CN118185824A - Serratia for antagonizing Pyricularia oryzae and application thereof - Google Patents
Serratia for antagonizing Pyricularia oryzae and application thereof Download PDFInfo
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- 108020004465 16S ribosomal RNA Proteins 0.000 claims abstract description 4
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- 239000003814 drug Substances 0.000 claims description 3
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- 239000005557 antagonist Substances 0.000 claims 1
- 241000894006 Bacteria Species 0.000 abstract description 53
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- 229920001817 Agar Polymers 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
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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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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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
- A01N63/20—Bacteria; 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
- 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
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/425—Serratia
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Abstract
The invention provides a Serratia GSRH-5-468 strain with good antagonistic effect on rice blast bacteria, which is preserved in China center for type culture collection, address: chinese, university of martial arts; post code 430072, with a preservation date of 2024, 3 months and 25 days, and a preservation number of: cctccc NO: m2024552; the 16S rDNA sequence of the strain is shown as SEQ ID NO. 1. The Serratia GSRH-468 strain has strong antibacterial property, and can be used for preparing a microbial preparation for treating/preventing rice blast.
Description
Technical Field
The invention relates to a Serratia strain which has a strong antibacterial antagonistic effect on rice blast bacteria, and belongs to the technical field of microorganisms.
Background
. Rice blast (Rice blast) is one of the most serious diseases in Rice production, and is harmful in various stages of Rice growth and development. Rice blast is a fungal disease that damages rice plants by rice blast bacteria (Magnaporthe oryzae). The disease becomes a key problem for ensuring the continuous high and stable yield of the rice. On rice leaves, rice blast causes leaf to fade, fade and fade; infection in the late growth stage of rice can lead to incomplete rice filling.
At present, chemical pesticides, biological control, cultivation management, disease resistance breeding and the like are main methods for controlling rice blast. Among them, chemical pesticides are the simplest and most effective methods for controlling, but chemical control is harmful to the environment, and can pollute surrounding animals, plants, etc., and can easily cause pathogenic bacteria to produce drug resistance to the chemical agents.
Microbial pesticides, which are biological agents, are prepared by further refining and further processing by means of bioactive components of microorganisms, and are effective components beneficial to plant pest control and agents capable of guaranteeing normal growth of plants. The microbial pesticide has a plurality of advantages, firstly, in the prevention and treatment of plant diseases and insect pests, the microbial pesticide can reduce or avoid pollution to the environment and also avoid harm to other animals and plants. Secondly, most of beneficial bacteria from plant rhizosphere soil or plant roots, leaves, fruits and other parts can be used as microbial pesticides, which can not destroy a soil micro-ecological system, can also effectively improve soil, promote the formation of organic matters in the soil, prevent or relieve salinization of the soil, prevent soil hardening and cultivate fertility. Most microbial pesticides consist of beneficial bacteria and metabolites thereof, so that the microbial pesticides can be decomposed by nature while being applied, further cyclic utilization is realized, and the microbial pesticides are the precondition of avoiding drug resistance of plant diseases and insects, thereby realizing efficient control effect.
The screening and application of rice blast bacteria antagonistic bacteria to biological control are the main measures for dealing with rice blast at present. Antagonistic bacteria with good prevention effect on rice blast can be obtained by screening, and more strain resources and measures can be provided for biological prevention and control of rice blast. And endophytic bacteria have the characteristics of easy culture and fermentation, shorter time consumption, simpler secondary metabolite extraction measures and the like, and can be better applied to large-scale prevention and treatment of rice blast. At present, biological control means gradually enter an important research range due to the advantages of low cost, high benefit, less pollution and the like. Under the condition of solving the problem of rice blast, the biological control of rice diseases by endophytes becomes a main research hot spot.
At present, related researches on rice blast bacteria antagonistic bacteria are more, for example, streptomyces with antagonism to rice blast of Chinese invention patent CN200710055933.0 and a preparation method thereof, bacillus berryis for antagonizing rice blast bacteria and producing gamma-polyglutamic acid of Chinese invention patent CN202011231527.7 and application thereof, a strain of rice endophyte OsiLf-2 for antagonizing rice blast bacteria in vitro and high efficiency, a strain of Bacillus subtilis GB519 for promoting rice growth and antagonizing rice blast bacteria of Chinese invention patent CN201810228606.9, application thereof and the like. But there are no reports in the art concerning Serratia sp.
Disclosure of Invention
The invention solves the problems in the background technology, and provides a Serratia (Serratia sp.) GSRH-468 strain with good antagonism effect on rice blast bacteria, which has strong antibacterial property and can be used for preparing microbial preparations for treating/preventing rice blast.
The applicant screens a novel strain from ginseng endophytes, namely a Serratia GSRH-5-468 strain which is preserved in China center for type culture collection, and addresses: chinese, university of martial arts; post code 430072, with a preservation date of 2024, 3 months and 25 days, and a preservation number of: cctccc NO: m2024552; the 16S rDNA sequence of the strain is shown as SEQ ID NO. 1.
The Serratia GSRH-468 strain can be used for preparing an antagonistic bacterium agent of rice blast bacteria and a microbial preparation for treating/preventing rice blast.
Compared with the prior art, the invention has the following advantages:
The antagonistic efficiency of GSRH-5-468 strain provided by the application is as high as 42.61%. The high antagonistic efficiency of the antagonistic bacteria indicates that the antagonistic bacteria has strong capability of antagonizing the rice blast bacteria, and can be used for developing microbial pesticides for preventing and treating the rice blast bacteria. Microscopic observation of the rice blast fungus hyphae at the antagonistic end shows that GSRH-5-468 strain can cause rupture and malformation of the rice blast fungus hyphae and also can cause reduction of the number of the hyphae, and GSRH-5-468 strain can effectively inhibit the rice blast fungus.
Drawings
FIG. 1 is a graph showing the results of activation of endophytic bacteria of ginseng in an embodiment of the present application;
FIG. 2 is a graph showing the result of activation of Pyricularia oryzae in the example of the present application;
FIG. 3 is a graph showing the results of primary screening for antagonistic bacteria in the example of the present application;
FIG. 4 is a graph showing the results of rescreening of antagonistic bacteria in an example of the application;
FIG. 5 is a bar graph showing antagonistic efficiency of 24 antagonistic bacteria according to an example of the present application;
FIG. 6 is a diagram showing the state of mycelia after antagonizing Pyricularia oryzae respectively in the strains selected in the examples of the present application.
Detailed Description
The present invention will be described in detail with reference to the following examples, but the scope of the present invention is not limited to the following examples.
Activation and cultivation of strains
Main reagent
(1) Culture medium: in this example, LB solid medium and oat solid medium are mainly used, and specific formulas are shown in the following table.
TABLE 1LB solid medium (500 mL) formulation
The preparation method comprises the following steps: 5.0g of tryptone (TRYPTONE) and 2.5g of YEAST EXTRACT powder (YEAST EXTRACT) and 5.0g of NaCl and 7.5g of agar are weighed first, then the weighed medicines are sequentially added into a clean conical flask (500 mL) prepared in advance, ultrapure water is added into the conical flask by using a cylinder, the volume is fixed to 500mL, the conical flask is shaken to enable the solute to be approximately dissolved, and the conical flask is sterilized for 15min at 115 ℃ in an autoclave.
TABLE 2 oat solid medium (1000 mL) formulation
The preparation method comprises the following steps: 30.0g of oat was weighed and boiled with a small fire of 1100mL of ultra-pure water for 7 minutes, filtered with a previously prepared sieve into a clean plastic beaker (2000 mL) to give about 950mL of oat juice, which was sized to 1000mL with ultra-pure water, then 15.0g of sucrose and 7.0g of agar were added to a prepared clean conical flask (2000 mL), followed by draining with a glass rod, and the sized oat juice was added to the conical flask and mixed until the drug was almost dissolved, and sterilized at 121℃for 20 minutes in an autoclave.
Activation of strains
Endophytic bacteria of 54 ginseng and rice blast bacteria (007) are provided by medicinal plant endophytic bacteria team of university of south China.
The activation of the endophytic bacteria of ginseng is as follows: 54 ginseng endophytes are activated on an ultra-clean workbench, and 4 endophytes are activated on a solid flat-plate culture medium of each LB. Bacteria in the seed-retaining plate are dipped with an inoculating loop, streaked and inoculated on the plate, sealed, marked with a sign, and inversely cultured in a constant temperature incubator 1d at 28 ℃. The results of activating endophytic bacteria of ginseng by dilution streaking are shown in FIG. 1.
Activation of Pyricularia oryzae was as follows: the hyphae of the rice blast bacteria cultured in advance are scraped by a cotton swab, bacterial blocks are picked by a sterile inoculating needle to the center of the oat solid culture medium, and 13 oat solid culture mediums are inoculated. The plate is subjected to film sealing treatment, marked and cultivated for 6 days in a constant temperature incubator at 28 ℃ in a dark place. The activation results are shown in FIG. 2, in which FIG. 2a shows activated Pyricularia oryzae b shows rice blast bacterium c: activated Pyricularia oryzae. The rice blast fungus hypha is colorless and transparent, gathers and forms gray filiform in the figure, has a diaphragm and branches; the black bottom is conidium of Pyricularia oryzae.
Screening of strains
Primary screening for antagonistic bacteria
Periodically observing the growth state of Pyricularia oryzae, when the colony diameter of Pyricularia oryzae reaches about 3cm, placing the Pyricularia oryzae in an ultra-clean workbench, inoculating 4 strains of activated ginseng endophytic bacteria of different types around the center of Pyricularia oryzae colony by using a sterilized inoculating needle, performing film sealing treatment, marking, placing in a constant temperature incubator at 28 ℃, culturing for 3 days in an inverted manner, periodically observing whether a transparent antibacterial zone or a antibacterial zone exists between endophytic bacteria and Pyricularia oryzae, and if the bacterial zone or the antibacterial zone exists, taking the bacterial zone as a primary selection antagonistic bacterial strain.
In this example, 54 endophytic strains were inoculated together, and 24 antagonistic strains with a zone of inhibition or zone were screened together, and as shown in the primary screening result, 2 relations exist between endophytic bacteria and rice blast bacteria in the present culture: namely nutritional competition and antagonism. FIG. 3 shows the results of primary screening of antagonistic bacteria (only part is shown), in FIG. 3, a is a control group, pyricularia oryzae without antagonistic bacteria grown at the same time as the experimental group, and b to f are experimental groups.
Compound screening antagonistic bacteria
The antagonistic bacteria and Pyricularia oryzae screened in the preliminary screening were again co-cultured, 3 repeated experiments were performed, and the antagonistic bacteria were inoculated to the left and right Pyricularia oryzae, and co-cultured for 3d. 3d observation was performed and the corresponding record was made: the radius R of the normal rice blast bacteria colony and the radius Ra of the rice blast bacteria colony at the antagonized end were calculated, and the antagonism ratio (%) (antagonism ratio= ((R-Ra)/R). Times.100%) was averaged. In the process of rescreening co-culture, observing the morphological change of rice blast fungus hypha under the continuous 3d action of antagonistic bacteria, picking out the hypha antagonized by two strains with strongest antagonistic action and the hypha which normally grows, and carrying out microscopic examination under an optical microscope.
After co-cultivation for 2d, antagonism phenomenon appears, the colony radius R of the normal growth rice blast bacteria and the colony radius R of the normal growth endophyte are also increased, the growth of hypha at the antagonism end is limited, and after 3d, the growth of the thallus is not changed obviously. FIG. 4 is a graph showing the results of co-cultivation of 24 antagonistic bacteria and fungi for 3d (only part is shown) in FIG. 4, wherein a is a control group, pyricularia oryzae without antagonistic bacteria grown at the same time as the experimental group, and b to f are experimental groups. As can be seen from the graph, the antagonism of the bacterial attack by the bacterial strain on the rice blast hypha on the side antagonized by the bacteria was evident, and the antagonism rate (%) of the 24 strains is shown in Table 3 below, wherein the 1 strain having the highest antagonism efficiency was GSRH-5-468 strain, and the antagonism rate was 42.61%.
TABLE 3 inhibition of Pyricularia oryzae hyphae by 24 antagonistic bacteria
The antagonism efficiency bar graph of 24 antagonistic bacteria in the re-screening test is shown in FIG. 5, and the highest antagonism efficiency of strain No. 24 GSRH-5-468 can be found from FIG. 5.
Antagonistic effects on pathogenic bacterial growth
On the 3 rd day of re-screening co-cultivation, respectively picking out rice blast fungus mycelia on the antagonistic side and the non-antagonistic side corresponding to 2 strains of fungus, and observing under an optical microscope after tabletting, wherein in the figure 6, a is the hypha on the non-antagonistic side of rice blast fungus co-cultivated with GSRHX-5-461, and the hypha is thin and long and grows normally; in FIG. 6b is antagonistic hyphae, the hyphae appear to be significantly broken and less. It is presumed that the antagonistic bacteria cause mutation of rice blast hyphae, and the action mechanism is that the strain can produce substances for inhibiting the growth of rice blast hyphae. In FIG. 6, the non-antagonistic hyphae of Pyricularia oryzae co-cultured with GSRH-5-468 are thin and long, and have no abnormal phenomena such as obvious distortion; in FIG. 6, d is the rice blast fungus hypha on the antagonistic side, which is small and short, and it is presumed that the strain may produce a substance that breaks and dissolves the rice blast fungus hypha.
Sequencing and sequence alignment, analysis
The strain is sent to a certain biotechnology company for sequencing, and the 16S rDNA sequence of the strain is shown as SEQ ID NO. 1. Sequencing results Blast similarity analysis was performed in GenBank nucleic acid database, and Blast sequence alignment and treeing analysis were performed to determine that they were Serratia sp.
Preservation of the strain:
the strain is preserved in China center for type culture Collection, address: chinese, university of martial arts; post code 430072, with a preservation date of 2024, 3 months and 25 days, and a preservation number of: cctccc NO: m2024552.
Claims (3)
1. Serratia sp.,. GSRH-5-468 strain, deposited under accession number: cctccc NO: m2024552, the 16S rDNA sequence of the strain is shown as SEQ ID NO. 1.
2. Use of Serratia GSRH-468 of claim 1 in the preparation of an antagonist of Pyricularia oryzae.
3. Use of Serratia GSRH-468 of claim 1 in the preparation of a medicament for controlling rice blast.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202410409221.8A CN118185824A (en) | 2024-04-07 | 2024-04-07 | Serratia for antagonizing Pyricularia oryzae and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202410409221.8A CN118185824A (en) | 2024-04-07 | 2024-04-07 | Serratia for antagonizing Pyricularia oryzae and application thereof |
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