CN116904322B - Biocontrol strain mould and application thereof - Google Patents
Biocontrol strain mould and application thereof Download PDFInfo
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- CN116904322B CN116904322B CN202310755231.2A CN202310755231A CN116904322B CN 116904322 B CN116904322 B CN 116904322B CN 202310755231 A CN202310755231 A CN 202310755231A CN 116904322 B CN116904322 B CN 116904322B
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- 230000000443 biocontrol Effects 0.000 title claims abstract description 33
- 241000222354 Trametes Species 0.000 claims abstract description 27
- 244000144730 Amygdalus persica Species 0.000 claims abstract description 16
- 235000006040 Prunus persica var persica Nutrition 0.000 claims abstract description 16
- 238000009629 microbiological culture Methods 0.000 claims abstract description 3
- 238000004321 preservation Methods 0.000 claims abstract 2
- 241001450911 Circinella Species 0.000 claims description 18
- 239000000725 suspension Substances 0.000 claims description 9
- 244000005700 microbiome Species 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 7
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 5
- 230000000813 microbial effect Effects 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 241000233866 Fungi Species 0.000 claims description 4
- 230000002401 inhibitory effect Effects 0.000 claims description 4
- 241000221079 Euphorbia <genus> Species 0.000 claims description 2
- 239000004480 active ingredient Substances 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 230000002262 irrigation Effects 0.000 claims description 2
- 238000003973 irrigation Methods 0.000 claims description 2
- 238000007790 scraping Methods 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 241000235527 Rhizopus Species 0.000 claims 1
- 239000002068 microbial inoculum Substances 0.000 claims 1
- 230000002265 prevention Effects 0.000 claims 1
- 241000196324 Embryophyta Species 0.000 abstract description 13
- 230000000694 effects Effects 0.000 abstract description 7
- 235000001018 Hibiscus sabdariffa Nutrition 0.000 abstract description 4
- 235000005291 Rumex acetosa Nutrition 0.000 abstract description 4
- 240000007001 Rumex acetosella Species 0.000 abstract description 4
- 230000005764 inhibitory process Effects 0.000 abstract description 4
- 235000003513 sheep sorrel Nutrition 0.000 abstract description 4
- 230000001737 promoting effect Effects 0.000 abstract description 3
- 241000321980 Trametes gibbosa Species 0.000 abstract description 2
- 241000894006 Bacteria Species 0.000 description 9
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- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 241000221198 Basidiomycota Species 0.000 description 2
- 241000222341 Polyporaceae Species 0.000 description 2
- 241000222383 Polyporales Species 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000003073 embolic effect Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 150000007523 nucleic acids Chemical group 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 239000006041 probiotic Substances 0.000 description 2
- 235000018291 probiotics Nutrition 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000021749 root development Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 241000222485 Agaricales Species 0.000 description 1
- 241000009794 Agaricomycetes Species 0.000 description 1
- 241000222382 Agaricomycotina Species 0.000 description 1
- 241000222518 Agaricus Species 0.000 description 1
- 241000186146 Brevibacterium Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 241000219071 Malvaceae Species 0.000 description 1
- 241000235395 Mucor Species 0.000 description 1
- 241000222385 Phanerochaete Species 0.000 description 1
- 241000227425 Pieris rapae crucivora Species 0.000 description 1
- 241000219492 Quercus Species 0.000 description 1
- 101100213970 Schizosaccharomyces pombe (strain 972 / ATCC 24843) ypt3 gene Proteins 0.000 description 1
- 241001106462 Ulmus Species 0.000 description 1
- 230000036579 abiotic stress Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000004790 biotic stress Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
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- 238000005286 illumination Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
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- 231100000419 toxicity Toxicity 0.000 description 1
- 230000035899 viability Effects 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
-
- 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
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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
- A01P21/00—Plant growth regulators
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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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Abstract
The invention discloses a biocontrol strain, namely, a strain number of the biocontrol strain, namely, a strain number of JSAFC and 1448, which is preserved in China general microbiological culture collection center (CGMCC) at the date of 31 of 05 month of 2023, wherein the preservation number is CGMCC No.40643. The strain can effectively inhibit the growth of the trametes maze (Trametesgibbosa), and the inhibition rate of the opposite culture of the flat plate reaches 71.04 percent. The biocontrol strain sorrel (circinellosp.) JSAFC 1448:1448 provided by the invention can effectively inhibit the growth of trametes maze, and has obvious effect of promoting the growth of peach plants.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and relates to a biocontrol strain sorrel and application thereof.
Background
The Trametes maze (Trametes gibbosa) belongs to Basidiomycota (Basidiomycota), agaricus subspecies (Agaricomycotina), agaricales (Agaricomycetes), polyporales (Polyporales), polyporaceae (Polyporaceae), and Trametes (Trametes). The trametes maze, also called trametes maze and Phanerochaete rupestris, is a common white rot fungus at home and abroad, grows on the inverted wood, dead wood, stump or standing tree of broad-leaved trees such as oak, elm, basswood, peach and the like, causes the wood to be white spongy decay, and affects the quality and economic value of the wood.
At present, chemical pesticides are common plant disease control methods, but a plurality of pathogenic bacteria can generate drug resistance after long-term use, the disease prevention effect is reduced, the environment is polluted after frequent and high-concentration use, the excessive residual toxicity threatens the health of human beings, the quality and the yield of agricultural products in China are limited, the sustainable development of agriculture is influenced, and the ecological balance is destroyed. Biological control is accepted worldwide as a promising alternative, and therefore, intensive development of biological control research is essential and urgent for sustainable development of agriculture.
Many beneficial microorganisms exist in the plant environment, which are not only non-pathogenic to the plant, but some even induce resistance to the plant, improving the viability of the plant under biotic or abiotic stress conditions. In addition, the microorganisms are derived from natural environment, have no pollution to the environment, and are beneficial to maintaining ecological balance when being used for preventing and treating plant diseases. Nowadays, more and more researchers are working to find probiotics as a method for biological control to treat plant diseases.
In view of the above, the prior art also lacks a green and efficient environmental probiotic for preventing wood rot.
Disclosure of Invention
A first object of the present invention is to provide a mould (circinella sp.).
A second object of the present invention is to provide a liquid microbial inoculant comprising sorrel (circinella sp.).
The third object of the invention is to provide the use of Mucor (CIRCINELLA sp.) in the control of peach tree rot caused by trametes maze.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
In a first aspect of the present invention, there is provided a biocontrol strain, the strain number of which is JSAFC 1448, the collection number of which is CGMCC No.40643, and the collection number of which is the collection of microorganisms from China general microbiological culture Collection center, the collection of microorganisms from the China Committee for culture Collection of microorganisms, the collection address of which is the national institute of microbiology, national institute 3, of the Chaoyang area North Star, beijing.
In a second aspect of the invention, the invention provides a biocontrol bacterium comprising the nucleic acid fragment of CIRCINELLA sp.jsafc 1448 of the first aspect of the invention.
Further, the base sequence of the nucleic acid fragment includes SEQ ID NO.1.
In a third aspect of the invention, the invention provides a biocontrol microbial agent, wherein the active ingredient of the biocontrol microbial agent is the biocontrol fungus, or the engineering bacteria.
In a fourth aspect, the invention provides the use of the biocontrol strain, the engineering bacteria or the biocontrol strain agent in inhibiting growth of trametes maze or in inhibiting growth of trametes maze.
In a fifth aspect, the invention provides the use of a biocontrol mould according to the invention (CIRCINELLA sp.) for promoting peach growth.
Further, the application is specifically: the prepared spore suspension of the biocontrol strain, namely JSAFC and 1448, is used for preventing and controlling peach tree rot caused by the trametes maze in a root irrigation mode.
Further, the preparation method of the spore suspension of the biocontrol strain, namely JSAFC and 1448, comprises the following steps:
(1) Taking biocontrol strain euphorbia JSAFC 1448, picking hypha by an inoculating needle under aseptic condition, inoculating sterilized PDA culture medium, and dark culturing at 25deg.C under 65% humidity for 5-7d;
(2) Scraping hyphae under aseptic condition, washing the hyphae with aseptic water and filtering with gauze;
(3) Under aseptic condition, the spore concentration is adjusted to 10 5/ml by using aseptic water.
Further, the spore suspension was applied at 5 mL/time/7 d for 30d.
The biocontrol strain sorrel (circinellosp.) JSAFC 1448:1448 provided by the invention can effectively inhibit the growth of trametes maze, and has obvious effect of promoting the growth of peach plants.
Drawings
Fig. 1: CIRCINELLA sp.jsafc 1448 phylogenetic tree.
Fig. 2: the biocontrol bacterium Circiella sp.JSAFC 1448 is cultivated in a way of being opposite to the trametes maze;
fig. 3: the effect of biocontrol bacterium CIRCINELLA sp.jsafc 1448 on peach tree rot caused by trametes maze in example 3.
Detailed Description
The following detailed description is exemplary and is intended to provide further explanation of the application. Unless defined otherwise, 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 application belongs.
As used herein, the term "biocontrol bacteria" refers to beneficial microorganisms, mainly bacteria, fungi, actinomycetes, which control plant diseases.
Example 1: isolation and identification of Circiella sp.JSAFC 1448
1. Separation of Circiella sp.JSAFC 1448
Taking soil with good Hainan Danzhou growth condition, separating from the soil to obtain CIRCINELLA sp.JSAFC 1448, wherein colony characteristics are as follows: culturing on PDA plate culture medium, and the colony grows fast and is in amorphous cotton flocculent shape with white surface and attached to the surface of the culture medium.
2. CIRCINELLA sp.jsafc 1448 molecular biology identification
(1) Genomic DNA of strain JSAFC 1448 was extracted using TIANGEN company kit.
(2) The ITS genes of the genomic DNA were amplified separately. The DNA was amplified using a reaction volume of 30. Mu.L containing 15. Mu.L of 2X EASYTAQ PCR Supermix (+dye), 1. Mu.L (10. Mu.M) of each of the primers ITS4/ITS5, 2. Mu.L of template DNA and 11. Mu.L of ddH 2 O, and the PCR amplification procedure was as follows: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 54℃for 45s, elongation at 72℃for 60s for 35 cycles; finally, the extension is carried out for 10min at 72 ℃.
(3) After electrophoresis of the amplified PCR product by 1% agarose gel, observing under an ultraviolet lamp, and sending the PCR product with the target band to Nanjing qingke biotechnology Co-Ltd for sequencing, wherein the sequencing result is as follows: the ITS gene sequence of the strain JSAFC 1448 in the step1 is SEQ ID NO.1.
(4) The SEQ ID NO.1 sequences were aligned on the NCBI database website and phylogenetic trees were established, see FIG. 1. The comparison results are shown in Table 1. From the results of table 1, it was estimated that biocontrol bacterium JSAFC 1448 of the present invention was a circumflex sp.
TABLE 1SEQ ID NO.1 sequence alignment
Example 2: inhibition effect of Circiella sp.JSAFC 1448 on trametes maze
(1) Inoculating a bacterial cake with the diameter of 4mm of the trametes maze to one side of a PDA culture medium flat plate on an ultra-clean workbench to serve as a control group, and simultaneously inoculating a bacterial cake with the diameter of 4mm of CIRCINELLA sp.JSAFC 1448 to one symmetrical side of the PDA culture medium to serve as an experimental group; and medium plates inoculated with only trametes maze were used as controls.
(2) Culturing the experimental group and the control group in the step (1) in an incubator at a constant temperature of 25 ℃ in an inversion manner under the condition of illumination for 8 hours and then in the dark for 16 hours.
(3) Culturing the culture medium of the experimental group and the control group in the step (2) artificially for 7 days according to the culture conditions, respectively measuring the colony radius of the trametes maze by a plate counter method, and observing the inhibition effect of CIRCINELLA sp.JSAFC 1448 on the trametes maze.
As shown in FIG. 2, the radius of the trametes maze colony of the blank control group is 2.97cm; in the experimental group, the average radius of the trametes maze pathogen is only 0.86cm, which shows that CIRCINELLA sp.JSAFC 1448 has remarkable inhibition effect on the trametes maze.
Example 3: CIRCINELLA influence of sp.jsafc 1448 on peach tree rot
(1) Strain CIRCINELLA sp.JSAFC 1448 and Brevibacterium maze JSAFC 1020 were inoculated into 9cm diameter dishes (15 ml PDA medium in each) and incubated at 25℃in the dark for 7d.
(2) CIRCINELLA sp.jsafc 1448 and trametes maze JSAFC 1020 cultured for 7 days were respectively scraped under aseptic conditions to obtain mycelia, the mycelia were rinsed with aseptic water and filtered with gauze, and then the spore concentration was adjusted to 10 5/ml.
(3) Pot with diameter of 12cm is selected, 1 healthy peach seedling (variety is sand red) with consistent growth condition is transplanted into each pot, 2 groups (control group and treatment group) are tested, and 10 replicates are set for each treatment. The control group was: 5mL of the suspension of the spores of the embolic material JSAFC 1020:1020 of the embolic material is irrigated into each pot; the experimental group is: each pot was irrigated with 5mL of CIRCINELLA sp.JSACA 1448 spore suspension and 5mL of trametes maze JSAFC spore suspension.
(4) And irrigating each group of peach seedlings every 2 days, counting the diameter and the plant height of the peach tree after 30 days, and observing the root development condition of the peach seedlings.
The results are shown in FIG. 3 and Table 2.
TABLE 2 statistical results of peach Tree height and diameter
Wherein, the numerical expression modes of the plant height and the diameter in the table are as follows: mean ± standard error.
From the data in fig. 3 and table 2, the plant height, diameter and root development of the experimental group are obviously better than those of the control group, and CIRCINELLA sp.jsafc 1448 can be seen to have an inhibitory effect on the peach saprophytic bacteria.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
Claims (6)
1. A biocontrol strain of Rhizopus (CIRCINELLA sp.) features that its strain number is JSAFC 1448 and its preservation number is CGMCC No.40643, which is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the month 31 of 2023.
2. The biocontrol microbial agent is characterized in that the active ingredient of the biocontrol microbial agent is the biocontrol fungus volume mould as claimed in claim 1.
3. Use of the biocontrol strain of claim 1 or the biocontrol strain of claim 2 for inhibiting growth of trametes maze.
4. The application of the biocontrol strain mildew and the biocontrol strain microbial inoculum of claim 1 to the prevention and treatment of peach tree rot caused by trametes maze, which is characterized in that the application is specifically as follows: the prepared spore suspension of the biocontrol strain, namely JSAFC and 1448, is used for preventing and controlling peach tree rot caused by the trametes maze in a root irrigation mode.
5. The use according to claim 4, wherein the spore suspension of biocontrol mould JSAFC 1448 is prepared as follows: (1) Taking biocontrol strain euphorbia JSAFC 1448, picking hypha by an inoculating needle under aseptic condition, inoculating sterilized PDA culture medium, and dark culturing at 25deg.C under 65% humidity for 5-7d; (2) Scraping hyphae under aseptic condition, washing the hyphae with aseptic water and filtering with gauze; (3) Under aseptic condition, the spore concentration is adjusted to 10 5/ml by using aseptic water.
6. The use according to claim 5, wherein the spore suspension is applied at a rate of 5 mL/time/7 d for 30d.
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| 辽宁碱蓬根际土壤真菌多样性对4种重金属胁迫的响应;官丽娜等;《辽宁农业科学》;20151231(第06期);第1-5页 * |
| 辽宁碱蓬根际土壤真菌多样性的季节变化及其耐盐性;邵璐等;《生态学报》;20150709;第36卷(第04期);第162-169页 * |
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