arrangement of 2-5 bottle peduncles, the bottle peduncles are straight or bent, the narrow flask shape or the middle part is expanded, and the secondary branches are often pairs. The stem size (3.0 to 5.2) μm× (9 to 10.5) μm (n=50), and the aspect ratio was 1.6 to 1.95 μm (B, C in fig. 2). Conidium is elliptic, single spore is green, aggregate is dark green, and wall is smooth. The diameter size (4 to 5.1) μm× (1.8 to 4) μm (n=50), and the aspect ratio is 1.1 μm to 2.2 μm (D in fig. 2).
3.2 molecular biological identification
ITS and TEF1-a sequence analysis
After extracting genomic DNA of the strain T0027 by a liquid nitrogen grinding method, adopting ITS sequence primers: ITS1 (5 '-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5 '-TCCTCCGCTTATTGATATGC-3') and TEF 1-alpha sequence primers: EF1-728F (5 '-CATCGAGAAGTTCGAGAAGG-3') and TEF1LLErev (5 '-AACTTGCAGGCAATGTGG-3'), nucleotide fragment amplification and sequencing were performed. Both primer synthesis and sequencing work was delegated to all biological engine Co. After the sequencing results of each strain are sheared and spliced, homology comparison is carried out in GenBank, sequences with the Sequence similarity higher than 99% are downloaded, 2 sequenced gene sequences of each pathogenic strain are spliced by using Sequence matrix1.7.8, the maximum reduction Method (MP) is based, the self-expanding value (booths trap) is set to be 1000, and a phylogenetic tree is constructed by using MEGA 7.
The nucleotide sequence of ITS ITS for strain T0027 is shown below:
GGAGTAGTAAACTCGGTAATGATCCCTCCGTAGGTGAACCTGCGGAGGGATCATTACCGAGTTTACAACTCCCAAACCCAATGTGAACCATACCAAACTGTTGCCTCGGCGGGGTCACGCCCCGGGTGCGTCGCAGCCCCGGAACCAGGCGCCCGCCGGAGGGACCAACCAAACTCTTTCTGTAGTCCCCTCGCGGACGTTATTTCTTACAGCTCTGAGCAAAAATTCAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCGGGGGGTCGGCGTTGGGGATCGGGAACCCCTAAGACGGGATCCCGGCCCCGAAATACAGTGGCGGTCTCGCCGCAGCCTCTCCTGCGCAGTAGTTTGCACAACTCGCACCGGGAGCGCGGCGCGTCCACGTCCGTAAAACACCCAACTTCTGAAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATA；
the nucleotide sequence of the strain T0027 TEF1-a is as follows:
CCGGCACCGACCTGGCCAGGGTGGTTCATGACGATGACCTGGGCGTTGAAAGAAGCGGCGCCCATGGGGGGGTCGTTCTTGGAGTCACCGGCAACGTTACCACGGCGGATATCCTTGACGGAGACGTTCTTGACGTTGAATCCAACGTTGTCACCGGGGACACCCTCAACGAGCTGCTCGTGGTGCATCTCGACGGACTTGACTTCAGTGGTGACGTTGGAGGGAGCGAAGGTAACGACCATACCGGGCTTGAGGACACCAGTCTCGATACGGCCGACAGGGACTGTTCCGATACCACCGATCTTGTAAACGTCCTGAAGGGGCAGACGGAGGGGCTTGTCTGTGGGACGCTTGGGGGGCTCAATGGCGTCAATGGCCTCGAGAAGGGTCTTGCCGGTGGACTTGCCAGCCTTGGTCTCCTTCTCCCAGCCCTTGTACCAGGGGCAGTTGGTGGAGGCCTGGAGCATGTTGTCGCCGTTGAAGCCGGAGATGGGGACGAAGGCAACGGTCTTGGGGTTGAAGCCGACCTTCTTGATGAAGTTGGAGGTCTCCTTGATGATCTCAAGGTAACGAGCCTCAGCCCAGTTGGCAGTGTCCATCTTGTTGATGGCAACGATGAGCTGCTTGACACCCAGGGTGTAGGCGAGCAGAGCGTGCTCACGGGTCTGGCCATCCTTGGAGATACCAGCCTCGAACTCACCAGTACCGGCAGCGATAATCAGGATAGCGCAGTCAGCCTGGGAGGTACCAGTGATCATGTTCTTGATGAAATCACGGTGACCGGGAGCGTCTGTAGTGGCACGTTAGAATGATGATTTCGACATGTCAAGAGCCAGGAATAACATACCAATGACGGTGACATAGTACTTGGGAGTCTCGAACTTCCAGAGGGCAATGTCGATGGTGATACCACGCTCACGCTCGGCCTTGAGCTTGTCAAGAACCCACGCATACTTGAAGGAACCCTTGCCGAGTTCGGCGGCTTCCTATTGATTGAAGCATGATTAGCACAATGAACCATAAATCGAGTCAAAAGACGATGCGATTGAATGAGTGCATTTTGTTGGGACGATGGATGAGGGACAGTGGCGATAGCGGGGTTGCCAGAAAAAAATTGGCACCCCACTAAAGTCAAACGAGACAGCAGAAAAATTTGCTGCCAAAGGAGGGGTAGACAGGTCAAGCGGGGTGTGACGCTGCGAGGAAAACAGACAAGACCGAGAACAGAATTGTCGGACACGATTGTGCCAAG。
as shown in FIG. 3, the T0027 strain and the model strain Trichoderma pseudokoningii (Trichoderma koningiopsis) GJS 93-20 were aggregated into one branch and the supporting rate was 99%, which was identified as Trichoderma pseudokoningii.
Trichoderma pseudokoningii (Trichoderma koningiopsis) T0027 is preserved in 2023, 7 and 17 days to China general microbiological culture Collection center (CGMCC) with a preservation unit address of Beijing city, chaoyang area, north Chen Xiyu No. 1, 3, and a preservation number: CGMCC No.40722.
Example 2 antibacterial Effect of Trichoderma pseudokoningii Strain fermentation broth on Kiwi berry Soft rot
Preparing a fermentation concentrated solution: inoculating 6 Trichoderma T0027 bacterial cakes with the diameter of 6mm into 120mL PDA liquid culture medium, shake culturing with PDA small blocks as control group, culturing at 28deg.C under 180r/min for 7d on a constant temperature shaking table to obtain Trichoderma fermentation broth, filtering the fermentation broth with 4 layers of gauze, concentrating to 10 times of the original volume, concentrating the fermentation broth at 10000 r.min -1 Centrifuging for 5min, sucking supernatant, and filtering with sterile microporous membrane with diameter of 0.22 μm for 2-3 times for sterilization to obtain sterile fermentation filtrate.
Preparing a medicine-containing flat plate: mixing the trichoderma T0027 fermentation concentrate with PDA culture medium to prepare a plate with fermentation liquor concentration of 20%, 25%, 30%, 35% and 40%, inoculating the vine fruit fungus in the center of the PDA plate, inoculating the vine fruit fungus in the control group which is a plate mixed by PD A small shaking culture liquid, repeating each group for 3 times, culturing at constant temperature of 25 ℃ for 4 days to determine colony diameters, calculating bacteriostasis rate, and calculating the bacteriostasis activity of the trichoderma pseudokoningii T0027 on the vine fruit fungus as shown in figure 4 and table 1:
TABLE 1 inhibition of the sterile fermentation filtrate of strain T0027 against Botrytis cinerea
Note that: the different lower case letters after the same column of numbers represent a significant difference (P.ltoreq.0.05).
As shown in Table 1, the experimental results of this example demonstrate that the effect of antagonizing Brevibacterium is gradually enhanced as the concentration of the fermentation broth of Trichoderma pseudokoningii strain of T0027 is increased. When the concentration of the fermentation liquor reaches 40%, the antagonism effect reaches 78.01%, which shows that the metabolite of the trichoderma pseudokoningii strain has a strong inhibition effect on kiwi fruit soft rot.
EXAMPLE 3 study of the biological Properties of Trichoderma pseudokoningii strains
1. Influence of different temperatures on the growth of Trichoderma pseudokoningii strain T0027
The puncher is used for inoculating 6mm fungus cake in the center of a Petri dish, and respectively culturing in incubator at 15 deg.C, 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C and 40 deg.C for 3d, and measuring mycelium diameter (mm) by crisscross method.
2. Effect of different pH on growth of Trichoderma pseudokoningii strain T0027
The pH value of the Chlamydomonas medium is respectively adjusted to be 3, 4, 5, 6, 7, 8, 9 and 10 by using 1.0mol/L HCl or NaOH for 8 gradients, and the plates are prepared for standby. The mycelia were collected by punching with a 6mm punch at the center of the dish and incubated at 25℃for 3 days, and the diameter (mm) of the mycelia was measured by the crisscross method.
3. Influence of different nitrogen sources on the growth of Trichoderma pseudokoningii strain T0027
And (3) taking a Chlamydia culture medium as a basic culture medium, respectively replacing sodium nitrate in the basic culture medium with urea, peptone, ammonium sulfate, yeast powder, beef extract and ammonium chloride by the same mass, regulating the pH value to 7, taking a 6mm fungus cake in the center of a culture dish with different carbon sources by a puncher, culturing at 25 ℃ for 3d, and measuring the diameter (mm) of hyphae by the same method.
4. Influence of different carbon sources on the growth of Trichoderma pseudokoningii strain T0027
Taking a Chlamydia medium as a basic medium, respectively replacing sucrose in the basic medium with starch, glucose, fructose, maltose, lactose and glycerol, regulating the pH value to 7, taking a 6mm fungus cake by a puncher at the center of a culture dish with different carbon sources, culturing at 25 ℃ for 3d, and measuring the diameter (mm) of hypha by the same method.
As shown in FIG. 5, the Trichoderma pseudokoningii strain has remarkable difference of hypha growth speed under different conditions of pH, temperature, carbon source and nitrogen source. The mycelium growth speed of the strain is faster than other pH gradients when the pH is 5-7, and increases with the increase of the pH when the pH is 3-7, and decreases with the increase of the pH when the pH exceeds 7; the growth speed is the fastest when the temperature is 25 ℃, and the mycelium growth amount is gradually increased along with the temperature rise when the temperature is 15-25 ℃, and the mycelium growth is inhibited when the temperature is gradually increased beyond 25 ℃; the bacterial strain takes beef extract as a nitrogen source, the mycelium grows most rapidly, sodium nitrate, peptone and yeast powder are used for times, and the mycelium grows most slowly when urea is taken as the nitrogen source; when starch, maltose and lactose are used as carbon sources, the growth speed of hyphae of the strain is obviously higher than that of other carbon sources, sucrose and glucose are inferior, and fructose is slowest. The optimal culture pH of the trichoderma pseudokoningii strain is 5-7, the optimal culture temperature is 25 ℃, the optimal growth nitrogen source is beef extract, and the optimal growth carbon source is starch, maltose and lactose.
Example 4 biocontrol Effect of Trichoderma pseudokoningii fermentation broth on Kiwi Soft rot
Pretreatment of kiwi fruits: fresh kiwi fruits with consistent sizes are taken, after the surface of 75% alcohol is disinfected, the kiwi fruits are washed 3 times by sterile water, and the surface moisture of the kiwi fruits is dried.
Disease prevention test: a sterilized toothpick was used to puncture a wound of about 6mm on the surface of each fruit, control: sticking 6mm soft rot fungi cake (Botrytis cinerea) on the wound, and culturing in a 28 deg.C incubator for one week; treatment group: the soft rot pathogen bacterial cake with the thickness of 6mm is stuck on a wound, is cultured for 24 hours in a 28 ℃ incubator, is taken out, is soaked in bacterial strain T0027 sterile fermentation broth (40%v/v) for 20 minutes, is taken out, is dried and is cultured in the 28 ℃ incubator. Each treatment was repeated 6 times and the plaque diameter (mm) was measured daily using the crisscross method. Inhibition (%) = [ (control fruit spot area-treated fruit spot area)/control fruit spot area]X 100, fruit spot area = pi (d/2) 2 D is the average value of the diameter of the fruit spot, and the control effect of the trichoderma pseudokoningii fermentation liquor on the actinidia soft rot is shown in fig. 6 and table 2.
TABLE 2 control effect of aseptic fermentation filtrate of strain T0027 on actinidia soft rot
Note that: the different lower case letters after the same column of numbers represent significant differences (P.ltoreq.0.05)
As shown in Table 2, at the time of inoculation of 4d, the treatment group started to appear lesions with a diameter of 5.83mm, whereas the control group had a diameter of 18.33mm. When the strain is inoculated to the 6d, the diameter of the lesion of the control group reaches 35.17mm, and the diameter of the lesion of the treatment group is 18.08mm because the treatment group is inhibited by the fermentation liquor of the strain T0027, and the control effect of the strain T0027 on the actinidia soft rot of the actinidia reaches 73.56% at the 6d, so that the strain T0027 fermentation liquor has remarkable control effect on the actinidia soft rot of the actinidia.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (9)
1. Trichoderma pseudokoningii (Trichoderma koningiopsis) T0027, which is characterized in that the trichoderma pseudokoningii is preserved in 2023, 7 and 17 days to the China general microbiological culture Collection center (China Committee) with a preservation address of Beijing, chaoyang, north Chen Xili No. 1, 3, and a preservation number: CGMCC No.40722.
2. A microbial agent comprising the strain, fermentation broth or metabolite of trichoderma pseudokoningii T0027 of claim 1.
3. The microbial agent of claim 2, wherein the trichoderma pseudokoningii T0027 is cultured in the following manner:
the trichoderma pseudokoningii T0027 is inoculated into a Chlamydia medium which takes starch, maltose and lactose as carbon sources and beef extract as nitrogen sources for culture.
4. A microbial agent according to claim 3, wherein the culturing conditions are: the pH is 5-7, and the culture temperature is 15-25 ℃.
5. Use of trichoderma pseudokoningii T0027 according to claim 1 for the preparation of an antibacterial agent.
6. The use of claim 5, wherein the antimicrobial agent comprises an plasmodium antimicrobial agent.
7. A method for controlling soft rot of kiwi fruits, comprising the step of applying the trichoderma pseudokoningii T0027 of claim 1 or the microbial inoculum of claim 2 to kiwi fruits.
8. Use of trichoderma pseudokoningii T0027 according to claim 1 or the microbial inoculum according to claim 2 for preparing a biocontrol medicament for preventing and treating soft rot of kiwi fruits.
9. The use according to claim 8, wherein trichoderma pseudokoningii T0027 exerts a biocontrol effect of controlling soft rot of kiwi fruits by inhibiting plasmopara viticola.
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Citations (2)
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|---|---|---|---|---|
| US5244680A (en) * | 1990-03-30 | 1993-09-14 | The United States Of America As Represented By The Secretary Of Agriculture | Biocontrol of postharvest rots in fruit |
| CN114933974A (en) * | 2022-05-11 | 2022-08-23 | 华北理工大学 | Trichoderma pseudokoningii C5-9 and application thereof in antagonism of botrytis cinerea |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5244680A (en) * | 1990-03-30 | 1993-09-14 | The United States Of America As Represented By The Secretary Of Agriculture | Biocontrol of postharvest rots in fruit |
| CN114933974A (en) * | 2022-05-11 | 2022-08-23 | 华北理工大学 | Trichoderma pseudokoningii C5-9 and application thereof in antagonism of botrytis cinerea |
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| ON-UMA RUANGWONG等: "Biocontrol Mechanisms of Trichoderma koningiopsis PSU3-2 against Postharvest Anthracnose of Chili Pepper", 《J FUNGI (BASEL)》, 7 April 2021 (2021-04-07) * |
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