CN117264851B - Streptomyces strain and application thereof - Google Patents
Streptomyces strain and application thereof Download PDFInfo
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- CN117264851B CN117264851B CN202311492599.0A CN202311492599A CN117264851B CN 117264851 B CN117264851 B CN 117264851B CN 202311492599 A CN202311492599 A CN 202311492599A CN 117264851 B CN117264851 B CN 117264851B
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- banana
- anthracnose
- streptomyces
- fusarium wilt
- pathogen
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Classifications
-
- 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
- A01P3/00—Fungicides
-
- 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
-
- 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
Abstract
The invention provides a streptomycete, a streptomycete namedStreptomyces fimicariusThe registration and preservation are carried out in the Guangdong province microorganism strain collection center, and the preservation number is GDMCC No:63831. the invention also provides the fermentation liquor of the streptomycete and an ethanol extract of the fermentation liquor. The streptomyces and the fermentation liquor and the crude extract of the fermentation liquor have broad-spectrum antibacterial activity, have good antagonism to various pathogenic bacteria, have the antibacterial rate of more than 57 percent, and especially have the antibacterial rate of more than 70 percent to strawberry anthracnose pathogen, chestnut epidemic pathogen, banana anthracnose pathogen, mango alternaria leaf spot pathogen, pear anthracnose pathogen and wheat gibberella, are potential biological agents for preventing and treating diseases such as anthracnose diseases and fusarium oxysporum, have wide development space and have good development and application prospects.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to a streptomycete strain and application thereof.
Background
Bananas are musaceae musa plants, are mainly distributed in tropical and subtropical zones, and are important fruit and grain crops. When the banana industry is continuously developed and a certain effect is gradually obtained, banana wilt is spread in a banana planting area, so that the traditional banana planting area is rapidly reduced, even phenomena of losing, barren and changing seeds occur, and great economic loss is brought to the banana industry. Banana wilt is characterized by comprising the step of specializing in fusarium oxysporumFusarium oxysporum f. sp. cubenseFoc) is infected with a soil-borne disease. The healthy development of the banana industry has been seriously threatened due to its high incidence, large destructiveness and rapid spread. Biological control by microorganisms is a relatively effective control strategy against banana vascular wilt by comparing different control strategies. Therefore, the biological control technology for preventing and controlling banana vascular wilt by adopting microbial agents or bacterial fertilizers becomes an important alternative prevention and control way.
Biological control is a control strategy for inhibiting pathogen infection by using antagonistic microorganisms, and mainly aims to prevent and control banana wilt by improving diversity of soil microorganisms and producing secondary metabolites to inhibit reproduction of fusarium. The biological control has the advantages of relatively less pollution to the environment, no side effect to bananas, simple application technology, no resistance to pathogenic bacteria and the like. Streptomyces can produce various secondary metabolites, and has the effects of resisting pathogenic bacteria, promoting plant growth and the like. The fermentation liquor prepared by the microorganisms not only can effectively promote the growth of crops, improve the quality of the crops and strengthen the disease resistance of plants, but also can not threaten the ecological environment of soil, and has the characteristics of high efficiency, environmental protection, low cost and the like compared with the traditional chemical fertilizers and medicaments. Due to the safety and green characteristics of prevention and control strategies, biological prevention and control have become research hotspots in recent years, but due to the complex variability of natural environments, biological prevention and control measures cannot meet the actual demands of various regions, and more safe and effective biological agents still need to be developed in real time and on the spot according to local conditions.
Medicinal plants and their endophytes are important sources of precious bioactive compounds and secondary metabolites. To date, the diversity of endophytes and the potential for secondary metabolites in medicinal plants have been relatively few. The endophytic streptomycete is widely existing in medicinal plant tissues, has rich species diversity, is an important component of a plant micro-ecological system, can secrete various novel bioactive metabolites including antibacterial, antifungal, antiviral and antitumor drugs, and is widely applied to industries such as medicines, agriculture and the like. At present, the medicinal plant of the green grass of bamboo leaves and the root of Chinese angelica is preparedCyclobalanopsis bambusaefolia) The endophyte actinomycetes of the formula (I) has not been researched, and the unknown resource to be developed has great application potential in the aspects of medicine and health, biological control and promotion of plant growth, and provides a new biocontrol resource for the control of banana wilt diseases.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a streptomycete strain and application thereof.
A first aspect of the present invention provides a Streptomyces, designatedStreptomyces fimicarius(test No. 43Y-GA-1), registered and deposited with the Guangdong province microorganism bacterium deposit center under the deposit No. GDMCC No:63831.
in a second aspect of the invention there is provided a fermentation broth of Streptomyces according to the first aspect of the invention.
In a third aspect, the invention provides an ethanol extract of a fermentation broth of Streptomyces according to the first aspect of the invention.
In a fourth aspect, the present invention provides a formulation comprising Streptomyces according to the first aspect of the invention, or a fermentation broth according to the second aspect of the invention, or an ethanol extract according to the third aspect of the invention.
In a fifth aspect, the invention provides the use of a Streptomyces according to the first aspect of the invention, or a fermentation broth according to the second aspect of the invention, or an ethanol extract according to the third aspect of the invention, or a formulation according to the fourth aspect of the invention, for antagonizing strawberry anthracnose, and/or chestnut blight, and/or banana anthracnose, and/or Alternaria mangiferum, and/or pear anthracnose, and/or wheat scab, and/or cucumber fusarium wilt, and/or tomato fusarium wilt, and/or banana leaf spot, and/or rice blast, and/or gum spore, and/or oil pear base rot, and/or banana gray spot, and/or banana fusarium wilt No. 1 physiological micro, and/or banana fusarium wilt No. 4 physiological micro.
In a sixth aspect, the invention provides the use of a Streptomyces according to the first aspect of the invention, or a fermentation broth according to the second aspect of the invention, or an ethanol extract according to the third aspect of the invention, or a formulation according to the fourth aspect of the invention, for controlling strawberry anthracnose and/or chestnut blight and/or banana anthracnose and/or Alternaria mangiferum leaf spot and/or Pyricularia oleander and/or Alternaria tricolor and/or cucumber fusarium wilt and/or tomato fusarium wilt and/or banana leaf spot and/or rice blast and/or colletotrichum gloeosporioides and/or Alternaria pyriformis and/or banana gray spot and/or banana fusarium wilt No. 1 physiological race and/or banana fusarium wilt No. 4 physiological race.
In a seventh aspect, the invention provides the use of Streptomyces according to the first aspect of the invention, or of a fermentation broth according to the second aspect of the invention, or of an ethanol extract according to the third aspect of the invention, or of a formulation according to the fourth aspect of the invention, for the crimping and collapsing of mycelia of Foc pathogens.
The streptomyces and the fermentation liquor and the crude extract of the fermentation liquor have broad-spectrum antibacterial activity, have good antagonism to strawberry anthracnose pathogen, chestnut blight pathogen, banana anthracnose pathogen, mango alternaria leaf spot pathogen, pear anthracnose pathogen, wheat red fungus pathogen, cucumber fusarium wilt pathogen, tomato fusarium wilt pathogen, banana long-shaped spot pathogen, rice blast pathogen, colletotrichum gloeosporioides, pyritinospora, banana gray spot pathogen, banana fusarium wilt 1 physiological micro-species, banana fusarium wilt pathogen 4 physiological micro-species and the like, have antibacterial rates of more than 57%, especially have antibacterial rates of more than 70% to strawberry anthracnose pathogen, chestnut blight pathogen, banana anthracnose pathogen, mango alternaria leaf spot pathogen, pyritic anthrax and wheat red fungus pathogen, are potential biological agents for preventing and controlling fusarium anthracnose and pointed spore bacteria, and the like, and have wide development and application prospects.
Drawings
FIG. 1 is a morphological observation Scanning Electron Microscope (SEM) of strain 43Y-GA-1.
FIG. 2 shows the bacteriostatic activity of strain 43Y-GA-1 against Foc pathogens.
FIG. 3 shows the bacteriostatic activity of the fermented extract of strain 43Y-GA-1 against Foc pathogens.
FIG. 4 is a scanning electron microscope observation of the mycelium morphology of banana vascular wilt after the fermentation extract treatment of the strain 43Y-GA-1.
FIG. 5 shows the control effect of strain 43Y-GA-1 on banana vascular wilt potted plants.
FIG. 6 shows the bulb blackening phenomenon.
Detailed Description
The invention will be further described with reference to specific embodiments in order to provide a better understanding of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
1. Experimental materials
1.1 Test strain
Test pathogenic fungi: no. 4 physiological race of banana fusarium wiltFusarium oxysporum f. sp. cubense race 4 (Foc, ATCC 76255) for bacteriostatic activity assays; strawberry anthracnose pathogenColletotrichum fragariae(ATCC 58718) Castanea mollissima epidemic pathogenCryphonectria parasitica（ATCC 38755) Colletotrichum gloeosporioides (L.) kurzColletotrchum musae(ACCC 37612), alternaria mangiferum leaf spotAlternaria tenuissima(ATCC 58124), pyricularia pyrifoliaColletotrichum gloeosporioides(ATCC 16330), wheat scabFusahum graminearum(ATCC 11696), cucumber fusarium wiltFusarium oxysporum f. sp cucumerinum(ATCC 36332), tomato fusarium wiltFusarium oxysporum f. sp. lycopersici(ATCC 16322), banana Spot-rot pathogenCurvularia fallax(ATCC 34598), pyricularia oryzaePyricularia oryae(ATCC 52083), colletotrichum gloeosporioidesColletotrichum gloeosporioides(ATCC 58222), puccinia aphanidermatumLasiodiplodia theobromaeLeptosphaeria gracilis (wall.) KuntzeCurvularia lunata(ATCC 60935), no. 1 physiological race of banana vascular wiltFusarium oxysporum f. sp. cubense race 1 (Foc, ACCC 31271) is used for broad-spectrum antibacterial activity detection of antagonistic bacteria, and the above plant pathogenic fungi are preserved in the national academy of tropical biotechnology institute of the national academy of tropical agriculture.
Test antagonistic bacteria: 43Y-GA-1 strain is prepared from five-finger mountain medicinal plant, bamboo leaf and cyclobalanopsis glaucaCyclobalanopsis bambusaefolia) Is obtained by the leaf separation of the above-mentioned materials.
1.2 Test medium
The culture medium for identifying the physiological biochemistry, the culture characteristics and the like of actinomycetes is referred to actinomycetes phylogenetic-principle, method and practice. PDA culture medium is used for antagonism test, and soybean meal fermentation culture medium is used for fermentation culture. The specific components are as follows.
Table 1 formulation of the Medium
| Name of the name | Formulation of |
| Yeast malt extract cultureBase (ISP 2) Or YE) | Yeast extract 4 g, malt extract 10 g, glucose 4 g, distilled water 1000 mL, agar 20 g, ph 7.2. |
| Oatmeal agar medium (ISP 3) | Oatmeal 20 g (1000 mL added to water for 20 min, filtered and supplemented with 1000 mL), trace salt solution 1 mL, agar 20 g, pH 7.2. |
| Inorganic salt starch agar culture medium （ISP4） | Soluble starch 10 g, K 2 HPO 4 1 g，MgSO 4 ·7H 2 O 1 g，NaCl 1 g，（NH 4 ） 2 SO 4 2 g，CaCO 3 2 g, trace saline solution 1 mL, distilled water 1000 mL, agar 20 g，pH 7.0-7.4。 |
| Glycerol-asparagine culture medium （ISP5） | Asparagine 1 g, glycerol 10 g, K 2 HPO 4 1 g, a trace salt solution 1 mL, distilled water 1000 mL, agar 20 g, pH 7.0-7.4. |
| Peptone-yeast extract agar culture Base (ISP 6) | Peptone 15 g, peptone 5 g, ferric ammonium citrate 0.5 g, K 2 HPO 4 1 g, sodium thiosulfate 0.08 g, yeast extract 1 g, distilled water 1000 mL, agar 20 g, pH 7.0 7.4。 |
| Tyrosine agar medium (ISP 7) | Glycerol 15 g, tyrosine 0.5 g, asparagine 1 g, K 2 HPO 4 0.5 g，MgSO 4 ·7H 2 O 0.5 g，NaCl 0.5 g，FeSO 4 ·7H 2 O0.01 and g, a trace amount of salt solution 1 and mL, distilled water 1000 mL, agar 20 g, pH 7.2-7.4. |
| Potato glucose agar medium （PDA） | Potato 200 g, glucose 20 g, agar 17-20 g, distilled water 1000 ml, ph:7.0. |
| gaoshi No. 1 culture medium | Soluble starch 20.0 g,NaCl 0.5 g, KNO 3 1.0 g， K 2 HPO 4 •3H 2 O 0.5 g，MgSO 4 •7H 2 O 0.5 g，FeSO 4 •7H 2 O0.01-g, agar 15-20 g, distilled water 1000 mL，pH：7.4-7.6。 |
| Soybean powder fermentation medium | Soluble starch 20 g, soybean meal 15 g, yeast powder 5 g, peptone 2 g,CaCO3 4 g,NaCl 4 g, distilled water 1000 ml, ph:7.0 |
2 experimental methods and results
The identification of antagonistic strains is mainly carried out by methods of culture characteristic observation, physiological and biochemical identification, spore morphology observation and the like.
2.1.1 scanning Electron microscope observation
Sample: actinomycetes 43Y-GA-1 strain is inoculated to a YE culture medium for culturing, and the actinomycetes are reversely cultured for about 7-10 days under the condition of 28 ℃. Cube patches containing actinomycete colonies were excised.
Front fixing: square pellets containing actinomycete colonies were immersed in glutaraldehyde at a volume fraction of 2.5% and allowed to stand at 4 ℃ overnight.
Rinsing: the immobilized samples were rinsed with 0.1 mol/L Phosphate Buffered Saline (PBS) for 15 min each, and repeated 3 times.
Dehydrating: sequentially dehydrating the rinsed sample with ethanol with volume fraction of 30%,50%,70%,80%,90% and 95% for 15 min each time; eluting with 100% ethanol for 2 times each for 20 min.
Intermediate liquid replacement: by usingV(ethanol):Vthe mixed solution of (isoamyl acetate) =1:1 is treated for 30 min to perform preliminary displacement, and then the sample 2 h with the volume fraction of 100% isoamyl acetate is treated to complete displacement of the intermediate solution.
And (3) naturally airing the treated sample in an aseptic super clean bench, plating gold, and observing the fine structures of hyphae and spore surfaces of actinomycetes by a scanning electron microscope.
The morphology of hyphae and spores of strain 43Y-GA-1 on YE medium was observed by scanning electron microscopy, as shown in FIG. 1. The strain 43Y-GA-1 has developed hypha, compact spiral spore chain and wrinkles on the surface.
2.1.2 physiological Biochemical characterization
The physiological and biochemical characteristics of actinomycetes are identified by referring to the common bacteria System identification handbook and actinomycetes System science.
(1) Strain 43Y-GA-1 culture characteristics
Strain 43Y-GA-1 was inoculated into ISP2, ISP3, ISP4, ISP5, ISP6 and ISP7 medium, and after culturing at 28 ℃ 7-14 d, the growth of the strain was observed.
(1) Experiment for carbon and nitrogen source utilization
The strain to be tested is inoculated into a culture medium containing different carbon sources and nitrogen sources, and after the strain to be tested is cultured at 28 ℃ for 7-14 and d, the growth condition of the strain to be tested is observed, and a negative control is set.
(2) PH experiment
The pH values of the liquid culture medium are respectively adjusted to 4, 5, 6, 7, 8, 9 and 10, strains to be detected are respectively inoculated into the culture mediums with different pH values, other culture conditions are consistent, the culture is carried out under shaking at 28 ℃, the culture is observed once a week for four weeks, and finally the growth condition of the strains on each culture medium is determined. And determining the upper limit and the lower limit of the pH value at which the strain can grow and the optimal pH value.
(3) Sodium chloride experiment
Media with different NaCl concentrations (1%, 3%, 5%, 7%, 9%, 11%, 13%, 15%) were prepared, the other components of the media were the same, strains to be identified were inoculated on these media, cultured at 28℃and observed once a week for four weeks, and finally whether the strains were grown on the media was recorded to determine the upper and lower limit concentrations of the tolerance of the strains to be identified to NaCl.
(4) Determination of enzymatic Properties
(1) Esterase (tween 20, tween 40, tween 80) experiments:
after the lipase medium was formulated, the individually encapsulated tween 20, 40, 80 was sterilized together and the tween was mixed with the medium to plate. The strain was inoculated on a plate, cultured for one to two weeks, and the plate was observed, and if a faint halo around the colony was generated, it was positive, whereas the other was negative.
(2) Urease assay:
the strain was inoculated on a urease medium, and after culturing at 28℃for 4 d, the medium was observed for discoloration. The test strain was tested for its ability to produce urease, and the medium became pink positive and negative without discoloration.
(3) Nitrate reduction
The strain to be tested is inoculated in a nitrate reduction culture medium, and is cultured at 28 ℃ for 7 and 14 and d, and the culture medium without bacteria inoculation is used as a control. A small amount of culture solution of 7d and 14 d was added to the test tube, and one drop of solution A and B was added dropwise, respectively, and the same was added dropwise in the control. When the solution turns into pink, rose red, orange or brown, etc., the nitrate is reduced positively; if no red color appears, 1 or 2 drops of diphenylamine reagent are added dropwise, and if blue color appears, the reduction effect is negative; if the color is not blue, the treatment is still treated as positive.
(4) Gelatin experiments
The strain to be identified was inoculated into a tube containing gelatin medium, then cultured at 28℃and observed for gelatin liquefaction at 5, 10, 20, 30 d. If the strain has the liquefaction phenomenon, the strain is positive, which indicates that the strain has the ability of liquefying gelatin, otherwise, the strain is negative.
The results showed that strain 43Y-GA-1 grew best on ISP2, ISP3 and ISP4 media, forming abundant endophytes, but grew poorly on ISP5 (Table 2). The strain has protease activity, can liquefy gelatin, and is positive for nitrate reduction, esterase test and urease test (figure 1); most of carbon sources can be well utilized, and the growth on the carbon sources such as sucrose, D-ribose, D-fructose, glucose and the like is good; nitrogen sources such as arginine, histidine and the like can be better utilized; can grow in the pH range of 4.0-8.0, and the optimal growth pH value is 7.0; the optimum growth salt concentration was less than or equal to 5% (table 3).
According to the morphological, culture and physiological-biochemical characteristics, the strain 43Y-GA-1 presents typical streptomycete morphological characteristics, namely streptomycete gray, and is named asStreptomyces fimicariusThe registration and preservation are carried out in the Guangdong province microorganism strain collection center, and the preservation number is GDMCC No:63831 the date of deposit is 2023, 09, 22 days, and the address of deposit is at the university of Mitsui, guangzhou, md.100, building 59, academy of microorganisms, guangdong, national institute of sciences.
TABLE 2 characterization of the culture of strain 43Y-GA-1 on 6 media
| Culture medium | Aerial hypha | Spore yarn | Intra-basal hypha | Soluble pigments | Growth status |
| ISP2 | White color | White color | Yellowish light yellow | Without any means for | Preferably, it is |
| ISP3 | Gray color | Gray color | Dark brown | Without any means for | Preferably, it is |
| ISP4 | White color | White color | Brown color | Without any means for | Preferably, it is |
| ISP5 | White color | White color | White color | Without any means for | Weaker and weaker |
| ISP6 | Apricot color | Off-white color | Pale brown | Without any means for | Moderate to moderate |
| ISP7 | Off-white color | White color | Pale brown | Without any means for | Moderate to moderate |
TABLE 3 physiological and biochemical characteristics of Strain 43Y-GA-1
Note that: in the results of carbon source and Nitrogen source' ++ + + + is shown the growth is good, the quality of the product is good, "++" indicates growth in general, "+" indicates growth possible, and "-" indicates no growth. In the physiological and biochemical test results, "+" indicates positive and "-" indicates negative.
2.2 Determination of bacteriostatic Activity
The antagonistic activity of the 43Y-GA-1 strain is measured by adopting a flat plate counter culture method by taking banana fusarium wilt as a target bacterium. A punch with the diameter of 5 mm is used for taking a bacterial cake of Foc bacteria with consistent growth vigor, the bacterial cake is connected to the middle of each PDA flat plate, 4 purified actinomycetes are respectively connected at the position 2.5 cm away from pathogenic bacteria colony, the bacterial cake is placed in a 28 ℃ incubator for inverted culture of 7d, and only Foc4 bacteria are inoculated as a control, and each treatment is repeated three times. The diameter of the colony of pathogenic fungi was measured by the crisscross method, and the inhibition rate of the growth of hypha was calculated according to the following formula.
Preparation of Strain 43Y-GA-1 fermentation extract: strain 43Y-GA-1 was inoculated into a triangular flask (250 mL) containing 100 mL of SLM liquid medium and cultured on a constant temperature shaker at 28℃and 200 r/min for 7d. Adding pure ethanol at a ratio of 1:1 (v: v), mixing, extracting to obtain active crude extract, filtering mycelium with filter paper, and rotary evaporating to obtain extract.
The antibacterial activity of the extract on Foc was measured by a filter paper sheet method. Soaking the filter paper sheet completely in the extract, taking out, and air drying. At a distance of 2.5. 2.5 cm from the cake of the test pathogenic bacteria, 4 filter paper sheets containing the fermentation liquid were attached, respectively, to inoculate only the test pathogenic bacteria as a control, and the inhibition rate of the hypha growth was calculated.
The antibacterial activity of the 43Y-GA-1 strain against Foc bacteria was measured by plate-counter culture. The test results showed 66.58% bacteriostatic activity against Foc pathogens (FIG. 2). The antibacterial activity of the 43Y-GA-1 strain fermentation extract on Foc bacteria is measured by adopting a filter paper diffusion method, the antibacterial effect on Foc bacteria is good, and the antibacterial activity reaches 72.57 percent (figure 3).
2.3 broad-spectrum antibacterial Activity detection
The broad-spectrum antibacterial activity of the strain 43Y-GA-1 is measured by adopting a plate counter culture method. The method for determining the antibacterial activity is the same as that of the method for determining the antibacterial activity of 2.2, and the antibacterial activity of the antibacterial activity is calculated.
The results are shown in Table 3. The antibacterial activity of the strain 43Y-GA-1 on 15 plant fungus pathogens is detected, and the bacterial inhibition effects on 15 test fungi of 9 species such as anthracnose (strawberry anthracnose, oil pear anthracnose, banana anthracnose and colletotrichum), cryptosporidium (chestnut blight), fusarium (banana fusarium wilt 1 physiological micro-seed, banana fusarium wilt 4 physiological micro-seed, tomato fusarium wilt and cucumber fusarium wilt), alternaria (mango alternaria leaf spot), fusarium (wheat red fungus), pseudocurvularia (banana long-shaped spot), pyriform (rice blast), crescent moon (banana large gray spot) and cocoa ball (oil pear pedicel rot) are all found, the antibacterial rates are all more than 57%, and especially the antibacterial rates of the 9 species such as strawberry anthracnose, chestnut blight, banana fusarium, mango alternaria leaf spot, oil pear and wheat red fungus are all more than 70%, and are respectively up to 70%, 67.67%, 35.74.77%, 35.74.72%, 35.72%. As can be seen, strain 43Y-GA-1 has broad-spectrum antibacterial activity.
TABLE 6 broad-spectrum antibacterial Activity assay
| Pathogenic bacteria | Antibacterial rate | Pathogenic bacteria | Antibacterial rate |
| Strawberry anthracnose pathogen | 84.77% | Tomato fusarium wilt | 66.08% |
| Chestnut epidemic disease germ | 76.67% | Banana alternaria leaf spot | 65.88% |
| Bacillus anthracis (Roxb.) kurz | 75.10% | Rice blast fungus | 65.63% |
| Alternaria mangiferum leaf spot | 74.67% | Colletotrichum gloeosporioides | 62.94% |
| Alternaria alternata (L.) Gaertn | 73.06% | Alternaria alternata (Thunb.) Boehringer | 61.37% |
| Wheat scab fungus | 72.94% | Leptosphaeria gracilis (Roxb.) Kuntze | 60.09% |
| Cucumber fusarium wilt | 68.04% | No. 1 physiological race of banana fusarium wilt | 57.52% |
| No. 4 physiological race of banana fusarium wilt | 66.58% |
2.4 Effect of extracts on Foc mycelium morphology
A punch was used to insert 0.5. 0.5 cm of the cake into the exact center of the plate containing 500ppm final extract at the edge of the Foc colony, and after incubation at 28℃for 5 d, square patches containing the edge of the colony were excised and untreated plates were used as controls. The sample processing method is the same as the method of 2.1.1 scanning electron microscope observation. A scanning electron microscope (SEM, model S-4800,Hitachi Limited,Japan) was used to observe Foc changes in the hyphae of the germ.
The effect of the extract on the mycelium surface of banana fusarium wilt at a concentration of 500ppm is shown in figure 4. The untreated mycelia of the control group Foc4 were observed to be normal in morphology, linear in structure and smooth in cell wall by a scanning electron microscope. The mycelium of Foc bacteria after the extract treatment is shriveled, and the like.
2.5 Prevention and treatment effect of bacterial strain 43Y-GA-1 on banana vascular wilt
Preparation of pathogenic bacteria spore suspension: freshly cultured Foc mycelia were picked and inoculated onto PDA solid medium and incubated at 28℃for 7d. Scraping spores with sterile distilled water, filtering with sterile filter paper, collecting pathogenic bacteria spore suspension, counting with a blood cell counting plate, and adjusting concentration to 6.0X10 with sterile water 5 cfu/mL for use.
Preparing bacterial strain 43Y-GA-1 bacterial liquid: streptomyces 43Y-GA-1 strain is inoculated in SLM culture solution and cultured for 7d at 28 ℃ for later use.
Selecting Brazilian banana seedlings with 3-4 leaf periods and consistent sizes to carry out a potting prevention effect test. The experiment was performed in 6 treatments, T1: only clear water was applied (control group); t2: applying a pathogen Foc; t3: after the bacterial strain 43Y-GA-1 was applied, pathogenic bacteria were applied. 10 plants each were treated.
After inoculation of pathogenic bacteria, the yellowing and withering of the stems and leaves on the overground part are observed, and the roots are cut to record the browning degree. The disease index grading standard of banana vascular wilt is grade 0: healthy plants; 1. stage: yellowing of the lower leaves; 3. stage:
20-40% of the yellow leaves; 4. stage: 40% -60% of the yellow disease leaves; 5. stage: 60% -80% of the yellow disease leaves; 6. stage: all plants were etiolated leaves and the plants died. And calculating the disease index and the prevention and control effects of banana vascular wilt.
The control effect of the 43Y-GA-1 strain on banana pot Miao Xiangjiao blight is shown in figures 5-6. The incidence rate of the T2 treatment group is 73.85%, the incidence rate is serious, and the bulbs are obviously blackened and the like (figure 6); the morbidity of the T3 treatment group is 28.36%, which shows that the 43Y-GA-1 strain has better control effect on banana vascular wilt and can inhibit infection of banana vascular wilt.
The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for this practical use will also occur to those skilled in the art, and are within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.
Claims (6)
1. Streptomyces species @Streptomyces fimicarius) The method is characterized in that the method is registered and preserved in the Guangdong province microorganism strain collection center, and the preservation number is GDMCC No:63831.
2. an ethanol extract of the fermentation broth of Streptomyces according to claim 1.
3. A formulation comprising the streptomyces sp according to claim 1 or the ethanol extract according to claim 2.
4. Use of a streptomyces according to claim 1, or an ethanol extract according to claim 2, or a formulation according to claim 3, for antagonizing strawberry anthracnose, and/or chestnut blight, and/or banana anthracnose, and/or alternaria mangiferum, and/or pear anthracnose, and/or wheat scab, and/or cucumber fusarium wilt, and/or tomato fusarium wilt, and/or banana blotch, and/or rice blast, and/or colletotrichum glomeratum, and/or pyritinospora, and/or banana gray spot, and/or banana fusarium wilt No. 1 physiological race, and/or banana fusarium wilt No. 4 physiological race.
5. Use of the streptomyces according to claim 1, or the ethanol extract according to claim 2, or the formulation according to claim 3, for controlling diseases caused by strawberry anthracnose, and/or chestnut blight, and/or banana anthracnose, and/or alternaria mangiferum, and/or pear anthracnose, and/or wheat scab, and/or cucumber fusarium wilt, and/or tomato fusarium wilt, and/or banana blotch, and/or rice blast, and/or colletotrichum gloeosporioides, and/or pyritic calix, and/or banana large gray spot, and/or banana fusarium wilt No. 1 physiological race, and/or banana fusarium wilt No. 4 physiological race.
6. Use of the ethanol extract of claim 2 for the crimping and collapsing of mycelia of Foc pathogens.
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| CN117264851A (en) | 2023-12-22 |
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