CN114134087B - Streptomyces alginolyticus and application thereof in prevention and control of wheat stem basal rot - Google Patents
Streptomyces alginolyticus and application thereof in prevention and control of wheat stem basal rot Download PDFInfo
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Classifications
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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
- A01N63/28—Streptomyces
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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
Abstract
The invention discloses streptomyces alginolyticus and application thereof in control of wheat stem basal rot, and belongs to the technical field of plant protection. The streptomyces alginolyticus is stored in China Center for Type Culture Collection (CCTCC) M2021455 in number WY3 and 2021, and 25 days of 4 years. The invention uses the streptomyces alginolyticus to be applied to the prevention and treatment of wheat stem basal rot for the first time; the data show that compared with the control group, the streptomyces alginolyticus (with the number of WY 3) has a promoting effect on the growth of wheat seedlings, and the plant height and root length are obviously increased; the inhibition rate of the streptomyces alginolyticus aseptic fermentation liquor to the wheat stem basal rot pathogen is 68.65%; the average control effect on the wheat stem basal rot reaches 60.87 percent.
Description
Technical Field
The invention belongs to the technical field of plant protection, and particularly relates to streptomyces alginolyticus and application thereof in prevention and control of wheat stem basal rot.
Background
Wheat is one of main grain crops in China, and the planting area reaches 2600 ten thousand hm 2 The disease problem faced in planting is the worldwide grain safety problem. Wheat stem rot is a worldwide soil-borne disease infested by a variety of fusarium species, and currently asian fusarium, graminearum and false graminearum are the main pathogenic bacteria in the Yangtze river basin and Huang-Huai-Mai region of China. Wherein Asian Fusarium (F. Aseptic) is used as the dominant population of winter wheat main producing area Fusarium culmorum in China,and huge loss is caused to the wheat production.
Fusarium asiaticum can harm all growth stages of wheat, and wheat stem basal rot can occur from the seedling stage to the mature stage of wheat, so that leaf sheaths and stems at the basal part of the stems can be browned, stem nodes can be necrotic, and plants can die when serious, and the wheat yield is affected. In recent years, under the two-harvest wheat-corn planting system, a large amount of corn and wheat straw are returned to the field to change the quantity of field-returning block pathogenic bacteria and the ecological small environment of the farmland, and the stalk-based Fusarium solani mainly survives on soil and disease plant residues in the forms of mycelium, chlamydospores and conidium, so that the damage of diseases is increased due to the fact that the stalk with bacteria remains in the field.
At present, chemical control is mainly used for controlling the wheat stem basal rot, agricultural control is assisted, and excellent varieties are bred, so that the chemical control is the most commonly used control method due to the long breeding period and the limitation of field management. Although the chemical pesticide can effectively control the disease condition, serious secondary pollution is caused to the ecological environment and wheat production, and meanwhile, the pesticide is reused to enhance the drug resistance of germs. Therefore, environmental-friendly biological control technologies are attracting attention, and research on fusarium antagonistic bacteria is also increasing. In the biocontrol microorganism group, actinomycetes are rich in resources, have larger development and application prospects, greatly promote the development of biological pesticide industry and other industries in China, and control wheat stem-based rot by utilizing beneficial microorganisms meets the requirements of green sustainable development of modern agriculture in China. At present, the research on actinomycetes in the screening of wheat stem-based rot biocontrol bacteria is relatively few, and the antagonism for effectively preventing and treating the wheat stem-based rot is still lacking at the present stage.
Disclosure of Invention
1. Problems to be solved
Aiming at the problems existing in the prior art, the invention provides the streptomyces alginolyticus and the application thereof in the prevention and treatment of the wheat stem basal rot, which can effectively prevent and treat the wheat stem basal rot.
2. Technical proposal
In order to solve the problems, the invention adopts the following technical scheme.
The Latin of the streptomyces alginolyticus is Streptomyces amritsarensis, the serial number of the streptomyces alginolyticus is WY3, the streptomyces alginolyticus is preserved in China Center for Type Culture Collection (CCTCC) M2021455 at the year of 2021 and the month of 25.
Among the above Streptomyces alginolyticus, the best fermentation conditions of the Streptomyces alginolyticus are: LB culture medium is adopted for shaking culture, the shaking culture speed is 180r/min, the culture temperature is 28 ℃, the initial pH is 8-9, and the fermentation time is 3d.
An application of the streptomyces alginolyticus in preventing and treating wheat stem basal rot.
The application of the streptomyces alginolyticus in the prevention and treatment of the wheat stem base rot is that the pathogenic bacteria of the wheat stem base rot is Fusarium asiaticum (F.aseptic).
The application of the streptomyces alginolyticus in preventing and controlling the wheat stem basal rot is that the streptomyces alginolyticus is in a pharmaceutically acceptable dosage form.
The application of the streptomyces alginolyticus in the prevention and treatment of wheat stem basal rot is that the streptomyces alginolyticus is in a bacterial suspension form.
3. Advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
the antagonistic bacterium WY3 of the Fusarium asiaticum is separated and screened from the soil of the root of the pathogenic wheat in a wheat producing area (Anqing city of Anhui province), and the inhibition rate is 64.35%. The result of researching the broad-spectrum antibacterial activity of WY3 shows that the antibacterial rate of streptomycete WY3 on corn ear rot germ (Fusarium proliferatum) reaches 55.38%; the antibacterial rate of the composition on various pathogenic bacteria such as Alternaria tabaci (Alternaria alternata) and Sclerotinia sclerotiorum (Sclerotinia sclerotiorum) is over 60 percent. Potted experiments show that the wheat irrigated by WY3 bacterial liquid has obviously increased wheat plant height by 2.14cm compared with the control group. It was shown that WY3 has a promoting effect on the growth of wheat seedlings. Wheat seedlings respectively irrigated with WY3 bacterial suspension and sterile water are inoculated with wheat straw stalk rot germ wheat grains at the two-leaf period, and the prevention effect reaches 60.87 percent. The WY3 has a protective effect on seedling-stage wheat stem basal rot.
Drawings
FIG. 1 is a diagram showing strain selection and opposing culture in example 1; in the figure: a: wheat rhizosphere soil bacteria separation, B: streptomyces WY3 streak culture, C: streptomyces WY3 is cultivated in opposition to F.a, D: streptomyces H103 is cultivated in opposition to F.a;
FIG. 2 is a tree diagram of the evolution of Streptomyces WY3 in the present invention.
FIG. 3 is a graph showing the effect of Streptomyces WY3 and Streptomyces H103 fermentation broth, sterile fermentation broth, and bacterial suspension on the growth of Rhizoctonia cerealis, respectively, in example 2;
FIG. 4 shows the mycelium morphology of pathogenic bacteria in example 2; in the figure: a: normal wheat stem rot germ germination morphology, B: pathogenic bacterial hypha morphology after antagonism of streptomyces WY3, C: pathogenic bacteria mycelium morphology after antagonism of streptomycete H103;
FIG. 5 is a graph showing the effect of WY3 on wheat seedling growth in example 3; in the figure: CK: inoculating no bacteria; WY3: accessing streptomyces WY3; h103: accessing streptomyces H103;
FIG. 6 is a graph showing the results of the potted plant control effect study of WY3 on wheat stem basal rot in example 4; in the figure: blank: inoculating no bacteria; f.a: inoculating stem basal rot germs; f.a +WY3: inoculating stem rot germ and streptomyces WY3; h103+ F.a: inoculating stem rot germ and streptomyces H103;
Detailed Description
The invention is further described below in connection with specific embodiments.
The test materials were as follows:
(1) Bacterial strain
Pathogenic bacteria of the wheat stem rot (fusarium f. Asepticum) tested are provided by the university of agriculture plant protection institute strain library of the Anhui.
(2) Main reagent
Agar, YEAST EXTRACT, TRYPTONE, sodium chloride, glucose, sucrose, 95% ethanol, glacial acetic acid, calcium carbonate, sodium hydroxide, hydrochloric acid, disodium bicarbonate, sodium bicarbonate, congo red, gelatin, potassium iodide, dipotassium phosphate, anhydrous sulfanilic acid, diphenylamine, sodium hydroxide glutaraldehyde, acetone, and ethyl acetate.
(3) Culture medium
Corn meal culture medium, gelatin liquefaction culture medium, nitrate liquid culture medium, gao's No. 1 culture medium, mung bean culture medium, milk coagulation peptone culture medium, gris reagent A liquid, gris reagent B liquid, starch hydrolysis culture medium, cellulose hydrolysis culture medium, LB culture medium, PDA culture medium and citrate culture medium.
Example 1
Screening and identification of antagonistic bacteria of wheat stem rot
Surface soil (soil at 10cm-15 cm) is collected from wheat stem rot disease incidence field blocks. After passing through a 2mm stainless steel sieve, 10g of soil sample is weighed, put into a triangular flask containing 90mL of sterile water and vibrated for 1h at 200rpm to prepare soil suspension. Sucking 1mL of soil suspension, and diluting 10 by adopting sterilized water gradient 1 -10 7 Multiple times. Subsequently, 0.1mL of each dilution of the soil suspension was aspirated, and the resulting suspension was spread on LB and Gao's solid medium plates uniformly, and incubated at 28℃for 2d-3d. After the colony grows out, the pure culture is obtained by continuous streak culture, and the culture is preserved at 4 ℃. Actinomycetes are selected and inoculated into a liquid culture medium of Gao's No. 1, and are placed in a shaking table at 28 ℃ and 180r/min for 3d of culture.
Inoculating the activated F.a bacterial block to the central position of a PDA flat plate, placing the flat plate in an incubator at 22 ℃, culturing for 1d, placing four sterile filter paper sheets (phi=6mm) at 2.5cm around the central position of the flat plate, sucking 5 mu L of bacterial liquid onto the filter paper sheets, placing a filter paper sheet dropwise added with 5 mu L of sterile water in a blank control manner, and culturing in the incubator at 22 ℃. When the hypha of the control group approaches to the full plate, the colony diameter is measured, each group of test is repeated 3 times, and the bacteriostasis rate is calculated.
The formula for calculating the inhibition rate of the wheat root system soil actinomycetes to the wheat stem basal rot is as follows:
wherein: a is the diameter of pathogenic bacteria colony of the control group;
b is the diameter of the colony of the pathogenic bacteria of the treatment group;
the Streptomyces alginolyticus strain is obtained through experimental separation and screening, and the number is WY3. The control strain was Streptomyces lilacinus (Strepto myces lavendulae), accession number H103. The Streptomyces plate-facing experiment shows that the Streptomyces WY3 has obvious antagonism on wheat stem rot, the inhibition rate on wheat stem rot reaches 64.35%, the inhibition rate of Streptomyces H103 is only 29.35%, and the screening and facing culture results of the strain are shown in figure 1.
Referring to FIG. 2, a Blast alignment was performed on the 16S rDNA sequence of Streptomyces WY3 in NCBI database, and strains having higher homology with the WY3 strain belong to Streptomyces; the MEGA11 software is used for constructing a phylogenetic tree, and actinomycetes WY3 and streptomyces alginolyticus (Streptomyces amritsarensis MH 482895.1) are on the same phylogenetic branch, and the similarity is 99.86%. Performing Blast comparison on a 16S rDNA sequence of streptomyces H103 in NCBI database, wherein strains with high homology with H103 strains belong to Streptomyces; the similarity of actinomycetes H103 and Streptomyces lilacinus is 99.50%.
Example 2
Streptomyces WY3 fermentation broth, sterile fermentation broth and bacterial suspension effect on growth of wheat stem rot germ
Picking up activated fresh Streptomyces WY3 lawn, inoculating into LB medium, culturing at 28deg.C in 180r/min shaking table for 3d, packaging the fermentation broth into centrifuge tube, placing into low temperature high speed centrifuge (4deg.C, 10000 r/min), collecting supernatant, and discarding precipitate. Filtration through a sterile bacterial filter (Φ=0.22 μm) gave a sterile fermentation broth of strain WY3.
The sterile broth was added to PDA medium to make plates containing 10% of the fermentation metabolite. And (3) taking activated wheat stem rot germ edge mycelium blocks by using a puncher with the inner diameter of 6mm, transferring the mycelium blocks to the central position of a PDA flat plate, taking the flat plate without the sterile fermentation liquid as a blank control, and culturing at the constant temperature of 22 ℃. When the control hyphae were spread over the whole petri dish, data were recorded and the bacteriostatic rate was calculated.
From example 1, it is found that the strain WY3 has a strong inhibitory effect on wheat stem rot. Therefore, the mycelia at the edge of the bacteriostasis area of the flat plate are picked up by using an inoculating needle, placed on a glass slide, covered with a cover glass, and observed under a microscope. As shown in FIG. 4, the hyphae of the control group grew normally, the surface was smooth, and the shape was uniform; after WY3 is acted, hyphae are expanded and deformed, distortion and abnormal branching occur, after H103 is acted, the hyphae grow normally, and the surface of the hyphae are slightly expanded.
The results show that the supernatant, the sterile fermentation liquor and the bacterial suspension of the streptomycete WY3 have obvious antagonism on the wheat stem basal rot, wherein the inhibition rate of the fermentation liquor reaches 84.55%, the inhibition rate of the sterile fermentation liquor reaches 68.65%, and the inhibition rate of the bacterial suspension reaches 82.56%; streptomyces H103 fermentation liquor inhibition rate is only 27.5%, sterile fermentation liquor inhibition rate is 6.62%, and bacterial suspension inhibition rate is only 24.61%.
TABLE 1 inhibition of the planar of wheat stem rot by WY3 fermentation broth, sterile fermentation broth, bacterial suspension
Example 3
Research on growth effect of WY3 on wheat seedlings
The single colony of the strain WY3 is picked and inoculated into LB culture medium, and the strain is placed in a shaking table at 28 ℃ and 180r/min for 3d of culture. Wheat is planted after disinfection and germination acceleration, the WY3 bacterial suspension is irrigated to the root of the wheat after wheat seedlings bud for 0d, 3d and 7d, and sterile water is irrigated to a control group.
The result shows that the bacterial liquid of WY3 has growth promoting effect on wheat seedling growth in the wheat growth process, the wheat irrigated with the bacterial liquid has a plant height of 2.14cm longer than that of untreated wheat, and the root length of the wheat is increased by 1.49cm (shown in Table 2). In the growth process of wheat, a certain amount of bacterial liquid is irrigated, the growth promoting effect is achieved, plants are stronger than wheat without the bacterial liquid, and under the same sample condition, the tillers of the plants irrigated with the bacterial liquid are more, and the growth is more vigorous (shown in figure 5). H103 has no obvious growth promoting effect.
TABLE 2 influence of Streptomyces WY3 on wheat seedling growth
Note that: inhibition is expressed as mean ± standard error, lower case english letters are significance of the inter-group differences at a significant level of P < 0.05.
Example 4
WY3 research on prevention effect of wheat stem-based rot potting
Wheat is planted, when wheat seedlings bud for 0d and 3d, WY3 bacterial liquid is irrigated to the root of the wheat, and sterile water is irrigated to a control group. Preparing diseased wheat grains in advance: after the seeds are disinfected, soaking the seeds for 12 hours, carrying out damp heat sterilization at 121 ℃ for 45 minutes, then inoculating 5-6 Fusarium asiaticum blocks, and culturing for 7 days at 25 ℃. The diseased kernels were inoculated to the basal part of the wheat stem in the two-leaf stage, and the control group was not inoculated with germs.
The results show that: wheat seedlings inoculated with Asian Fusarium are severely dwarfed, the stem base is blackened, the average disease index is 51.11, the average disease index of the wheat seedlings treated by WY3 root irrigation is only 18.57, and the prevention effect reaches 60.87%; the wheat seedlings treated by the root irrigation of H103 have no obvious prevention effect.
TABLE 3 control effect of WY3 on F.a wheat stem basal rot
The foregoing is a further elaboration of the present invention in connection with the detailed description, and it is not intended that the invention be limited to the specific embodiments shown, but rather that a number of simple deductions or substitutions be made by one of ordinary skill in the art without departing from the spirit of the invention, should be considered as falling within the scope of the invention as defined in the appended claims.
Claims (5)
1. A streptomyces alginolyticus, characterized in that: the Latin theory of the streptomyces alginolyticus isStreptomyces amritsarensisThe serial number of the strain is WY3, and the strain is preserved in China center for type culture Collection (CCTCC M2021455) in the year 2021 and the month 25.
2. A fermentation method of the Streptomyces alginolyticus according to claim 1, characterized in that: LB culture medium is adopted for shaking culture, the shaking culture speed is 180r/min, the culture temperature is 28 ℃, the initial pH is 8-9, and the fermentation time is 3d.
3. Use of the streptomyces alginolyticus according to claim 1 in the prevention and treatment of wheat stem basal rot, characterized in that: the pathogenic bacteria of the wheat stem-based rot are Fusarium asiaticumFusarium asiaticum）。
4. The use of streptomyces alginolyticus in the prevention and treatment of wheat stem basal rot according to claim 3, characterized in that: the streptomyces alginolyticus is in a pharmaceutically acceptable dosage form.
5. The use of the streptomyces alginolyticus according to claim 4 in the prevention and treatment of wheat stem basal rot, characterized in that: the streptomyces alginolyticus is in a bacterial suspension form.
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| CN111500501A (en) * | 2020-05-07 | 2020-08-07 | 中国地质大学（北京） | Streptomyces misonii strain and application thereof in preventing and treating wheat root rot and stem basal rot |
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| CN111500501A (en) * | 2020-05-07 | 2020-08-07 | 中国地质大学（北京） | Streptomyces misonii strain and application thereof in preventing and treating wheat root rot and stem basal rot |
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