CN116694505A - Streptomyces crimson capable of preventing and treating clubroot and application thereof - Google Patents
Streptomyces crimson capable of preventing and treating clubroot and application thereof Download PDFInfo
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- 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
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Abstract
The invention relates to Streptomyces crimson CC2-6 capable of preventing and treating clubroot, which is preserved in China general microbiological culture Collection center (CGMCC) No.26458 in 2023 and 1-12 days, and also relates to application of the Streptomyces crimson CC2-6 in clubroot. The Streptomyces roseoformis CC2-6 has strong inhibition effect on various plant pathogenic bacteria on a flat plate, and the fermentation liquor metabolite has good prevention and control effects on clubroot. The germination inhibition rate of the dormant spores of the plasmodiophora radicis is 51.01%, the root and root hair infection inhibition rate of the Chinese cabbage is 77.41%, the pot control effect of the Chinese cabbage treated by the CC2-6 fermentation liquor is 27.46%, and the pot control effect of the Chinese cabbage is 53.20% when the Chinese cabbage is treated in 7d in advance. In addition, the strain has natural harmony and compatibility with natural ecology, and has no toxic or side effect and no residue on soil ecology compared with chemical pesticides. Therefore, the method has potential commercial development and application value in biological control practice of diseases.
Description
Technical Field
The invention relates to the field of crop disease control, in particular to Streptomyces roseoformus capable of controlling clubroot and application thereof.
Background
Clubroot is a soil-borne disease that mainly infects some commercial crops in cruciferae, such as rape, radish, mustard tuber, cabbage, and the like. Clubroot is widely distributed in more than fifty countries and regions of the world, including africa, south america, north america, europe, asia and oceangoin, and crop yield losses of up to 10-15% each year around the world due to clubroot, and therefore clubroot is regarded as one of the important cruciferous crop diseases. According to statistics, the occurrence area of the clubroot in China can reach 4800-6000 ten thousand mu in the lighter year, and the hazard area is higher than 1.35 hundred million mu in the outbreak year.
Clubroot disease occurs and produces a hazard at the root of the plant and can be infested by the clubroot bacteria throughout the growth period of the plant, but the seedling stage is the most predominant disease-sensing stage. After the underground root is infected by the plasmodiophora, the hormone change in the plant body is caused, the plant cell division is accelerated, and the main root and the lateral root expand to form tumors. The root surface is smooth in the initial stage of the formation of the swollen root, the root rot in the later stage is changed into dark brown in color, odor is emitted, tumors are enlarged, root hairs are reduced or even absent, and the absorption and the transmission of moisture and nutrients of plants are inhibited, so that the overground parts can show symptoms such as water deficiency wilting leaves yellowing and the like, and the whole plant is withered when serious.
At present, the prevention and treatment of the clubroot still takes chemical agents as main agents, the operation is simple and quick, the cost is low, the effect is quick, but the use of chemical bactericides is easy to cause serious consequences such as drug resistance of pathogenic bacteria, pollution to the environment, residual toxicity and the like, and finally the problems of poor prevention and treatment effect, ecological safety hazard, human health and the like occur.
With further development of scientific research, the biological pesticide prepared by using the secondary metabolite of the biological pesticide has the characteristics of no pollution, no residue, difficult generation of drug resistance of harmful organisms, high environmental compatibility, safety to people and livestock and the like; and the metabolic products generated by microorganisms are utilized to treat plant pathogenic bacteria, so that the research of biological disease control is a current hot spot. The biocontrol potential of secondary metabolites produced by actinomycetes mainly shows the inhibition effect on the growth of hyphae, spore germination and infection of pathogenic bacteria, more and more actinomycetes which are derived from various plant endophytes or different extreme environments are discovered, and the types of the metabolites produced by actinomycetes are also becoming more and more abundant. Therefore, the development of biological control research is an important approach for sustainable development of agriculture, and has important practical significance in production.
Disclosure of Invention
In order to solve the problems, the invention provides the Streptomyces roseoformus which can prevent and treat clubroot, and the Streptomyces roseoformus is preserved in China general microbiological culture collection center (CGMCC) No.26458 in the year 2023, month 1 and day 12.
The invention also provides application of the streptomyces crimson-brown fungus in preventing and treating clubroot.
The invention also provides a method for preventing and treating clubroot, which comprises the step of applying the Streptomyces roseoformis culture product to soil for crop planting.
In a specific embodiment, the method comprises the steps of:
s1: culturing the streptomyces crimson and preparing a streptomyces crimson fermentation broth;
s2: applying the Streptomyces roseoformus fermentation broth to the soil in which the crop is planted.
In a specific embodiment, in S1, the streptomyces roseoformis is cultured in medium No. 1.
In a specific embodiment, S1 comprises the steps of:
s11: inoculating the Streptomyces crimson to a liquid culture medium of Gao's No. 1 for culture;
s12: culturing at 28deg.C and 180rpm for 7 days to obtain the Streptomyces roseoformis fermentation broth.
In a specific embodiment, in S2, the crop is subjected to a periodic rooting treatment by the fermentation broth of streptomyces roseoformis.
In a specific embodiment, the time interval between every two root irrigation treatments is 7 days.
The Streptomyces roseoformis CC2-6 strain has strong inhibition effect on various plant pathogenic bacteria on a flat plate, and fermentation liquor metabolite has good prevention and control effects on clubroot. The germination inhibition rate of the dormant spores of the rhizomatous bacteria is 51.01%, the water-cultured seedling test shows that after the treatment of the fermentation liquor of CC2-6 for 9d, the root hair infection inhibition rate of the Chinese cabbage root is 77.41%, the living body inoculation test shows that after the fermentation liquor of the rhizomatous bacteria and the actinomyces are inoculated for 50d simultaneously, the pot control effect of the Chinese cabbage treated by the fermentation liquor of CC2-6 is 27.46%, and the pot control effect of the Chinese cabbage is 53.20% after the treatment of the pot control effect of the Chinese cabbage is advanced for 7d. In addition, the strain is obtained by separating from soil, has natural harmony and compatibility with natural ecology, and has no toxic or side effect and no residue on the soil ecology compared with chemical pesticides. Therefore, the method has potential commercial development and application value in biological control practice of diseases.
Preservation of biological materials
The purified Streptomyces crimson and brown fungus CC2-6 is preserved in China general microbiological culture Collection center (CGMCC) of China national institute of microbiological culture Collection center (CGMCC) of national academy of sciences of China, no. 3, national area of the Korean, beijing, 1 month and 12 days in 2023, with a preservation number of CGMCC No.26458.
Drawings
FIG. 1 is a photograph of a plate morphology of Streptomyces roseoformus CC 2-6;
FIG. 2 is a photograph of a single colony morphology of Streptomyces roseoformis CC 2-6;
FIG. 3 is a photomicrograph of spore chains and aerial hyphae of Streptomyces roseoformis CC 2-6;
FIG. 4 shows the identification of Streptomyces crimson CC2-6 for the production of various hydrolases;
FIG. 5 shows the inhibition of Streptomyces crimson CC2-6 against pathogenic bacteria of cotton fusarium wilt;
fig. 6 shows the disease controlling effect of Streptomyces roseoformus CC2-6 clubroot.
Detailed Description
The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.
1. Identification and preservation of Streptomyces crimson CC2-6
The group collects rhizosphere soil of non-farmland ecological systems such as lawns, bamboo forests, street trees and the like from 4 areas of north, sand-ground dams, banan and south banks of Chongqing, a plurality of strains obtained by separation are subjected to primary screening and secondary screening, one strain CC2-6 is separated, and the strain is determined to have better prevention and treatment effects on clubroot through in-vivo plant indoor potting inoculation tests.
Strains CC2-6 were cultured on a culture plate of Gao's No. 1 at 28℃for 5-7d, and the morphology of the plate was shown in FIG. 1, and the single colony was shown in FIG. 2.
The slide glass sterilized in advance is obliquely inserted into a flat plate at 30 ℃, after being inversely cultured for 7d in a 28 ℃ incubator, the cover glass is taken out, a little distilled water is dripped on the slide glass, and the morphology of the morphological characteristics of aerial hyphae and spore chains of the strain is observed by using an optical microscope, as shown in figure 3.
Genomic DNA of the strain was extracted with bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO: 1) and 1492R (5'-TACGGCTACC TTGACGAC-3', SEQ ID NO: 2). The genome DNA of the strain CC2-6 is used as a template, and after PCR amplification, the 16S rDNA sequence is obtained through electrophoresis detection and sequencing, and is shown as SEQ ID NO. 3. Blast comparison is carried out on the obtained sequence result in NCBI, a 16S rDNA sequence of a related strain is selected, a Neighbor-Joining method (Neighbor-Joining) of MEGA 7.0 software is used for tree construction, and the strain is identified to belong to Streptomyces roseoformis by combining morphological characteristics (Streptomyces purpeofuscus).
The strain is preserved in China general microbiological culture Collection center (CGMCC) of China national institute of culture Collection of microorganisms, national academy of sciences of China No. 3, including North Chen and West Lu No. 1, the Korean region of Beijing, at 1 month 12 of 2023, with a preservation number: CGMCC No.26458.
2. Streptomyces roseofaciens CC2-6 fermentation broth preparation and metabolite analysis
The antagonistic actinomycetes XDS3-6 was inoculated into a sterile liquid medium for Kyoho synthesis No. 1, and subjected to shaking fermentation at 28℃and 180rpm for 7d. The fermentation broth was centrifuged at 7500rpm for 10min, and the supernatant was discarded. Freezing at-80deg.C, and lyophilizing in a lyophilizing machine for 12 hr.
3. Streptomyces crimson CC2-6 for preventing and controlling pathogenic bacteria
3.1 bacteriostasis of Streptomyces crimson CC2-6 against pathogenic bacteria
Antagonistic actinomycetes were inoculated in some hydrolase selection media. The plates were placed upside down in a 28℃incubator and after 7d the strain growth was observed and the radius of the transparent circle was recorded. Streptomyces roseofaciens CC2-6 was detected to produce various hydrolases (FIG. 4). It is presumed that it has a good antibacterial effect against some pathogenic bacteria.
Further, the effect of antagonistic bacteria on the growth of hyphae of pathogenic fungi (fusarium wilt of cotton) was measured by a plate-counter streaking method. The center of the plate is inoculated with a fungus cake of 8mm, antagonistic actinomycetes are inoculated at two sides 25mm away from the fungus cake, two straight lines of 30mm are drawn, actinomycetes are inoculated 3d earlier than the pathogenic fungi, the control group is inoculated with the pathogenic fungi at the center of the plate, actinomycetes are not inoculated, 3 replicates are treated each, and the culture is carried out in a constant temperature incubator at 28 ℃. And when the control group pathogenic fungi grow on the flat plate, observing whether the treatment group generates a zone of inhibition, measuring the colony diameter of the pathogenic fungi, and calculating the inhibition rate. Determining the inhibition effect of the CC2-6 strain on pathogenic fungi. The results are shown in FIG. 5, and the CC2-6 strain has remarkable antibacterial effect.
3.2 Streptomyces crimson CC2-6 for preventing and treating clubroot
Taking root secretion of Chinese cabbage as a culture medium, adding 5mL of root secretion, 0.5mL of suspension of dormant spores of plasmodium and 0.5mL of fermentation liquor of bacterial strain CC2-6 into a sterilized triangular flask, culturing in the dark at 24 ℃, and taking sterilized liquid culture medium as a control, wherein each treatment is repeated for 3 groups. And 5d, observing germination of dormant spores by a microscope, staining with 1% lichen red (dissolved in 45% acetic acid) for 10-15s, and calculating spore germination rate and germination inhibition rate. The results showed that the germination inhibition rate of the dormant spores of the plasmodium rhizomatosis was 51.01%.
Placing Chinese cabbage seeds in a culture dish filled with wet filter paper, accelerating germination under 24 deg.C illumination and wetting condition, transplanting seedlings into 10mL centrifuge tube filled with Hoagland nutrient solution for water culture after 3d, inoculating rhizomatous resting spores and CC2-6 strain fermentation liquor after 5d, wherein the final concentration of rhizomatous resting spores is 10 7 The treated group was added to the broth at a ratio of 1:30 per mL, and the control group was added to Hoagland nutrient solution. And after water planting for 9d, taking the root of the seedling, washing with clear water, putting the seedling into fluorescent pink dye liquor for dyeing for 30min, and moving under a microscope to observe the infection condition of the root hair of the seedling. The result shows that after the Chinese cabbage is treated by the CC2-6 fermentation liquor for 9 days, the root hair infection inhibition rate of the Chinese cabbage root is 77.41%.
Inoculating Chinese cabbage by root irrigation. Taking out the root from refrigerator, placing at 25deg.C, rotting in dark environment for 5 days, mixing the root with water at a ratio of 1:5, intermittently stirring with juicer, filtering, and preparing and adjusting the concentration of the dormant spore suspension of the root of the Amycolatopsis to 2.5X10 8 And each mL. Inoculating antagonistic bacterial cake into Gao's first liquid culture medium at 28deg.C and 180r.min -1 Culturing under the condition for 7d to obtain the product with concentration of 10 7 CFU/mL actinomycete fermentation broth. Root irrigation treatment of each Chinese cabbage with 30mL of dormant spore suspension of plasmodiophora radicis to obtain final bacterial content of 3×10 per gram of soil 7 And each. Treatment a: antagonistic bacteria and plasmodiophora radicis are inoculated simultaneously, 20mL of actinomycete fermentation liquor is irrigated every 7d, and the total irrigation is carried out five times, and the treatment B: the antagonistic bacteria are inoculated in advance for 7 days, 20mL of actinomycete fermentation liquor is irrigated every 7 days, and the total irrigation is five times. Inoculating the suspension of dormant spores of the rhizomatous bacteria and irrigating a liquid culture medium of Gao's first as a disease control, and repeating 20 white strains each treatmentThe disease was investigated 50d after inoculation and statistically recorded. The results show that after the plasmodiophora radicis and the CC2-6 fermentation liquor are inoculated for 50 days simultaneously, the control effect of the Chinese cabbage potting treated by the CC2-6 fermentation liquor is 27.46%, and the control effect of the Chinese cabbage potting treated by the CC2-6 fermentation liquor is 53.20% when the CC2-6 fermentation liquor is treated for 7 days in advance (figure 6).
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (8)
1. Streptomyces crimson for preventing and treating clubroot is characterized in that the Streptomyces crimson is preserved in China general microbiological culture Collection center (CGMCC No. 26458) in 1-12-year 2023.
2. The use of streptomyces crimson according to claim 1 for controlling clubroot.
3. A method for controlling clubroot, comprising the step of applying the streptomyces crimson culture product of claim 1 to the soil of crop plants.
4. A method according to claim 3, comprising the steps of:
s1: culturing the streptomyces crimson and preparing a streptomyces crimson fermentation broth;
s2: applying the Streptomyces roseoformus fermentation broth to the soil in which the crop is planted.
5. The method of claim 4, wherein in S1, the streptomyces crimson-brown mold is cultured in medium No. one of gao.
6. The method of claim 5, wherein S1 comprises the steps of:
s11: inoculating the Streptomyces crimson to a liquid culture medium of Gao's No. 1 for culture;
s12: culturing at 28deg.C and 180rpm for 7 days to obtain the Streptomyces roseoformis fermentation broth.
7. The method of claim 4, wherein in S2 the crop is subjected to periodic rooting with the fermentation broth of streptomyces roseoformis.
8. The method of claim 7, wherein the time interval between each root irrigation treatment is 7 days.
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| CN116064288A (en) * | 2022-08-30 | 2023-05-05 | 内蒙古农业大学 | Streptomyces roseoformis HC7-22 for plant iron-removing and growth-promoting and application thereof |
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