CN117467573A - Streptomyces manipulus Sm-28 and prevention effect and growth promoting effect thereof on soybean bacterial maculopathy - Google Patents
Streptomyces manipulus Sm-28 and prevention effect and growth promoting effect thereof on soybean bacterial maculopathy Download PDFInfo
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- CN117467573A CN117467573A CN202311411355.5A CN202311411355A CN117467573A CN 117467573 A CN117467573 A CN 117467573A CN 202311411355 A CN202311411355 A CN 202311411355A CN 117467573 A CN117467573 A CN 117467573A
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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
-
- 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
-
- 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
- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
-
- 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
-
- 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
- C12R2001/465—Streptomyces
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Environmental Sciences (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Virology (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Agronomy & Crop Science (AREA)
- General Engineering & Computer Science (AREA)
- Dentistry (AREA)
- Biomedical Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
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Abstract
The invention belongs to the field of microbial biocontrol, and provides Streptomyces manipulus (Streptomyces manipurensis) Sm-28 with a preservation number of CCTCC NO: m2023906. The invention also provides application of the Streptomyces manipulus (Streptomyces manipurensis) Sm-28 in preventing and treating soybean bacterial maculopathy and promoting seedling growth.
Description
Technical Field
The invention belongs to the field of microbial biocontrol, and relates to a Streptomyces manipulus (Streptomyces manipurensis) Sm-28 strain for antagonizing soybean maculosa and application thereof in plant disease control and growth promotion.
Background
Soybean (Glycine max) is an important oil crop and plant protein source, which is used as food, feed and fuel worldwide due to the high nutritional content (proteins, sugars, oils, fatty acids and amino acids etc.) in seeds. As an important crop, soybeans are widely used worldwide, play a vital role in human and animal nutrition, and have a rapidly growing economic importance, with a global yield of about 3.065 hundred million tons and a harvest area of 1.175 hundred million hectares. Soybean has important roles in global scope and plays an important role in agricultural production and social development in China. China is one of main soybean production countries, the main production areas are concentrated in the three-provinces of northeast, the plain of Huang-Huai-Hai and the areas downstream of Yangtze river, the perennial planting area accounts for 8-10% of the cultivated land area of grains, and the main production areas occupy important positions in national economy.
Leaf diseases affect soybean growth and development, often resulting in reduced soybean yield and quality. Bacterial plaque caused by Xanthomonas (Xanthomonas), bacterial plaque caused by Pseudomonas (Pseudomonas) is the primary bacterial disease affecting soybean production. These bacterial diseases often occur in areas with warm and humid climates, and bacteria enter plants through pores and wounds by water or wind-blown rainwater, thereby achieving disease transmission. The pathogenic bacteria of bacterial leaf spot disease of soybean is carpet yellow soybean pathogenic variety (Xanthomonas axonopodis pv. Glycons, xag), soybean leaves infected with Xag fall off prematurely, so photosynthesis efficiency is obviously reduced, so that the seed volume is reduced or immature seeds are produced, and the number of seeds is reduced, so that the soybean yield is directly affected. The disease is most prevalent in the late season, where the soybean is planted, and in the early stage where the leaves produce small bluish spots surrounding an unclear yellow halo, after which the spots become irregular lesions with dark brown centers and broken epidermis. In the final stage of the disease, some lesions merge with each other, evolving into lesions of larger area, and eventually the leaves dry and necrotize. These symptoms result in a great reduction in soybean yield due to the reduced leaf surface area where photosynthesis is active.
Agriculture has been affected by many diseases, such as pathogenic bacteria, weeds, insects, etc., from ancient times to date, resulting in a dramatic drop in yield. With the advent of chemical pesticides, this crisis has been largely resolved. However, excessive reliance and unrestricted use of chemical pesticides can degrade soil, contaminate groundwater and cause land nutrient imbalances and impoverishments. Pesticide residue exceeding also causes consumer concerns about food safety and causes trade barriers to export crops. Therefore, it is urgent to find an environmentally friendly alternative.
Biopesticides are a broad range of beneficial microorganisms (bacteria, fungi, viruses, protozoans or algae) and their metabolites that are used to inhibit infection by plant pathogens and reduce the incidence of plant diseases. In recent years, biopesticides have received increasing attention for their eco-friendly properties as compared with chemical pesticides. Biopesticides are not easily tolerant to the pathogen of interest, are less harmful to other beneficial microorganisms, and exhibit prolonged persistence due to their natural reproductive ability.
Actinomycetes are an important group of biocontrol microorganisms widely used in agricultural production, contain abundant GC linear genomes, have strong biosynthesis potential, are producers of natural active substances (including two-thirds of known antibiotics and many anticancer, antifungal and immunosuppressant agents), and are vital to human health, agriculture and biotechnology development. Gaoni and the like (2019) separate actinomycetes from ginkgo tree forest soil, and the inhibition rate of the actinomycetes to apple rot pathogens and apple alternaria leaf spot pathogens measured by a growth rate method reaches more than 98 percent, and the inhibition rate of the actinomycetes to other pathogenic bacteria such as apple brown spot pathogens, wheat gibberella, rice blast and the like is more than 60 percent. Zhang Yanjun and the like (2014) from 88 strains obtained by separation and purification from different environments, the bacterial strain No. 12 with obvious antagonism to rice bacterial leaf blight is obtained by screening, the diameter of a bacteriostasis circle is 60.9mm, and the bacterial strain has certain application value and development prospect as a microbial pesticide.
The currently known uses of Streptomyces manipulus (Streptomyces manipurensis) are as follows:
1. CN115380922a provides a microbial plant growth regulator with growth promoting effect, comprising, in parts by weight: 1-20 parts of pineapple leaf, 1-50 parts of cassava residue, 0.1-3 parts of bacillus licheniformis and 0.5-5 parts of Streptomyces manniprature s-1 (streptomyces manipurensis s-1); the preservation number of the Streptomyces manneplerosis s-1 is CCTCC No: m2017261.
2. CN108102944B provides an actinomycete which is streptomyces mannipus s-1 (streptomyces manipurensis s-1) with a preservation number of cctccc No: m2017261. The anti-bacterial agent has antagonism to banana fusarium wilt 1 and 4 seeds, especially has remarkable antagonism to banana fusarium wilt 4, and has wide development space in the control of banana fusarium wilt.
Disclosure of Invention
The invention aims to solve the technical problem of providing Streptomyces manipulus Sm-28 and an effect of preventing and promoting growth of soybean bacterial maculopathy.
In order to solve the technical problems, the invention provides Streptomyces manipulus (Streptomyces manipurensis) Sm-28 with a preservation number of CCTCC NO: m2023906.
The invention also provides application of the Streptomyces manipulus (Streptomyces manipurensis) Sm-28: has inhibiting effect on plant pathogenic bacteria and plant pathogenic fungi.
The plant pathogenic bacteria are: common bacterial blight germs of kidney beans (Xanthomonas axonopodis pv.phaseoli), soybean bacterial spot germs (Pseudomonas syringae pv.glyconea), rice bacterial strip germs (Xanthomonas oryzae pv.oryzicola), rice bacterial leaf blight germs (Xanthomonas oryzae pv.oryzae), rice bacterial basal rot germs (Dickeya zeae);
the plant pathogenic fungi are: poplar Fusarium wilt (Fusarium solani), tomato early blight (Alternaria solani), watermelon anthracnose (Colletotrichum orbiculare), balsam pear Fusarium wilt (Fusarium oxysporum f.sp.moldingcae), fusarium pseudograminearum (Fusarium pseudograminearum), rice blast (Magnaporthe oryzae).
The invention also provides another application of the Streptomyces manipulus (Streptomyces manipurensis) Sm-28: has effects of preventing and treating bacterial macula disease of soybean.
The invention also provides another application of the Streptomyces manipulus (Streptomyces manipurensis) Sm-28: has growth promoting effect on plants (young plants). The plant is soybean. The growth promotion comprises increasing the plant height, root length, stem thickness and weight (including fresh weight and dry weight) of soybean seedlings.
Aiming at the problem that the pesticide control of the bacterial leaf spot of the soybean is increasingly exposed, the biological control of the bacterial leaf spot of the soybean by utilizing microorganisms and secondary metabolites thereof becomes a research direction. The invention aims to provide 1 strain of Streptomyces manipulus (Streptomyces manipurensis) Sm-28 with high antagonism to soybean maculosa bacteria.
According to the invention, 98 actinomycetes are separated from different habitat soil, 1 antagonistic Sm-28 strain with strong inhibition effect on soybean maculosa bacteria (Xanthomonas axonopodis pv. Glycoses, xag) is obtained through primary screening and secondary screening, and the strain is selected from soil under park vegetation in Jiaxing Jianjia Zhejiang. So as to provide new antagonistic actinomycete resources for biological control of soybean bacterial maculopathy.
The Sm-28 strain was deposited as follows:
preservation name: streptomyces manneplerosis Sm-28 Streptomyces manipurensis Sm-28, accession number: china center for type culture Collection; preservation address: chinese university of armed chinese, deposit number: cctccc NO: m2023906, date of preservation: 2023, 06, 05.
The invention relates to Sm-28 strain screening soil under park vegetation in Jiaxing willow Zhejiang. Streptomyces manipulus (Streptomyces manipurensis) was identified based on morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis.
The fermentation filtrate of the Streptomyces manipulus (Streptomyces manipurensis) Sm-28 strain has remarkable inhibition effect on soybean maculopathy, and can be used for preventing and treating soybean bacterial maculopathy and the like. The Sm-28 strain is fermented for 7d (the temperature is 28 ℃) in a Gao's No. 1 liquid culture medium, a 0.22 mu m filter membrane is used for filtering to obtain fermentation filtrate, the inhibition effect of the fermentation filtrate on soybean macula bacteria (Xanthomonas axonopodis pv. Glycones, xag) is measured by an oxford cup method, and the diameter of a bacteria inhibition zone can reach 34.0+/-0.26 mm (figure 5). The antibacterial spectrum test result shows that: the Sm-28 strain fermentation filtrate has strong inhibition effect on rice bacterial leaf spot bacteria (Xanthomonas oryzae pv. Oryzicola), rice bacterial leaf blight bacteria (Xanthomonas oryzae pv. Oryzae) and rice bacterial basal rot bacteria (Dickeya zeae) (table 3, figure 7); also has better inhibition effect on the growth of 6 plant pathogenic fungi hyphae, wherein the hyphae inhibition rate on fusarium pseudograminearum (Fusarium pseudograminearum) and fusarium solani (Alternaria solani) reaches 68.57+/-2.86% and 66.33 +/-3.36% respectively (table 4, fig. 8). The potted plant prevention effect test shows that: the relative prevention effect of the fermentation filtrate of the Sm-28 strain on the bacterial maculopathy of the soybean can reach 88.14 percent (see table 5 and figure 9); experiments also show that the Sm-28 strain fermentation filtrate diluent has obvious growth promotion effect on a plurality of growth indexes of soybean seedlings (see Table 6). The Sm-28 strain can provide excellent strain for the development of microbial pesticide, and has better application prospect in biological control of crops such as soybean and the like.
In summary, the invention provides 1 Streptomyces manipulus (Streptomyces manipurensis) Sm-28 strain antagonizing soybean maculosa bacteria (Xanthomonas axonopodis pv. Glyces, xag) and application thereof in prevention and control of soybean bacterial maculosa and promotion of seedling growth.
Drawings
The following describes the embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows colony morphology of 28 representative actinomycete pure strains (culture medium 1 Gao's, culture 7 d);
FIG. 2 shows the colony and microscopic morphological characteristics of Sm-28 strain (Gao's No. 1 medium, 7 d);
FIG. 3 shows the results of a physiological biochemical part of Sm-28 strain;
FIG. 4 is a Sm-28 strain evolutionary tree constructed based on the 16S rDNA sequence;
FIG. 5 shows the inhibition of growth of the soybean macula bacteria Xag by the Sm-28 strain fermentation filtrate (Oxford cup method);
FIG. 6 is an electron micrograph of the effect of Sm-28 strain fermentation filtrate on the cells of the soybean maculosa bacteria Xag;
in fig. 6:
a: gao's No. 1 liquid medium control; b: treating the fermentation filtrate of the Sm-28 strain;
FIG. 7 shows the inhibitory effect of Sm-28 strain fermentation filtrate on 5 plant pathogenic bacteria;
FIG. 8 shows the inhibitory effect of Sm-28 strain fermentation filtrate on 6 plant pathogenic fungi;
FIG. 9 shows the control effect of Sm-28 strain fermentation filtrate on soybean bacterial maculopathy;
in fig. 9: CK1: inoculating a negative control of Gaoshan No. 1 culture solution; CK2: inoculating an Xag positive control; t1: spraying Sm-28 fermentation filtrate stock solution, and inoculating Xag; t2: spraying 4 times of dilution of Sm-28 fermentation filtrate, and inoculating Xag; t3: spraying 8 times of dilution of Sm-28 fermentation filtrate, and inoculating Xag; t4: after inoculation of the Xag, 4-fold dilutions of the Sm-28 fermentation filtrate were sprayed.
Detailed Description
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
the culture mediums related to the invention are as follows:
(1) Solid medium No. 1 gao: soluble starch 20g, KNO 3 1g、K 2 HPO 4 0.5g、MgSO 4 ·7H 2 O 0.5g、NaCl 0.5g、FeSO 4 ·7H 2 O0.01 g, agar 20g, 1000mL of water, pH 7.2-7.4.
(2) Culture broth No. 1 gao (i.e., liquid medium No. 1 gao): the other ingredients were the same as in (1) except that no agar was added.
(3) NA medium (NA solid medium): beef extract 3g, peptone 5g, naCl 5g, agar 20g, water 1000mL, pH 7.2.
(4) NA culture solution: the other ingredients were the same as in (3) except that no agar was added.
(5) PDA solid medium: 200g of potato, 20g of glucose, 18g of agar and 1000mL of water, and the pH value is 6.0-6.5.
EXAMPLE 1 screening and identification of Streptomyces manipulus (Streptomyces manipurensis) Sm-28 Strain
Strain 1
(1) Actinomycete strain: isolation, purification, screening and identification of the deposited actinomycete strains from the soil of different habitats.
(2) The soybean maculosis (Xanthomonas axonopodis pv. Glyces, xag, hereinafter abbreviated as Xag) is an existing conventional strain, for example, NEAU001 strain, which is a microorganism belonging to the national academy of sciences.
2 Medium
Comprises the following culture mediums:
the solid culture medium of Gao's No. 1 is used for separation, purification, identification and the like of actinomycete strains.
Culture solution 1 of Gao's No. 1 is used for liquid fermentation culture of actinomycetes strain, etc.
NA medium is used for culturing Xag germ.
NA culture solution is used for activating Xag germ.
3 Experimental method
3.1 isolation, purification and preservation of actinomycete Strain
Actinomycetes were isolated from different habitat soil samples by dilution coating. Weighing 5g of soil in a conical flask, adding 45mL of sterile water, and shake culturing for 0.5h by a shaking table, wherein the concentration of the soil suspension is 10 -1 1mL of the suspension with the concentration is taken in a test tube, 9mL of sterile water is added, and the concentration of the soil suspension is 10 -2 Repeating the above operation to dilute the soil sample to 10 -5 . 100 mu L of the suspensions with different concentrations are uniformly coated on a Gao's No. 1 solid culture medium plate (Gao's No. 1 solid culture medium) containing potassium dichromate (50 mu g/mL), the culture is inverted at 28 ℃, when bacterial colonies with actinomycete characteristics are observed, the bacterial colonies are picked and inoculated on a new Gao's No. 1 solid culture medium plate by an inoculating needle, and the transferring operation is repeated until the bacterial colonies grow in a sterile mode. The purified actinomycete strain was numbered and stored in 25% glycerolIn a refrigerator at 80 ℃.
3.2 screening of antagonistic Xag actinomycete strains
3.2.1 Co-cultivation Primary screening
Actinomycetes stored in a refrigerator at the temperature of-80 ℃ are inoculated on a Gao's No. 1 plate, and are cultivated at the constant temperature of 28 ℃ for 2 times of activation, and when colonies grow to 7d, the actinomycetes are used for primary screening; cutting actinomycete cakes with the diameter of 6mm and growing vigorously.
1mL OD 600 Xag bacteria solution=0.6 was added to 99mL NA solid medium, and the mixture was poured into a plate. The actinomycete cake is poured into the center of the pathogen-containing flat plate. Culturing at 28 ℃ for 2-3 d, observing whether a bacteriostasis ring appears, and primarily screening to obtain antagonistic actinomycetes.
3.2.2 oxford cup process re-screening
(1) Fermentation culture of actinomycete strains: inoculating antagonistic actinomycetes into a solid culture medium plate of Gaoshi No. 1, culturing at 28deg.C for 3d, punching with a sterile puncher with diameter of 6mm on the actinomycete plate to obtain bacterial blocks, inoculating into a 250mL triangular flask containing 100mL of culture solution of Gaoshi No. 1, shake culturing at 160r/min and 28deg.C for 7d, centrifuging the fermentation broth 10000r/min for 3min, and filtering with 0.22 μm filter membrane to obtain fermentation filtrate.
(2) Culturing of Xag: the Xag is inoculated into NA culture solution, shake bed activation culture is carried out (160 r/min,28 ℃), and when the bacterial density reaches OD 600 When=0.6, 1mL of Xag bacteria liquid is added into 99mL of melted NA culture medium, the mixture is poured into a plate, after solidification, a sterilized oxford cup is placed in the center of the plate, 200 μl of fermentation filtrate is added, the temperature is kept constant for 3 days at 28 ℃, the size of a bacteriostasis zone is measured by a crisscross method, and 3 repetitions are set for each experiment. And selecting actinomycete strains with strong antagonism according to the size of the inhibition zone.
3.2.3 scanning Electron microscope observation of morphological changes of Xag cells after treatment of fermentation filtrate of target Strain
(1) Culturing of Xag: inoculating Xag into NA culture solution, shake-culturing (160 r/min,28 ℃) until bacterial density is OD 600 ＝0.6。
(2) Fermentation culture of actinomycete strains: a fermentation filtrate of antagonistic actinomycetes was obtained by the method of 3.2.2 (1).
(3) Scanning electron microscope observation: after 5mL of the Xag bacterial liquid is centrifuged to obtain thalli, 5mL of fermentation filtrate diluent (prepared by mixing equal volume of fermentation filtrate and Gao's No. 1 culture liquid) is added for common shaking culture for 4h (160 r/min and 28 ℃), the Xag bacterial liquid treated by the Gao's No. 1 culture liquid is used as a control group, and after 4h of culture, 10000r/min is centrifuged for 5min to collect thalli. Gently washing the collected thalli for 3 times by using a phosphate buffer solution, fixing the thalli in a refrigerator with the temperature of 4 ℃ by using 2.5% glutaraldehyde overnight, and then soaking and washing the thalli for three times by using the phosphate buffer solution for 10 minutes each time; gradient dehydrating with 30%, 50%, 70%, 80%, 90%, 95% ethanol for 2 times and 15min each; soaking with tertiary butanol for three times, each for 10 minutes, and then placing into a vacuum freeze dryer for vacuum drying; and (5) performing scanning electron microscope observation after the metal spraying of the sample.
3.3 identification of Sm-28 Strain
3.3.1 morphological feature observations
Taking a Sm-28 strain 1 bacterial cake (diameter is 6 mm), and inoculating on a Gao's No. 1 culture medium plate for activation culture for 3d; the colonies were inoculated onto a new Gao's No. 1 medium plate with an inoculating needle, cultured at 28℃for 7 days, and observed for their colony size, morphology, texture, surface, color, etc. Morphological characteristics of the endomycelia, aerial mycelia, spore filaments and the like of the Sm-28 strain are observed under an optical microscope.
3.3.2 physiological Biochemical characterization experiments
And performing physiological and biochemical tests on the Sm-28 strain, such as enzymatic properties, utilization of carbon source nitrogen source and the like.
3.3.3 16S rDNA sequence analysis
Extracting the genomic DNA of Sm-28 strain, amplifying 16S rDNA sequence, and sequencing by Shanghai bioengineering company. And submitting the 16S rDNA sequence obtained by sequencing to GenBank, comparing by utilizing BLAST, analyzing, and constructing a phylogenetic tree by adopting a Neighbor-Joing method in MEGA-X software to determine the actinomycete species.
4. Experimental results
4.1 Obtaining pure strains of actinomycetes
98 pure actinomycetes strains are obtained in total by separation and purification from soil samples of different habitats, and the colony of 28 representative pure actinomycetes strains cultured for 7d in a culture medium of Gao's No. 1 is shown in FIG. 1.
4.2 screening of antagonistic Xag actinomycete strains
Preliminary screening and rescreening of the Xag inhibition effect are carried out on 98 actinomycete strains by using a co-culture method and an oxford cup method, and the inhibition effect of different actinomycete strains is greatly different. 1 actinomycete strain with strong antagonism is obtained through screening, the number of the actinomycete strain is Sm-28, the diameter of a bacteriostasis circle measured by an oxford cup method reaches 34.0+/-0.26 mm (figure 5), and the Sm-28 strain is separated from soil under park vegetation in Jiaxing willow in Zhejiang.
Sm-28 strain was preserved with the following information:
preservation name: streptomyces manneplerosis Sm-28 Streptomyces manipurensis Sm-28, accession number: china center for type culture Collection; preservation address: chinese university of armed chinese, deposit number: cctccc NO: m2023906, date of preservation: 2023, 06, 05.
4.3 changes in the morphology of the Xag cells after treatment of the fermentation filtrate of the Sm-28 Strain
The scanning electron microscope observation result shows that: after the strain Sm-28 fermentation filtrate was treated with the Xag cells, the contents of the Xag cells were leaked out, and the cells were shriveled and dissolved, further indicating that the strain Sm-28 fermentation filtrate had a strong lethal effect on the Xag cells (FIG. 6).
4.4 identification results of Sm-28 Strain
Morphological characteristics of Sm-28 strain: culturing on Gaoshi No. 1 culture medium at 28deg.C, and growing to obtain white mycelium and gas mycelium, and slowly growing to pink. After 7d of culture, pink velvet colonies are formed, the middle of the velvet colonies are raised, thalli are fluffy, the edges of the velvet colonies are irregularly fluff, and no soluble pigment is produced; aerial hyphae are loose and branched, and are broken and differentiated into spore filaments after maturation, and the spore filaments are linear and spiral, and the aerial hyphae are first-order rotagenesis (figure 2).
The results of the physiological and biochemical tests show that: the Sm-28 strain can produce amylase, cellulase, lipase, urease, gelatinase, hydrogen sulfide and melanin (Table 1, figure 3); various carbon sources can be utilized, and glycerin and starch (table 2) are preferable for the utilization effect; a variety of nitrogen sources may be utilized, with preferred nitrogen sources being peptone and yeast powder.
TABLE 1 physiological and biochemical test results of Sm-28 strains
Note that: ++ means that the capacity is high and, in the ++ representation capability, + indicates weaker capacity, -indicates no
TABLE 2 carbon source and Nitrogen source utilization test results of Sm-28 Strain
Note that: ++ represents the growth is vigorous and the growth is carried out, ++ means that the growth is good and that, + indicates general growth, -indicates no growth
The nucleotide sequence of the 16S rDNA sequence analysis is shown as SEQ ID NO. 1 of the sequence table.
The evolutionary distance from Streptomyces manipulus (Streptomyces manipurensis) was closest (FIG. 4). Based on morphological characteristics of the Sm-28 strain, physiological and biochemical test results and 16S rDNA sequence analysis, the Sm-28 strain was identified as Streptomyces mannopropionii (Streptomyces manipurensis) in combination with Streptomyces (Streptomyces) species search and tree analysis.
Description: the Streptomyces manneplerosis s-1 (streptomyces manipurensis s-1) described in the background art has no inhibition effect basically when the diameter of the inhibition zone of Xag is smaller than 10mm according to the oxford cup method.
EXAMPLE 2 determination of the antibacterial spectrum of Streptomyces manipulus (Streptomyces manipurensis) Sm-28 Strain
Strain 1
(1) Streptomyces manipulus (Streptomyces manipurensis) strain Sm-28.
(2) 5 representative phytopathogenic bacteria: the bacterial strain comprises common bacterial blight germs of kidney beans (Xanthomonas axonopodis pv.phaseoli), bacterial spot germs of soybean (Pseudomonas syringae pv.glyconea), bacterial spot germs of rice (Xanthomonas oryzae pv.oryzicola), bacterial blight germs of rice (Xanthomonas oryzae pv.oryzae) and bacterial basal rot germs of rice (Dickeya zeae), and is used for the determination of bacterial spectrum of fermentation filtrate of Sm-28 strains.
(3) 6 representative plant pathogenic fungi: the fungus spectrum of the fermentation filtrate of the Sm-28 strain is determined by using poplar Fusarium wilt (Fusarium solani), tomato early blight (Alternaria solani), watermelon anthracnose (Colletotrichum orbiculare), balsam pear Fusarium wilt (Fusarium oxysporum f.sp. moldinger), fusarium pseudograminearum (Fusarium pseudograminearum) and rice blast fungus (Magnaporthe oryzae).
2 Medium
NA medium (NA solid medium), NA broth: used for culturing plant pathogenic bacteria;
culture broth No. 1 gao (i.e., liquid medium No. 1 gao): used for culturing the fermentation broth of Sm-28 strain.
PDA solid medium: is used for culturing plant pathogenic fungi.
3 Experimental method
3.1 cultivation of Sm-28 Strain and preparation of fermentation filtrate
3 bacterial cakes (diameter is 6 mm) of Sm-28 bacterial strain are taken by a puncher and inoculated into Gaoshi No. 1 culture solution, the liquid loading amount is 100/250mL,160r/min and the constant temperature shaking table culture is carried out for 7d at 28 ℃. And (3) placing the fermentation liquor into a 50mL centrifuge tube, centrifuging at 10000r/min for 3min, filtering the supernatant with a 0.22 mu m filter membrane, and removing residual spores to obtain fermentation filtrate for later use.
3.2 activation of phytopathogenic bacteria
The 5 pathogenic bacteria preserved at 4 ℃ are respectively inoculated into NA culture medium, activated and cultured for 2d at 28 ℃, and then transferred onto new NA culture medium and cultured for 2d at 28 ℃ for bacterial antimicrobial spectrum measurement.
3.3 determination of antibacterial Spectrum of Sm-28 Strain
Inoculating 5 plant pathogenic bacteria into NA culture solution, shake culturing (160 r/min,28 deg.C), and culturing when bacterial density reaches OD 600 When the bacteria is=0.6, 1mL of pathogenic bacteria liquid is respectively added into 99mL of melted NA culture medium, the mixture is uniformly mixed and poured into a plate, and after solidification, the sterilized oxford cup is usedPlaced in the center of the plate, 200. Mu.L of fermentation filtrate was added, the culture solution of Gaoshi No. 1 was used as a control, the culture was carried out at a constant temperature of 28℃for 3 days, and the size of the inhibition zone was measured by the crisscross method, and 3 replicates were set for each set of experiments.
3.4 determination of the antifungal Spectrum of the Sm-28 Strain
And 6 plant pathogenic fungi are clamped by sterile forceps and activated in PDA solid culture medium, and are cultured for 7d at 28 ℃ for standby. 4 plant pathogenic fungi bacterial cakes (with the diameter of 6 mm) are cut by a puncher, 4 bacterial cakes (with the diameter of 6 mm) of a target strain are placed on 4 opposite corners of a PDA flat plate in a reverse buckling mode at the center of the flat plate, a Gaoshi No. 1 solid culture medium with the same diameter is used as a control, when pathogenic fungi hyphae of a control group grow on the whole flat plate, the diameters of pathogenic fungi bacterial colonies of each group are recorded, and the hyphae growth inhibition rate is calculated. Hypha growth inhibition ratio= (control group colony diameter-treatment group colony diameter)/control group colony diameter. Experiments were repeated 3 times.
4 experimental results
4.1 inhibition effect of Sm-28 Strain fermentation filtrate on 5 plant pathogenic bacteria
The experimental results show that: the Sm-28 strain fermentation filtrate has the strongest inhibition effect on rice bacterial leaf spot germ (Xanthomonas oryzae pv. Oryzicola); secondly, bacterial blight bacteria (Xanthomonas oryzae pv. Oryzae) and bacterial basal rot bacteria (Dickeya zeae) of rice are treated; the inhibition effect on common bacterial blight of kidney beans (Xanthomonas axonopodis pv.phaseoli) and soybean bacterial spot pathogens (Pseudomonas syringae pv.glycoea) was weak (see table 3, fig. 7).
TABLE 3 inhibition of 5 phytopathogenic bacteria by Sm-28 Strain fermentation filtrate
Note that: different lowercase letters indicate that there is a significant difference at the P <0.05 level
4.2 inhibition effect of Sm-28 Strain fermentation filtrate on 6 plant pathogenic fungi
The inhibition effect of the fermentation filtrate of the Sm-28 strain on 6 plant pathogenic fungi is measured by a hypha inhibition rate method and is shown in Table 4 and FIG. 8. The inhibition effect on Fusarium pseudograminearum (Fusarium pseudograminearum) and Fusarium solani (Alternaria solani) is strongest, the hypha inhibition rate reaches 68.57+/-2.86% and 66.33 +/-3.36%, and the hypha inhibition rate on rice blast bacteria (Magnaporthe oryzae) is 52.33 +/-2.31%.
TABLE 4 inhibition of 6 phytopathogenic fungi by Sm-28 Strain fermentation filtrate
Note that: different lower case letters represent significant differences between treatments, P <0.05
EXAMPLE 3 control and growth promoting Effect of Sm-28 Strain fermentation filtrate on bacterial Zebra disease of soybean
1 strain and soybean variety
(1) Actinomycetes for controlling and promoting growth: streptomyces manipulus (Streptomyces manipurensis) strain Sm-28.
(2) Pathogenic bacteria: leptodermia sojae (Xanthomonas axonopodis pv. Glycines, xag).
(3) Soybean variety: zhou Dou 25 No. 5225 was chosen as the experimental material.
2 Medium
Culture broth No. 1 gao (i.e., liquid medium No. 1 gao): the method is used for preparing the culture fermentation filtrate of the Sm-28 strain.
NA medium, NA broth: is used for culturing Xag germ.
3. Experimental method
3.1 Cultivation of soybeans
Selecting full soybean seeds, sowing the seeds in a seedling raising tray, transplanting the soybean seeds in an independent small basin after the soybean seedlings emerge, culturing the soybean seeds in an illumination incubator, and illuminating for 18 hours and dark for 6 hours at 26 ℃ and 23 ℃ in day and night. And (5) when the soybean true leaves are fully unfolded for standby.
3.2 cultivation of Xag pathogens
Inoculating Xag germ preserved at 4 ℃ on an NA culture medium plate, and performing activation culture for 3d at 28 ℃; picking single colony, inoculating into conical flask (250 mL) containing 100mL NA culture solution, shake culturing at 28deg.C under 160r/min to OD 600 ＝0.6。
3.3 preparation of Sm-28 Strain fermentation filtrate
3 bacterial cakes (diameter is 6 mm) of Sm-28 bacterial strain are taken by a puncher and inoculated into Gaoshi No. 1 culture solution, the liquid loading amount is 100/250mL,160r/min and the constant temperature shaking table culture is carried out for 7d at 28 ℃. And (3) placing the fermentation liquor into a 50mL centrifuge tube, centrifuging at 10000r/min for 3min, filtering the supernatant with a 0.22 mu m filter membrane, and removing residual spores to obtain fermentation filtrate for later use.
The fermentation filtrate was diluted with 3 volumes of sterile water to obtain a 4-fold dilution of the fermentation filtrate.
The fermentation filtrate was diluted with 7-fold volume of sterile water to obtain an 8-fold dilution of the fermentation filtrate.
3.4 preliminary study of potted plant control Effect
(1) Negative control group (CK 1): a sterile brush is used for dipping the blank Gaoshi No. 1 culture solution, and the culture solution is uniformly brushed on the front and back surfaces of the leaves and is moisturized for 24 hours.
(2) Positive control group (CK 2): dipping the Xag bacterial liquid by using a sterile brush, uniformly brushing the Xag bacterial liquid on the front and back surfaces of the blades, and preserving moisture for 24 hours.
(3) Treatment group 1 (T1): uniformly spraying Sm-28 fermentation filtrate on front and back sides (100 μL/cm) of soybean leaf with spray pot 2 ) Spraying every 0.5h for 6 times; after 12h of the last spraying, the same CK2 treatment method is used for inoculating Xag and preserving moisture.
(4) Treatment group 2 (T2): uniformly spraying 4-time dilution of the fermentation filtrate of Sm-28 on the front and back surfaces of soybean leaves according to the method described in T1, and inoculating Xag and preserving moisture by using a CK2 treatment method after 12 hours of last spraying.
(5) Treatment group 3 (T3): and uniformly spraying 8-time diluent of the fermentation filtrate of Sm-28 on the front and back surfaces of soybean leaves according to the method described in T1, and inoculating Xag and preserving moisture by using a CK2 treatment method after 12 hours of last spraying.
(6) Treatment group 4 (T4): the same CK2 method is used for inoculating Xag, preserving moisture, and uniformly spraying 4-time diluent of the fermentation filtrate of Sm-28 to the front and back surfaces of soybean leaves according to the method described by T1 after 12 hours of inoculation, wherein the spraying is performed once every 0.5 hour for 6 times.
And (5) after the treatment is finished, continuously placing the soybeans in an illumination incubator for cultivation, recording the disease condition after the disease is cured, and calculating the disease index and the prevention and treatment effect. The whole experiment was repeated 3 times for each group of 5 soybeans.
Disease grading standard: 0 grade, leaf no disease spot; stage 1, the area of the disease spots occupies less than 1/4 of the area of the leaves; 2, the area of the disease spots occupies about 1/4 to 1/2 of the area of the leaves; 3, the area of the disease spots occupies 1/2 to 3/4 of the area of the leaves, and nearly half of the leaves die; stage 4, the area of the disease spots occupies more than 3/4 of the area of the leaves, and more than half or all of the leaves die.
Incidence (%) = number of diseased leaves/total number of investigation leaves x 100%
Disease index = Σ (number of disease plants at each stage×number of disease stages corresponding to each stage)/(total number of investigation×4) ×100
Relative control (%) = (positive control disease index-treatment group disease index)/positive control disease index x 100%
3.5 Effect of Sm-28 Strain fermentation filtrate on Soybean seedling growth
Transplanting the soybean with seedling in small basin, and measuring the growth indexes of soybean plant. The soybeans were divided into three groups of 10 pots each, each: control group (CK), root-irrigating culture solution Gaoshi No. 1 is applied for 3 times, 20mL each time, and interval is 4d; treatment group 1 (T1), root irrigation, applying 50-fold dilution of Sm-28 fermentation filtrate 3 times, 20mL each time, and 4d intervals; treatment group 2 (T2), roots were irrigated with 100-fold dilutions of Sm-28 fermentation filtrate 3 times, 20mL each, 4d apart. After all treatments are completed, soybean plants with consistent growth vigor are selected in 15d, and the fresh weight, dry weight, stem thickness, plant height and root length of the soybean plants are measured, and growth promotion and amplification are calculated.
4 experimental results
4.1 prevention and treatment effect of Sm-28 Strain fermentation filtrate on soybean bacterial maculopathy
The experimental results are shown in table 5 and fig. 9: the disease index of the positive control group (CK 2) is up to 98.33, and the occurrence of diseases can be slowed down after fermentation liquor treatment is applied. When Sm-28 strain fermentation filtrate is used for prevention treatment (T1, T2 and T3 treatment groups), the relative prevention effect is up to 88.14%, the morbidity and the disease index are increased along with the reduction of the concentration of the fermentation liquor, and when the fermentation filtrate (T3) diluted by 8 times is used for treatment, the disease index is reduced to 61.02% compared with the primary liquor treatment group (T1); the prevention treatment (T2) with 4-fold dilution of the fermentation filtrate has a relative prevention effect of 74.58 percent, which is superior to the treatment (T4, prevention effect of 52.54 percent) with the same concentration of fermentation filtrate. The control effect test shows that the control effect and concentration of the Sm-28 strain fermentation filtrate are positively correlated, and the control effect on the bacterial maculopathy of the soybean is better than the treatment effect.
TABLE 5 control of bacterial leaf spot of soybean by fermentation filtrate of Sm-28 strain
Note that: different lowercase letters indicate that there is a significant difference at the P <0.05 level
4.2 Effect of Sm-28 Strain fermentation filtrate on Soybean seedling growth
The influence of the fermentation filtrate of the Sm-28 strain on the growth of the soybean seedlings is discussed by measuring 5 growth indexes of fresh weight, dry weight, stem thickness, plant height and root length of the soybean seedlings. The results are shown in Table 6. Other growth indexes of soybean seedlings except root length and plant height are promoted by the fermentation filtrate of the Sm-28 strain. After applying Sm-28 strain fermentation filtrate diluent, the stem thickness of soybean seedlings is increased from 3.00mm to 3.45mm and 3.94mm, and the growth promotion and amplification are respectively 15.1% and 31.4%; the dilution of the fermentation filtrate of 100 times promotes the fresh weight to increase by 64.41 percent; the dry weight of soybean seedlings is increased by 1.66 times and 2 times respectively. Experiments show that the Sm-28 strain fermentation filtrate diluent has obvious growth promotion effect on a plurality of growth indexes of soybean seedlings, and is beneficial to the accumulation of soybean biomass.
TABLE 6 Effect of different concentrations of Sm-28 fermentation filtrate on soybean seedling growth
Description: the Streptomyces mannopropan s-1 (streptomyces manipurensis s-1) described in the background art above was examined for the growth-promoting effect on the growth of soybean seedlings according to the above, and it was found that there was little obvious growth-promoting effect on the growth of soybean seedlings.
Finally, it should also be noted that the above list is merely a few specific embodiments of the present invention. Obviously, the invention is not limited to the above embodiments, but many variations are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.
Claims (7)
1. Streptomyces manipulus (Streptomyces manipurensis) Sm-28 is characterized by: the preservation number is CCTCCNO: m2023906.
2. The use of Streptomyces manipulus (Streptomyces manipurensis) Sm-28 is characterized in that: has inhibiting effect on plant pathogenic bacteria and plant pathogenic fungi.
3. Use of Sm-28 according to claim 2, characterized in that:
the plant pathogenic bacteria are: common bacterial blight germs of kidney beans (Xanthomonas axonopodis pv.phaseoli), soybean bacterial spot germs (Pseudomonas syringae pv.glyconea), rice bacterial strip germs (Xanthomonas oryzae pv.oryzicola), rice bacterial leaf blight germs (Xanthomonas oryzae pv.oryzae), rice bacterial basal rot germs (Dickeya zeae);
the plant pathogenic fungi are: poplar Fusarium wilt (Fusarium solani), tomato early blight (Alternaria solani), watermelon anthracnose (Colletotrichum orbiculare), balsam pear Fusarium wilt (Fusarium oxysporum f.sp).
momordica), fusarium pseudograminearum (Fusarium pseudograminearum), rice blast bacteria (Magnaporthe oryzae).
4. The use of Streptomyces manipulus (Streptomyces manipurensis) Sm-28 is characterized in that: has effects of preventing and treating bacterial macula disease of soybean.
5. Use of Sm-28 according to any one of claims 2 to 4, characterized in that: has growth promoting effect on plants.
6. Use of Sm-28 according to claim 5, characterized in that: the plant is soybean.
7. Use of Sm-28 according to claim 6, characterized in that: the growth promotion comprises the steps of increasing the plant height, root length, stem thickness and weight of soybean seedlings.
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