CN117887589B - Trichoderma FJ059 capable of parasitizing sclerotium of southern blight and application thereof - Google Patents
Trichoderma FJ059 capable of parasitizing sclerotium of southern blight and application thereof Download PDFInfo
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- CN117887589B CN117887589B CN202410089252.XA CN202410089252A CN117887589B CN 117887589 B CN117887589 B CN 117887589B CN 202410089252 A CN202410089252 A CN 202410089252A CN 117887589 B CN117887589 B CN 117887589B
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- 241000223259 Trichoderma Species 0.000 title abstract description 47
- 241001558929 Sclerotium <basidiomycota> Species 0.000 title description 23
- 235000002566 Capsicum Nutrition 0.000 claims abstract description 11
- 239000006002 Pepper Substances 0.000 claims abstract description 8
- 235000016761 Piper aduncum Nutrition 0.000 claims abstract description 8
- 235000017804 Piper guineense Nutrition 0.000 claims abstract description 8
- 235000008184 Piper nigrum Nutrition 0.000 claims abstract description 8
- 240000008574 Capsicum frutescens Species 0.000 claims abstract description 3
- 239000001390 capsicum minimum Substances 0.000 claims abstract description 3
- 238000004321 preservation Methods 0.000 claims description 6
- 241001530056 Athelia rolfsii Species 0.000 claims description 3
- 241000302866 Trichoderma semiorbis Species 0.000 claims 5
- 244000203593 Piper nigrum Species 0.000 claims 1
- 241000221662 Sclerotinia Species 0.000 claims 1
- 239000002689 soil Substances 0.000 abstract description 24
- 241000722363 Piper Species 0.000 abstract description 7
- 201000010099 disease Diseases 0.000 abstract description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 4
- 238000000034 method Methods 0.000 abstract description 4
- 230000035784 germination Effects 0.000 description 12
- 238000012258 culturing Methods 0.000 description 11
- 239000000725 suspension Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 241000192705 Aphanothece Species 0.000 description 3
- 241001460073 Trichoderma asperellum Species 0.000 description 3
- 238000012271 agricultural production Methods 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 239000008223 sterile water Substances 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000013043 chemical agent Substances 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 235000000832 Ayote Nutrition 0.000 description 1
- 240000004244 Cucurbita moschata Species 0.000 description 1
- 235000009854 Cucurbita moschata Nutrition 0.000 description 1
- 235000009804 Cucurbita pepo subsp pepo Nutrition 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 244000183278 Nephelium litchi Species 0.000 description 1
- 241000219000 Populus Species 0.000 description 1
- 241001646398 Pseudomonas chlororaphis Species 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 244000000005 bacterial plant pathogen Species 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 210000004262 dental pulp cavity Anatomy 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000005553 drilling Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- 230000000243 photosynthetic effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 235000015136 pumpkin Nutrition 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
Classifications
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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
-
- 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
-
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/885—Trichoderma
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- Health & Medical Sciences (AREA)
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- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Mycology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Pest Control & Pesticides (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Plant Pathology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Environmental Sciences (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Botany (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
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- Biomedical Technology (AREA)
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Abstract
The invention provides trichoderma FJ059 and an application method thereof for preventing and treating pepper southern blight. Wherein the trichoderma FJ059 can inhibit the growth of the southern blight by 100% when being cultivated on a PDA flat plate in a way of being opposite to the southern blight for 15 days; the trichoderma FJ059 can kill 100% of the southern blight propagules after being co-cultured with the southern blight propagules on the soil surface for 15 days; the trichoderma FJ059 and the southern blight propagules are co-cultured in soil for 15 days, so that 100% of the southern blight propagules can be killed; the pepper seedlings inoculated with trichoderma FJ059 did not find the phenomenon of southern blight within one week. The invention provides possibility for preventing and controlling the southern blight germ and the capsicum southern blight, and has important significance for effectively preventing and controlling the disease.
Description
Technical Field
The invention relates to a trichoderma strain for agricultural production, in particular to trichoderma FJ059 capable of parasitizing sclerotium of southern blight and application thereof, and the Latin classification of the strain is named as: trichodema semiorbis.
Background
At present, the prevention and control of plant diseases are mainly performed by chemical agents, the use of the chemical agents causes plant pathogenic bacteria to generate drug resistance, the prevention and control difficulty is increased, the chemical pesticides pollute and destroy the environment, the biological chain is destroyed, and the pesticide residues cause great harm to human bodies. Therefore, finding an environmentally friendly and pollution-free control technology is increasingly important.
Trichoderma belongs to the phylum of Deuteromycotina (Deuteromycotina), class Cellularomyces (Hyphomycetes), order Cellulare (Hyphomycetales), family Comametaceae (Moniliales), genus Trichoderma (Trichoderma), is a widely-distributed saprophytic fungus, exists in ecological environments such as soil, root canal, leaf canal and seeds, and it is reported that more than 300 Trichoderma strains are found in natural environments at present. Studies have found that trichoderma is an important species in agricultural production, e.g., CN108207944a discloses wettable powders of trichoderma asperellum HN compositions, liu Zhaoying et al (plant research, 2018, 38 (1): 64-74) reported the effect of trichoderma asperellum combination application on poplar growth and photosynthetic characteristics, CN107142213a discloses the growth promoting effect of trichoderma asperellum. Therefore, the search for new trichoderma strains has important significance for agricultural production, especially for disease control of crops.
Disclosure of Invention
It is an object of the present invention to provide trichoderma FJ059, which trichoderma FJ059 is used for controlling pepper southern blight caused by southern blight germ (Sclerotium rolfsii) and the propagule structure (Sclertia) thereof.
Detailed Description
The invention is described in detail below in connection with specific embodiments, which are intended to be illustrative rather than limiting.
Example 1: isolation and preservation of Trichoderma FJ059
1. Trichoderma FJ059, collected from pumpkin land in Fuzhou, fujian province.
Collecting a soil sample: the soil is collected by a five-point diagonal sampling method. Selecting 5 equal division points on the diagonal line of the collected land, drilling about 15-20cm of soil samples at each point by using a soil sampler with the depth of 25cm, uniformly mixing 5 parts of soil, and filling about 50g of soil samples into a self-sealing bag by a four-division method. Subpackaging into 50mL plastic bottles in a laboratory, and preserving at 4 ℃ for later use.
Isolation of strains: 10g of soil sample was taken from a refrigerator by the dilution plate method and placed in a triangular flask containing 90mL of sterilized water and 5-10 glass beads having a diameter of 4mm, followed by shaking for 30min on a shaking table of 200 r/min. Pouring 5mL into a triangular flask containing 45mL of sterilized water, uniformly mixing, then sucking 5mL, transferring into the triangular flask containing 45mL of sterilized water, fully uniformly mixing, sucking 0.2mL on a flat plate with the diameter of 9cm, uniformly coating by using a coater, and culturing in a constant-temperature incubator at 28 ℃. And (3) picking colonies producing green conidia after 48-72 hours, transferring to a PDA plate, continuously culturing in a constant temperature incubator at 28 ℃, and transferring to the PDA plate again for constant temperature culturing at 28 ℃ when the diameters of the colonies are 4-5 cm.
2. Trichoderma FJ059 strain preservation
Trichoderma FJ059 is subjected to vacuum freeze-drying preservation in China general microbiological culture Collection center (CGMCC) on 11 and 13 days of 2023, the preservation number is CGMCC No.40920, and the preservation period is 30 years.
3. The identification of binding molecule biology (Gene sequencing) by morphological identification was Trichodema semiorbis, which has been published in ：The Highly Contiguous Genome Resource ofTrichoderma semiorbis FJ059,a Biological ControlAgent for Litchi DownyBlight.
Example 2: plate counter culture
Inoculating Trichoderma FJ059 and southern blight onto PDA plates, culturing at 28deg.C for 72 hr, respectively picking bacterial cakes from colony edges by 5mm puncher, and simultaneously transferring onto PDA plates with diameter of 90mm, wherein the distance between two bacterial cakes is 70mm; the growth radius of trichoderma FJ059 is observed and measured after the plate is placed at 28 ℃ for 15 days for culture, the bacteriostasis rate is calculated, a PDA plate which is not connected with trichoderma and only connected with southern blight is used as a control, and the test is repeated for 5 times. The bacteriostasis rate is calculated according to the following formula:
Antibacterial ratio (%) = ((radius of control pathogen-radius of counter pathogen)/radius of control pathogen) ×100%
Calculated as follows: the growth inhibition rate of trichoderma FJ059 to southern blight is 100%.
Example 3: co-culturing Trichoderma FJ059 and southern blight propagule on soil surface
Inoculating southern blight bacteria on a blank PDA plate, and culturing at 30 ℃ for 15 days to obtain a propagule (sclerotium); trichoderma FJ059 strain was inoculated on PDA plate, cultured at 28℃for 5 days, and then Trichoderma spores on PDA plate were scraped off with a coater to prepare spore suspension having a concentration of 1X 10 7 CFU/mL.
20G of moist soil with the water content of 80% is filled in a culture dish with the diameter of 90mm, 9 sclerotium are placed in a mode of 3 multiplied by 3, 10 mu L of trichoderma spore suspension is dripped into each sclerotium, the sclerotium is cultured for 15 days at the temperature of 28 ℃, the sclerotium is taken out, the surface is sterilized, the germination rate of the sclerotium is counted in a blank PDA plate singly, and a soil plate without the trichoderma spore suspension and only the sclerotium is placed is used as a control. The test was repeated 5 times. The bacteriostasis rate is calculated according to the following formula:
germination rate (%) = ((control germination amount-treated germination amount)/control germination amount) ×100%
The statistics are carried out to obtain: the germination rate of sclerotium was 0% after 15 days of inoculation of the trichoderma spore suspension.
Example 4: co-culturing Trichoderma FJ059 and Aphanothece propagule in soil
Inoculating southern blight bacteria on a blank PDA plate, and culturing at 30 ℃ for 15 days to obtain a propagule (sclerotium); trichoderma FJ059 strain was inoculated on PDA plate, cultured at 28℃for 5 days, and then Trichoderma spores on PDA plate were scraped off with a coater to prepare spore suspension having a concentration of 1X 10 7 CFU/mL.
A culture dish with a diameter of 90mm is filled with 20g of moist soil with a water content of 80%, 9 sclerotium are buried in the soil in a 3X 3 mode, 10 mu L of trichoderma spore suspension is dripped into the soil above each sclerotium, the sclerotium is taken out after 15 days of culture at 28 ℃, the sclerotium germination rate is counted in a blank PDA plate after surface sterilization, and a soil plate in which only the sclerotium is buried without dripping the trichoderma spore suspension is used as a control. The test was repeated 5 times. The bacteriostasis rate is calculated according to the following formula:
germination rate (%) = ((control germination amount-treated germination amount)/control germination amount) ×100%
The statistics are carried out to obtain: the germination rate of sclerotium was 0% after 15 days of inoculation of the trichoderma spore suspension.
Example 5: trichoderma FJ059 pot experiment for preventing and treating capsicum southern blight
Inoculating southern blight bacteria on a blank PDA plate, and culturing at 30 ℃ for 15 days to obtain a propagule (sclerotium); trichoderma FJ059 strain was inoculated on PDA plate, cultured at 28℃for 5 days, and then Trichoderma spores on PDA plate were scraped off with a coater to prepare spore suspension having a concentration of 1X 10 7 CFU/mL.
Mixing soil with the Aphanothece curcas propagule, wherein the density is based on 10g of soil with 1 sclerotium. And (5) inoculating pepper seedlings according to the eight-leaf period. The treatments were as follows:
(1) CK: 2000. Mu.L of sterile water;
(2) M: 1000. Mu.L of sterile water was added with 100g of sclerotium soil;
(3) T: 1000. Mu.L of sterile water was added to 1000. Mu.L of 1X 10 7 Trichoderma spore liquid;
(4) M+T:100g of sclerotium soil is added with 1000 mu L of 1X 10 7 trichoderma spore liquid;
(5) M+tl: 100g of sclerotium soil was inoculated 72h before 1000. Mu.L of 1X 10 7 Trichoderma spore liquid was added.
Observing and counting the disease condition of each treated pepper seedling after one week of inoculation, and controlling the pepper southern blight by using trichoderma spore liquid. The morbidity is calculated according to the following formula:
Incidence (%) = ((control healthy seedling number-treated healthy seedling number)/control healthy seedling number) ×100%
The statistics are carried out to obtain: CK incidence was 0%; the incidence of M is 65%; the incidence of T is 0%; the incidence of M+T is 0%; the incidence of m+tl was 0%.
Drawings
FIG. 1 is a graph showing the results of culturing Trichoderma FJ059 against a plate of southern blight bacteria for 15 days.
FIG. 2 is a graph showing the results of co-culturing Trichoderma FJ059 and Aphanothece propagules on soil surface for 15 days.
FIG. 3 is a graph showing the result of controlling the pepper southern blight by trichoderma FJ 059.
Claims (3)
1. Trichoderma semiorbis strain FJ059, the preservation number of which is CGMCC No.40920.
2. Use of Trichoderma semiorbis strain FJ059 according to claim 1, characterized in that: trichoderma semiorbis strain FJ059 is used for preventing and treating pepper southern blight caused by southern blight germ (Sclerotium rolfsii).
3. Use of Trichoderma semiorbis strain FJ059 according to claim 2, characterised in that: trichoderma semiorbis strain FJ059 is used for preventing and controlling capsicum sclerotinia caused by a southern blight germ (Sclerotium rolfsii) propagule structure (Sclertia).
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| CN117887589B true CN117887589B (en) | 2024-09-20 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101928673A (en) * | 2010-07-14 | 2010-12-29 | 洪亚辉 | Trichoderma harzianum |
| CN110272832A (en) * | 2019-08-05 | 2019-09-24 | 海南大学 | Trichoderma asperellum FJ069 and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3691264B2 (en) * | 1997-11-13 | 2005-09-07 | クミアイ化学工業株式会社 | A new strain of Trichoderma atrobide |
| CN115975816A (en) * | 2022-10-12 | 2023-04-18 | 广西壮族自治区农业科学院 | Trichoderma viride strain RM9 for antagonizing jasmine southern blight and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101928673A (en) * | 2010-07-14 | 2010-12-29 | 洪亚辉 | Trichoderma harzianum |
| CN110272832A (en) * | 2019-08-05 | 2019-09-24 | 海南大学 | Trichoderma asperellum FJ069 and its application |
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