CN114134087A - Streptomyces algicidal and application thereof in prevention and treatment of wheat stem basal rot - Google Patents
Streptomyces algicidal and application thereof in prevention and treatment of wheat stem basal rot Download PDFInfo
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- 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
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Abstract
The invention discloses streptomyces algicidal and application thereof in prevention and treatment of wheat stem basal rot, and belongs to the technical field of plant protection. The Streptomyces algicidal strain is preserved in China center for type culture Collection in No. 4-25.2021 with the number WY3 and the preservation number is CCTCC M2021455. The streptomyces algicidal is applied to the prevention and treatment of the wheat stem basal rot for the first time; the data show that compared with a control group, the streptomyces algicidal (with the number of WY 3) has a promoting effect on the growth of wheat seedlings, and the plant height and the root length are obviously increased; the inhibition rate of the streptomyces alginolyticus sterile fermentation liquid on wheat stem basal rot pathogenic bacteria is 68.65%; the average control effect on the wheat stem base rot is 60.87%.
Description
Technical Field
The invention belongs to the technical field of plant protection, and particularly relates to streptomyces algicidal and application thereof in prevention and treatment of wheat stem basal rot.
Background
Wheat is one of the main grain crops in China, and the planting area reaches 2600 kilohm2The disease problem in planting is the worldwide food safety problem. Wheat stem base rot is a worldwide soil-borne disease infected by various fusarium, and currently Asian fusarium, fusarium graminearum and pseudofusarium graminearum are main pathogenic bacteria in Yangtze river basin and Huang-Huai-wheat area in China. Wherein Asian Fusarium (F.asiaticum) is used as the dominant population of Fusarium solani in main winter wheat producing area, and causes huge loss to wheat production.
Asian fusarium can damage each growth stage of wheat, wheat stem basal rot can occur from seedling stage to mature stage of wheat, leaf sheath and stem at stem base can be browned, stem node is necrotic, and plant death can be caused in serious conditions, thus wheat yield is influenced. In recent years, under a wheat-corn double cropping system, a large amount of corn and wheat straws are returned to the field, so that the number of pathogenic bacteria in the field and the ecological microenvironment of the field are changed, fusarium solani mainly survives in the soil and diseased plant residues in the forms of mycelium, chlamydospores and conidia, and the bacterial-bearing straws remain in the field, so that the diseases are more harmful.
At present, the control of the wheat stem base rot is mainly chemical control, and agricultural control and breeding of good varieties are assisted, and due to the long breeding period and the limitation of field management, the chemical control is the most common control method. Although the chemical pesticide can effectively control the disease condition, serious secondary pollution is caused to the ecological environment and the wheat production, and meanwhile, the pesticide resistance of germs is enhanced by repeatedly using the pesticide. Accordingly, environmental friendly biocontrol technologies are gaining attention, and research on antagonistic bacteria of fusarium is increasing. In the biocontrol microorganism group, the actinomycetes has rich resources and a wide development and application prospect, greatly promotes the development of the biopesticide industry and other industries in China, and utilizes beneficial microorganisms to control the wheat stem basal rot to meet the requirement of green sustainable development of modern agriculture in China. The research on actinomycetes in the existing biocontrol strain screening of wheat stem basal rot is relatively few in fruit, and antagonistic bacteria for effectively preventing and treating the wheat stem basal rot are still lacking at the present stage.
Disclosure of Invention
1. Problems to be solved
Aiming at the problems in the prior art, the invention provides streptomyces algicidal and an application thereof in the prevention and treatment of wheat stem basal rot, which can effectively prevent and treat the wheat stem basal rot.
2. Technical scheme
In order to solve the above problems, the present invention adopts the following technical solutions.
The Streptomyces algicidal is Streptomyces amyrosarensis with the Latin chemical name, the number of the Streptomyces amydasensis is WY3, the Streptomyces algicidal is preserved in China center for type culture collection (CCTCC M2021455) at 25 months and 4 months in 2021.
In the streptomyces algicidal, the optimal fermentation conditions of the streptomyces algicidal are as follows: adopting LB culture medium to carry out shaking culture, wherein the shaking culture speed is 180r/min, the culture temperature is 28 ℃, the initial pH is 8-9, and the fermentation time is 3 d.
An application of the streptomyces algicidal in the prevention and treatment of wheat stem basal rot.
The streptomyces algicidal is applied to the prevention and treatment of wheat stem basal rot, and the pathogenic bacteria of the wheat stem basal rot is Fusarium Asiatica (F.
The streptomyces algicidal is applied to the prevention and treatment of wheat stem basal rot, and the dosage form of the streptomyces algicidal is a pharmaceutically acceptable dosage form.
The streptomyces algicidal is applied to the prevention and treatment of wheat stem basal rot, and the dosage form of the streptomyces algicidal is bacterial suspension.
3. Advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
the antagonistic bacterium WY3 of Fusarium Asiatica is separated and screened from soil of rhizosphere of diseased wheat in a wheat production area (Anhui, Anqing city), and the inhibition rate is 64.35%. The broad-spectrum antibacterial activity of WY3 is researched, and the result shows that the antibacterial rate of streptomycete WY3 on maize ear rot (Fusarium proliferatum) reaches 55.38%; the bacteriostasis rate of the composite bacterial preparation on various pathogenic bacteria such as Alternaria alternata (Alternaria alternata), Sclerotinia sclerotiorum (Sclerotinia sclerotiorum) and the like is over 60 percent. The pot experiment shows that the plant height of the wheat irrigated by the WY3 bacterial liquid is obviously increased by 2.14cm compared with that of a control group. The WY3 shows that the wheat seedling growth promoter has the function of promoting the growth of wheat seedlings. The wheat seedlings whose roots are respectively irrigated with WY3 bacterial suspension and sterile water are simultaneously inoculated with wheat stem-base blight wheat grains in the two-leaf period, and the prevention effect reaches 60.87%. The WY3 shows that the wheat stem base rot in the seedling stage has a protective effect.
Drawings
FIG. 1 is a diagram showing screening and confrontation culture of strains in example 1; in the figure: a: separating the rhizosphere soil bacteria of wheat, B: streptomycete WY3 streak culture, C: culturing streptomycete WY3 and F.a in opposition, and culturing the strain D: culturing streptomycete H103 and F.a in opposition;
FIG. 2 is a tree diagram of the evolution of Streptomyces WY3 of the present invention.
FIG. 3 is a graph showing the respective effects of fermentation broth, sterile fermentation broth, and bacterial suspension of Streptomyces streptomyces WY3 and Streptomyces H103 on the growth of Rhizoctonia cerealis in example 2;
FIG. 4 shows the hyphal morphology of the pathogenic bacteria in example 2; in the figure: a: normal wheat basal stem rot germination morphology, B: pathogenic bacteria hypha morphology after streptomycete WY3 antagonism, C: the hypha form of pathogenic bacteria after streptomycete H103 antagonism;
FIG. 5 is a graph showing the results of the effect of WY3 on wheat seedling growth in example 3; in the figure: CK: inoculating no bacteria; WY 3: inoculating streptomyces WY 3; h103: inoculating streptomyces H103;
FIG. 6 is a graph showing the results of the study on the prevention effect of WY3 on the potting of stem rot of wheat in example 4; in the figure: blank: inoculating no bacteria; f.a: inoculating rhizoctonia rot; f.a + WY 3: inoculating rhizoctonia solani and streptomycete WY 3; h103+ F.a: inoculating rhizoctonia solani and streptomycete H103;
Detailed Description
The invention is further described with reference to specific examples.
The test materials were as follows:
(1) bacterial strain
The pathogenic bacteria of wheat stem basal rot (fusarium asiaticum) were provided by the institute of plant protection, university of agriculture, anhui.
(2) Primary reagent
Agar, YEAST EXTRACT, TRYPTONE trypton, sodium chloride, glucose, sucrose, 95% ethanol, glacial acetic acid, calcium carbonate, sodium hydroxide, hydrochloric acid, disodium hydrogen carbonate, sodium dihydrogen carbonate, congo red, gelatin, potassium iodide, dipotassium hydrogen phosphate, anhydrous sulfanilic acid, diphenylamine, sodium hydroxide glutaraldehyde, acetone, and ethyl acetate.
(3) Culture medium
Corn flour culture medium, gelatin liquefaction culture medium, nitrate liquid culture medium, Gauss No. 1 culture medium, mung bean culture medium, milk peptonization culture medium, Grignard reagent A liquid, Grignard 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 basal rot
Surface soil (10cm-15cm soil) was collected from a field with a stem rot disease of wheat. After passing through a stainless steel sieve of 2mm, 10g of a soil sample is weighed and placed into a triangular flask containing 90mL of sterile water, and the mixture is shaken at 200rpm for 1h to prepare a soil suspension. Absorbing 1mL of soil suspension, and diluting 10 percent by adopting sterilized water in a gradient way1-107And (4) doubling. Then, 0.1mL of each dilution soil suspension is respectively sucked and evenly coated on LB and Gao's I solid culture medium plates, and the mixture is cultured for 2d-3d at the constant temperature of 28 ℃. After the colony grows out, the pure culture is obtained by continuous streak culture and is preserved at 4 ℃. Picking actinomycetes and inoculating the actinomycetes into Gao's bacteriumThe culture medium 1 is cultured in a shaking table at 28 ℃ and 180r/min for 3 d.
Inoculating the activated F.a bacterial block to the center of a PDA plate, placing in an incubator at 22 ℃, culturing for 1d, placing four sterile filter paper sheets (phi is 6mm) at 2.5cm around the center of the plate, sucking 5 mu L of bacterial liquid to the filter paper sheets, placing filter paper sheets dropwise added with 5 mu L of sterile water in blank control, and placing in the incubator at 22 ℃ for culturing. When the mycelium of the control group is close to the full plate, the diameter of the bacterial colony is measured, each group of experiments is repeated for 3 times, and the bacteriostasis rate is calculated.
The formula for calculating the inhibition rate of wheat root system soil actinomycetes on wheat stem-based pythium aphanidermatum is as follows:
in the formula: a is the diameter of the pathogenic bacteria colony of the control group;
b is the diameter of the pathogenic bacteria colony in the treatment group;
and (4) separating and screening an experiment to obtain the alga lysing streptomyces strain with the number WY 3. The comparative strain was Streptomyces lavendulae (Streptomyces lavendulae), accession number H103. The streptomycete WY3 has obvious antagonism on the wheat stem basal rot through a plate confronting experiment, the inhibition rate on the wheat stem basal rot bacteria reaches 64.35 percent, the inhibition rate on the streptomycete H103 is only 29.35 percent, and the results of strain screening and confronting culture are shown in figure 1.
Referring to FIG. 2, the 16S rDNA sequence of Streptomyces WY3 was Blast-compared in NCBI database, and all strains with high homology to WY3 strain belong to Streptomyces genus; a phylogenetic tree is constructed by MEGA11 software, and the actinomycete WY3 and the Streptomyces algicidal (Streptomyces amyrsarensis MH482895.1) are on the same phylogenetic branch, and the similarity is 99.86%. Performing Blast comparison on a 16S rDNA sequence of the streptomycete H103 in an NCBI database, wherein strains with higher homology with the H103 strain belong to streptomycete; the similarity of the actinomycete H103 and the streptomyces lavendulae is 99.50%.
Example 2
Influence of streptomycete WY3 fermentation liquor, sterile fermentation liquor and bacterial suspension on growth of phomopsis graminis
Selecting activated streptomycete WY3 fresh lawn, inoculating into LB culture medium, culturing in a shaking table at 28 deg.C and 180r/min for 3d, subpackaging the fermentation liquid into centrifuge tubes, placing into a low-temperature high-speed centrifuge (4 deg.C and 10000r/min), taking supernatant, and discarding precipitate. The filtrate was filtered through a sterile bacterial filter (. PHI. ═ 0.22 μm) to obtain a sterile fermentation broth of the strain WY 3.
Adding the sterile fermentation liquor into a PDA culture medium to prepare a flat plate containing 10% of fermentation metabolites. Using a puncher with the inner diameter of 6mm to punch activated wheat stem basal rot fungi marginal mycelium blocks, transferring the mycelium blocks to the center of a PDA flat plate, taking the flat plate without adding sterile fermentation liquor as a blank control, and culturing at constant temperature of 22 ℃. And when the reference group hyphae are fully paved on the whole culture dish, recording data and calculating the bacteriostasis rate.
As can be seen from example 1, the strain WY3 has a strong inhibitory effect on Phomopsis tritici. Therefore, the plate zone edge hyphae were picked with an inoculating needle, placed on a glass slide, covered with a cover glass, and observed under a microscope. As shown in FIG. 4, the control group had normal hypha growth, smooth surface and uniform shape; after WY3 acts, hypha expands and malformates, and has the phenomena of distortion and abnormal branching, and after H103 acts, hypha grows normally and slightly expands on the surface.
The results show that the supernatant, the sterile fermentation liquor and the bacterial suspension of the streptomycete WY3 have obvious antagonistic action on the basal rot of wheat stems, wherein the inhibition rate of the fermentation liquor reaches 84.55 percent, the inhibition rate of the sterile fermentation liquor reaches 68.65 percent, and the inhibition rate of the bacterial suspension reaches 82.56 percent; the inhibition rate of the streptomycete H103 fermentation liquor is only 27.5 percent, the inhibition rate of the sterile fermentation liquor is 6.62 percent, and the inhibition rate of the bacterial suspension is only 24.61 percent.
TABLE 1 inhibition rate of WY3 fermentation liquid, sterile fermentation liquid and bacterial suspension on wheat stem-base rot germ plate
Example 3
Research on growth effect of WY3 on wheat seedlings
A single colony of the strain WY3 is selected and inoculated into an LB culture medium, and is cultured for 3d in a shaking table at 28 ℃ and 180 r/min. And (3) after the wheat is sterilized and sprouted, planting, after wheat seedlings sprout for 0d, 3d and 7d, irrigating the WY3 bacterial suspension on the roots of the wheat, and irrigating sterile water on a control group.
The result shows that the bacterial liquid of WY3 has growth promoting effect on wheat seedling growth in the growth process of wheat, the plant height of the wheat with the inoculated bacterial liquid is 2.14cm longer than that of the untreated wheat on average, and the root length of the wheat on average is increased by 1.49cm (shown in Table 2). In the growth process of wheat, a certain amount of bacteria liquid is poured, a certain growth promotion effect is achieved, plants are sturdier than wheat without the bacteria liquid, and under the same sample condition, the plants with the bacteria liquid are more tillered and grow more vigorously (as shown in figure 5). H103 has no obvious growth promoting effect.
TABLE 2 influence of Streptomyces WY3 on wheat seedling growth
Note: inhibition was expressed as mean ± standard error, and lower case english letters were significance of differences between groups at the significance level P < 0.05.
Example 4
Research on prevention effect of WY3 on wheat stem basal rot potted plant
And (3) planting the wheat, irrigating the WY3 bacterial liquid to the root of the wheat when the wheat seedlings bud for 0d and 3d, and irrigating sterile water to the control group. Preparing diseased wheat grains in advance: after the seeds are disinfected, soaking the seeds for 12h, carrying out moist heat sterilization at 121 ℃ for 45min, then inoculating 5-6 Fusarium Asiatica blocks, and culturing at 25 ℃ for 7 d. The diseased wheat grains are connected to the basal part of the wheat stem in the two-leaf period, and the control group is not inoculated with germs.
The results show that: wheat seedlings inoculated with Asian Fusarium are seriously dwarfed, the stem base is blackened, the average disease index is 51.11, the average disease index of the wheat seedlings treated by root irrigation with WY3 is only 18.57, and the control effect reaches 60.87%; the wheat seedlings treated by H103 root irrigation have no obvious control effect.
TABLE 3 prevention and treatment effects of WY3 on F.a wheat stalk base rot
While the invention has been described in further detail in connection with specific embodiments thereof, it will be understood that the invention is not limited thereto, and that various other modifications and substitutions may be made by those skilled in the art without departing from the spirit of the invention, which should be considered to be within the scope of the invention as defined by the appended claims.
Claims (6)
1. A Streptomyces algicidal is characterized in that: the Latin chemical name of the streptomyces algicidal isStreptomyces amritsarensisThe number of the culture medium is WY3, and the culture medium is preserved in China center for type culture Collection in 2021, 4 months and 25 days, and the preservation number is CCTCC M2021455.
2. The streptomyces algicidal strain of claim 1, wherein: the optimal fermentation conditions of the streptomyces algicidal are as follows: adopting LB culture medium to carry out shaking culture, wherein the shaking culture speed is 180r/min, the culture temperature is 28 ℃, the initial pH is 8-9, and the fermentation time is 3 d.
3. Use of streptomyces algicidal as claimed in claim 1 or 2 for the control of wheat stem basal rot.
4. The use of streptomyces algicidal for the control of wheat stem basal rot according to claim 3, characterized in that: the pathogenic bacteria of the wheat stem basal rot is Fusarium Asiaticum (F.) (Fusariumasiaticum）。
5. The use of streptomyces algicidal for the control of wheat stem basal rot according to claim 3, characterized in that: the dosage form of the streptomyces algicidal is a pharmaceutically acceptable dosage form.
6. The use of streptomyces algicidal for the control of wheat stem basal rot according to claim 5, characterized in that: the dosage form of the streptomyces algicidal is bacterial suspension.
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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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