CN116656509A - Paenispira rosea strain, microbial preparation and application thereof - Google Patents
Paenispira rosea strain, microbial preparation and application thereof Download PDFInfo
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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
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal 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
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
-
- 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
-
- 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
- A01P5/00—Nematocides
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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
- C12N1/14—Fungi; Culture media therefor
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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
- C12R2001/645—Fungi ; Processes using fungi
-
- 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
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Pest Control & Pesticides (AREA)
- Virology (AREA)
- Plant Pathology (AREA)
- Environmental Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Botany (AREA)
- General Chemical & Material Sciences (AREA)
- Agronomy & Crop Science (AREA)
- Dentistry (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention provides a Paenibacillus roseus strain with a preservation registration number of CGMCC No.18153. The invention also provides a microbial agent obtained by using the Paenibacillus roseus strain, and the microbial agent is in the form of liquid, powder or granule. The microbial inoculum is prepared by the following method: fermenting the Paenibacillus roseus strain with liquid to obtain liquid, or adding carrier and adjuvant, granulating or pulverizing. The strain SYP-1-1 of the invention has strong parasitic capability and antagonism, and can effectively prevent and treat various plant soil-borne diseases such as crop sclerotinia, fusarium wilt, root rot, verticillium wilt, root knot nematode and the like.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a Paenibacillus roseus strain, a microbial inoculum containing the strain and application of the strain.
Background
Plant soil-borne diseases are important plant diseases, pathogens can survive in soil for a long time, germinate when the environment is proper, plant roots are infected to cause plant morbidity and even death, and the soil-borne diseases cause great economic loss to agricultural production. For a long time, due to ecological unbalance of soil and reduction of the types and the quantity of beneficial microorganisms in the soil caused by a large amount of pesticide and fertilizer application, soil-borne diseases such as fusarium wilt, rhizoctonia rot, root rot, sclerotinia sclerotiorum, epidemic diseases, root knot nematode and the like are frequently and seriously aggravated year by year, the crop yield and the quality are seriously affected, and the sustainable development of agricultural production is restricted.
Soil-borne diseases are of a wide variety, with fungal and nematode diseases being the most serious two types of diseases. Soil-borne diseases have the characteristics of concealment, outbreak, destruction and strong infectivity, and are extremely difficult to radically cure. The control methods in the current production, such as agriculture control, physical control, chemical control, disease-resistant varieties and the like, have a certain limit. Biological control has the advantages of no toxicity, no residue, environmental friendliness, long disease-preventing lasting period and the like, and has great potential in the control of plant soil-borne diseases in recent years. The Pachyrhizus roseus (Clonostachys rosea) belongs to the phylum of Deuteromycotina, class Cephalosporium, order Condiophora and family Costules, is an important bacterial parasitic fungus, and can parasitize various plant pathogenic fungi and plant nematodes such as Rhizoctonia solani, fusarium oxysporum, sclerotinia sclerotiorum, botrytis cinerea, verticillium dahliae and the like, and the Pachyrhizus roseus has good biocontrol effect on various plant diseases in greenhouse and field experiments. The efficient biocontrol strain of the Paenidromyces roseus is excavated, and the method has important significance for realizing green prevention and control of soil-borne diseases, realizing weight losing and drug reducing and guaranteeing safe production of agricultural products.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides a Paenibacillus roseus strain and a preparation thereof.
The second purpose of the invention is to provide application of the strain and the microbial inoculum of the Paenibacillus roseus.
The technical scheme for realizing the purposes of the invention is as follows:
the strain of the Paenibacillus roseus has a preservation registration number of CGMCC No.18153 and a preservation date of 2019, 8 and 9. The strain of Paenibacillus roseus is designated SYP-1-1. The microbial strain is preserved in the common microorganism center of China Committee for microbial strain management, and the address is North Chenxi Lu 1 in the Beijing Chaoyang area of China.
The microbial agent is prepared from the Paenibacillus roseus strain, and the microbial agent is in the form of liquid, powder or granule.
The microbial agent is characterized by being prepared by the following method:
1) Spore suspension preparation: inoculating the strain of the Paenibacillus roseus on a PDA culture medium, culturing for 6-8 days at 25-28 ℃, adding sterile water to elute spores, and preparing spore suspension;
2) Seed culture: adding a seed culture medium into a triangular flask, sterilizing at high temperature and moist heat, inoculating spore suspension into the triangular flask when cooling to room temperature, and placing the triangular flask on a shaking table for shaking and dark culture at 25-28 ℃ for 48-84 hours to obtain seed liquid;
3) Liquid fermentation culture: preparing a fermentation medium, sterilizing, cooling, inoculating seed liquid, and culturing at 25-28 ℃ for 48-96 hours to obtain fermentation liquor.
Wherein in the step 2), the seed culture medium is potato dextrose(PD) medium or complex medium comprising the following components in a volume of 1000 ml: 15-30 g glucose, 5-15 g yeast extract powder and K 2 HPO 4 0.5-2 g of MgSO 4 ·7H 2 O0.5-1.0 g, water to 1000ml, and HCl solution of 1M to adjust pH to 5.5-6.5. The compound culture medium is more suitable for the growth and spore production of the strain than the PD culture medium based on the nutrition requirement of the Paenispira rosea.
Wherein, in the step 3), the culture medium components of the liquid fermentation culture according to the volume of 1000 milliliters comprise: 10-25 g of glucose, 10-25 g of corn meal, 5-15 g of yeast extract powder, 5-15 g of bean cake powder and K 2 HPO 4 0.5-2 g of MgSO 4 0.2-1 g, adding water to 1000ml, adjusting pH to 5.5-6.5, sterilizing to obtain the fermentation medium.
The pH can be adjusted by using 0.1-2M HCl solution or other common organic acid or inorganic acid solution.
Wherein, the liquid fermentation culture conditions of the step 3) are as follows: the inoculation amount is 0.5-5%, the rotating speed is 150-220 r/min, the temperature is 25-28 ℃, and the culture time is 48-96 hours.
Further, fermenting the Paenibacillus roseus strain by liquid to obtain a fermentation liquor, and adding or not adding a stabilizer to obtain a liquid; or directly adding a carrier into the fermentation broth according to the mass ratio of the fermentation broth to the carrier of 1-3:1, or adding the carrier after centrifugally concentrating the fermentation broth, or adding a combination of the carrier and an auxiliary agent, wherein the addition amount of the carrier is 10-30 percent (mass percent) of the fermentation broth, mixing, granulating, drying at 35-45 ℃ or crushing to obtain powder; the carrier is one or more of diatomite, light calcium carbonate, talcum powder, turf, rice hull powder and straw powder.
Because diatomite has good adsorptivity and strong water absorption, rice hull powder is easy to disperse after being adsorbed, the filler in the step 4) is preferably a combination of diatomite and rice hull powder, and the weight ratio of the diatomite to the rice hull powder is 1-3:1.
Preferably, the microbial agent has a bacterial content of (1-10) x 10 8 CFU/g。
The application of the Paenibacillus roseus strain in preventing and treating crop nematode disease, sclerotinia sclerotiorum, fusarium wilt, root rot and verticillium wilt.
The microbial agent is applied to prevention and treatment of crop nematode disease, sclerotinia rot, fusarium wilt, root rot and verticillium wilt.
The invention has the beneficial effects that:
the strain SYP-1-1 provided by the invention can parasitic sclerotinia sclerotiorum, fusarium, botrytis cinerea, root knot nematode and other plant pathogenic fungi and pathogenic nematodes, can generate metabolites with antibacterial and nematicidal activities, antagonize pathogenic fungi and kill plant nematodes, and simultaneously, the strain SYP-1-1 can also generate a large amount of cell wall hydrolases such as beta-1, 3-glucanase, chitinase and the like to digest the cell walls of the pathogenic fungi and the wall of the nematodes, so that plant fungi and nematode diseases are prevented and controlled.
Experiments research the control effect of the strain of the Paenibacillus roseus SYP-1-1 on various plant fungi and nematode diseases. The result of the prevention and treatment effect on the soybean sclerotinia shows that the soybean plants treated by the white-blood-spot polyporus pinsitus are healthy and have little disease spots. The result of the control effect determination of SYP-1-1 microbial inoculum on cucumber fusarium wilt shows that when the dosage of the microbial inoculum is 1 multiplied by 10 8 When CFU/plant is used, the control effect on wilt reaches 70.8%, which is equivalent to 30% carbendazim wettable powder. In addition, the SYP-1-1 fermentation liquid has better control effect on cucumber root knot nematode disease, and the control effect reaches 53.5%.
Drawings
FIG. 1 shows the colony morphology of the strain of S.roseus SYP-1-1 on PDA medium.
FIG. 2 is an ITS rDNA sequence homology evolutionary tree of the strain of the Saprolegnia rosea SYP-1-1.
FIG. 3 shows sclerotinia sclerotiorum of the strain Pyveromyces roseus SYP-1-1.
FIG. 4 is an electron micrograph of the mycelium of Fusarium oxysporum (S.roseum) of the strain SyP-1-1.
FIG. 5 shows the results of the measurement of the antagonistic capacity of the strain of Paenibacillus roseus SYP-1-1 against Fusarium oxysporum.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
Unless otherwise indicated, all approaches used in this specification are known in the art.
Example 1
The Paenispira rosea strain is isolated from suburban corn fields in Shanxi province and is preserved in China general microbiological culture Collection center (CGMCC) No.18153, and the preservation date is 2019, 8, 9. The strain of Paenibacillus roseus is designated SYP-1-1. The strain of the Paenibacillus roseus was inoculated into a PDA medium and cultured at 26℃for 7 days, and the colony morphology is shown in FIG. 1. The left panel shows the colony back morphology of SYP-1-1 strain, and the right panel shows the colony front morphology of SYP-1-1 strain.
Molecular identification of Paenidromyces roseus SYP-1-1 strain
DNA sequencing is carried out by utilizing fungus ITS sequences, and the sequence obtained by sequencing is compared with ITS sequences of known fungi, so that molecular identification is carried out on the strain SYP-1-1 of the Paenispira rosea. Primer sequence ITS1:5'-TCCGTAGGTGAACCTGCGG-3', ITS4:5'-TCCTCCGCTTATTGATATGC-3', PCR amplification system: DNA (80 ng/. Mu.L) template 2. Mu.L, ITS1 (10. Mu.M) 1. Mu.L, ITS4 (10. Mu.M) 1. Mu.L, 1.1X1.3 Super PCR Mix enzyme 21. Mu.L. Amplification procedure: pre-denaturation at 98℃for 3min, denaturation at 98℃for 10s, annealing at 55℃for 10s, extension at 72℃for 10s,35 cycles; extending at 72℃for 10min. The PCR products were electrophoretically detected and sequenced. Sequencing results are shown in a sequence table.
By comparing the highest homology of SYP-1-1 strain and the Saprolegnia rosea, the homology reaches 100%, and the homology comparison evolutionary tree is shown in figure 2.
Example 2
Test of sclerotium of Sclerotinia sclerotiorum of Paenichips polyporus roseus SYP-1-1 strain
The Saponaria pinnatifida with the preservation number of CGMCC No.18153 is inoculated into a PDA culture medium and cultured for 7 days at 26 ℃. Adding 5mL of sterile water, aseptically scraping spores to obtain spore suspension, and adjusting concentration to 1×10 7 spores/mL. Taking a proper amount of sclerotium, sterilizing with 1% NaClO surface for 3min, and washing with sterile waterAnd (3) placing the mixture on sterilized filter paper for drying. Soaking equal amount of sclerotium in spore suspension for 10min, taking out sclerotium, sucking excessive water with sterilized filter paper, and placing in culture dish (9 cm) with wet filter paper for 25 sclerotium per dish. The bacterial nuclei were cultured in a wet state at 26℃for 10 days, and the bacterial nuclei were observed to be parasitic. Sterile water-soaked sclerotium was set as a blank control, with 3 replicates per treatment. The parasitic capacities were classified into 5 grades according to the growth of the strain and the rotting degree of the sclerotium, as shown in Table 1.
TABLE 1 division of sclerotium class of Leptosphaeria roseosporus parasiticus
The measurement result shows that the strain SYP-1-1 of the Paenibacillus roseus has strong parasitism on sclerotinia sclerotiorum, and the parasitism rate reaches more than 90% in 24 hours. After the moisturizing culture for 7 days, the surface of the sclerotium is covered with parasitic fungus hypha, and the parasitic level reaches level 4 (figure 3A); dissecting the sclerotium found that the internal tissue of healthy sclerotium appeared white and hard in texture, while the infected sclerotium turned black brown internally and soft rot (fig. 3B); SYP-1-1 strain is marked by Green Fluorescent Protein (GFP), and the successful infection of the sclerotium by the Polysporotrichum roseum strain can be clearly seen under a scanning electron microscope, and a large amount of spore is grown and produced in the sclerotium (figure 3C).
Example 3
Test of Fusarium roseum SYP-1-1 Strain
A sterilized glass slide is placed in the middle of a PDA flat plate, one side is inoculated with a fresh bacterial cake of a strain SYP-1-1 of the Paenibacillus roseus, after 2 days of culture at 25 ℃, the other side is inoculated with a fresh bacterial cake of Fusarium oxysporum by adopting the same method, and when two hyphae grow together on the glass slide, the glass slide is taken down and placed under a scanning electron microscope to observe the interaction condition of the two. The results show that the S.roseus SYP-1-1 hyphae are attached to fusarium oxysporum hyphae, and special infection structures such as attachment cells and infection nails are generated, invade host cells (figure 4), absorb a large amount of nutrition, and finally cause digestion and death of pathogenic bacteria.
Example 4
Antagonistic capability determination of Paenidromyces roseus SYP-1-1 strain on Fusarium oxysporum
Inoculating and activating the strain SYP-1-1 of the Paenispira rosea in a PDA plate, inoculating a bacterial cake with uniform size to a position 2cm away from the edge of the PDA plate (with the diameter of 9 cm) after fresh hyphae grow out, culturing at 26 ℃ for 5 days, inoculating a bacterial cake of Fusarium oxysporum at the same distance at the opposite end of the plate, culturing at 26 ℃ for 20 days, and measuring the expansion distance of SYP-1-1 to pathogenic bacteria. Each experiment was repeated 3 times. The results show that the strain SYP-1-1 of the Paenispira rosea has strong antagonism and can effectively inhibit the growth and the expansion of fusarium oxysporum hyphae (figure 5).
Example 5
Chitinase Activity assay of Paenibacillus roseus SYP-1-1 Strain
SYP-1-1 strain spore suspension is inoculated into a 250mL triangular flask filled with 60mL sclerotium powder culture medium according to the inoculation amount of 4%, shake culture is carried out for 3 days at 28 ℃ and 180r/min, the fermentation liquor is centrifuged at 10000r/min for 15min at 4 ℃, and the supernatant is taken as crude enzyme liquid.
Drawing an N-acetylglucosamine standard curve: taking 8 test tubes, adding 0, 50, 100, 150, 200, 250, 300 and 350 mu L of N-acetylglucosamine standard solution into each test tube, adding distilled water to 0.5mL, adding 0.5mL of DNS solution, uniformly mixing, carrying out water bath at 100 ℃ for 10min, rapidly using flowing cold water to room temperature, adding distilled water to 5mL, measuring absorbance at 540nm, measuring 3 times of each concentration, and drawing a standard curve.
Enzymatic reaction of crude enzyme solution of SYP-1-1 Strain: adding 1% colloidal chitin and crude enzyme solution into 5mL test tube, accurately reacting at 40deg.C for 30min, centrifuging at 10000r/min for 15min, collecting supernatant, adding 0.5mL DNS solution, water-bathing at 100deg.C for 10min, flowing cold water to room temperature, adding distilled water to constant volume of 5mL, mixing, and determining OD 540 . The N-acetylglucosamine content and the enzyme activity in the crude enzyme solution were calculated according to a standard curve.
The results show that the SYP-1-1 strain has stronger chitinase activity of 6.56U. Chitin is an important component of plant pathogenic fungi cell walls and nematode walls, and SYP-1-1 strain can counteract pathogenic fungi cell walls and nematode walls by producing a large amount of chitinase, so that plant fungi and nematode diseases are prevented and controlled.
Example 6
Determination of the beta-1, 3-glucanase Activity of the Syp-1-1 Strain of Paenibacillus roseus
Sclerotium powder medium: taking sclerotium of sclerotium cultured in laboratory, pulverizing with pulverizer, sieving with 25 mesh sieve, and preparing sclerotium powder culture medium containing 1% sclerotium powder, 0.3% NaCl, 0.3% K 2 HPO 4 And 0.3% MgSO 4 。
Glucose solution: accurately weighing 100mg of glucose, dissolving in 100mL of water, and refrigerating at 4 ℃ for later use.
N-acetylglucosamine solution: accurately weighing 100mg of N-acetylglucosamine, dissolving in 100mL of water, and refrigerating at 4 ℃ for later use.
DNS solution: 192g of potassium sodium tartrate was weighed and dissolved in 500mL of distilled water heated to 70℃and 6.3g of DNS (3, 5-dinitrosalicylic acid), 21g of NaOH and anhydrous Na were added in sequence 2 SO 4 5g, stirring until dissolved, cooling, fixing the volume to 1000mL with distilled water, storing in a brown bottle, and storing in a dark place.
Beta-1, 3-glucan enzymatic reaction substrate: 100mg laminarin was dissolved in 100mL of 0.2mol/L pH 5 acetic acid-sodium acetate buffer.
SYP-1-1 strain spore suspension was inoculated in an amount of 4% into a 250mL Erlenmeyer flask containing 60mL of sclerotium powder medium, and cultured at 28℃under shaking at 180r/min for 3 days. Centrifuging the fermentation broth at 4deg.C and 10000r/min for 15min, and collecting supernatant as crude enzyme solution. Glucose standard curves were plotted as follows: taking 10 test tubes, respectively adding 0 mu L,50 mu L,100 mu L,200 mu L,400 mu L,600 mu L,800 mu L,1000 mu L,1200 mu L and 1400 mu L of glucose standard solution into each test tube, adding distilled water to 2.0mL, adding 2.0mL of DNS solution, uniformly mixing, carrying out water bath at 100 ℃ for 10min, rapidly using flowing cold water to room temperature, adding distilled water to a constant volume of 15mL, measuring absorbance at 540nm, measuring 3 parallel concentrations, taking glucose concentration as an abscissa, taking absorbance value as an ordinate, and drawing a standard curve.
Adding dextran substrate and crude enzyme solution into 15mL test tube, respectively 1mL, accurately reacting at 40deg.C for 30min, adding 2.0mL DNS solution, water-bathing at 100deg.C for 10min, flowing cold water to room temperature, adding distilled water to volume of 15mL, mixing, and measuring OD 540 . And calculating the glucose content and the enzyme activity in the crude enzyme liquid according to a standard curve. The amount of glucose produced at 1. Mu.g per minute per mL of enzyme solution was defined as one enzyme activity unit.
The measurement result shows that the beta-1, 3-glucanase activity of the S.roseus SYP-1-1 strain is higher and is 69.89U. Dextran is an important component of plant pathogenic fungi cell walls, and SYP-1-1 strain can degrade pathogenic fungi cell walls by producing a large amount of beta-1, 3-glucanase and other cell wall hydrolases, inhibit the growth and the expansion of pathogenic fungi and prevent and treat fungal diseases.
Example 7
Liquid fermentation of the strain Paenibacillus roseus SYP-1-1
Inoculating the Saponaria pinnatifida with the preservation number of CGMCC No.18153 into a PDA culture medium from a preservation inclined plane, culturing for 7 days at 28 ℃, adding sterile water, eluting spores, and preparing spore suspension; adding a seed culture medium into a triangular flask, sterilizing under high pressure and moist heat, inoculating the spore suspension into the seed culture medium when cooling to room temperature, and performing shake culture at 28 ℃ for 48 hours at 180r/min to prepare seed liquid. Then inoculating the strain into a fermentation medium with an inoculum size of 2%, and carrying out shaking culture at 28 ℃ and 180r/min for 72 hours to obtain the Paenispira rosea fermentation broth.
The formula of the seed culture medium is as follows: 1000ml of water contains 20 g of glucose, 6 g of yeast extract powder and K 2 HPO 4 1g of MgSO 4 ·7H 2 0.5 g of O;
the culture medium components (in 1000ml volume) of the liquid fermentation culture include: 15 g of glucose, 15 g of corn meal, 5g of yeast extract powder, 5g of bean cake powder and K 2 HPO 4 1g of MgSO 4 0.5 g;
the pH of the 2 culture mediums is regulated to 6.0 by 1M hydrochloric acid, and the seed culture medium and the fermentation culture medium are obtained after sterilization.
Example 8
Preparation of Paenispira rosea SYP-1-1 microbial inoculum
The same mass of carrier which is diatomite and rice hull powder (1:1, mass ratio) is added into the fermentation broth of the Paenimolding rosea obtained in the example 7, and the mixture is adsorbed, dried at 35 ℃ and crushed to prepare SYP-1-1 powder.
Weighing 1g of microbial inoculum, adding sterile water, diluting in a column, coating on PDA culture medium, and measuring bacterial content to 2.1X10% 8 CFU/g。
Example 9
Control effect of spore suspension of Paenibacillus roseus SYP-1-1 strain and other strains on soybean sclerotinia
Inoculating different strains of Saprolegnia rosea from the preservation inclined plane into PDA culture medium, culturing at 26deg.C for 2 weeks, adding sterile water to elute spores to obtain 1×10 strain 7 spore/mL spore suspension. Sterile soybean seeds (medium yellow No. 13) were sown into pots of 11cm diameter, 6 grains per pot. After emergence of the seeds, each pot was reduced to 3 plants. When soybean seedlings were grown to 9 compound leaves, experimental treatment was started. The spore suspension is sprayed on the front and back surfaces of each compound leaf uniformly, an equal amount of soybean sclerotinia fermentation liquor is inoculated after 2 hours, and experimental plants are placed in a greenhouse for moisture preservation and culture (the temperature is controlled to be 26-28 ℃, the relative humidity is 60%, and all flowerpots are randomly placed). After 7 days of inoculation, the disease condition of soybean leaves is investigated, and the disease index and the prevention and treatment effect of each treatment are counted. According to the disease conditions, the disease severity is divided into four grades, and the disease grading standards are as follows: 0 = no lesions on soybean leaves; 1 = spot area less than 10%; 2=the area of the lesion is 10-30%; 3 = 30-50% of the area of the lesion; 4 = lesion area greater than 50%. Each treatment was repeated 3 times, 12 pot plants each, to inoculate only sclerotinia sojae and clear water as positive and negative controls, respectively.
The results showed that 7 days after inoculation of sclerotinia sojae, a large number of severe lesions appeared on the soybean leaves of the clear water control group, while soybean plants treated with the S.rosea were healthy and less lesions appeared, wherein the treatment with SYP-1-1 strain spore suspension had the highest control effect, the control effect reached 76.3%, the control effect of CBS227.8 strain (purchased from the Netherlands general microbiological strain collection center) was 52.0%, the control effect of 3.3655 strain (purchased from the China general microbiological strain collection center) was 50.6%, and the control effect of SYP-1-1 strain was significantly different from that of other 2 strains of S.rosea (P < 0.05).
Example 10
Determination of control effect of Paenibacillus roseus SYP-1-1 microbial inoculum on cucumber fusarium wilt
Selecting cucumber seeds with full seeds and uniform size, baking seeds in an oven at 68 ℃ for 5 hours, soaking in 1% NaClO, sterilizing for 3min, repeatedly washing with sterile water, airing on sterile filter paper, and sowing. After 14 days, the seedlings were transplanted to the seedlings inoculated with Fusarium oxysporum (concentration 10) 6 CFU/g soil) was used in a flowerpot (diameter: 10 cm) and the trichostrongylus rosea mold prepared in example 8 was applied simultaneously, 3 plants per pot, and the incidence and index of bacterial wilt were investigated after 28 days. Disease conditions were graded on a 5-grade scale, grade 0: healthy disease-free plants, grade 1: the disease is slight, and less than 1/4 of leaves of the disease plant show wilting; 2 stages: the disease is serious, and 1/4 to 1/2 of the leaves of the disease plant show wilting; 3 stages: the disease is serious, and more than 1/2 of leaves of the disease plant appear wilting; 4 stages: the disease strain dies or nearly dies. The treatment with no microbial inoculum and the common chemical pesticide carbendazim are used as controls, and each treatment is carried out in 4 basins and 3 times. Plant disease index = [ Σ (number of plants at each stage x number of corresponding stages)/survey total number of plants x highest number of stages]X 100; disease preventing effect (%) = (control disease index-treatment disease index)/control disease index x 100%.
The result shows that the S.roseus SYP-1-1 microbial inoculum has good control effect on cucumber fusarium wilt, and the control effect is improved along with the increase of the concentration of the microbial inoculum. Cucumber leaf wilting and yellowing of control group, and when the microbial inoculum application concentration is 1.0X10 7 When CFU/plant is used, the disease spots on the leaves are obviously reduced, the disease symptoms are obviously lightened, and the prevention effect is 50.0%; when the dosage of the microbial inoculum is 1.0X10 8 When CFU/plant is used, the control effect on cucumber fusarium wilt reaches 70.8%, and the effect is equivalent to that of 30% carbendazim wettable powder.
Table 2 effect of Phosphaeria rosea SYP-1-1 microbial inoculum on controlling cucumber fusarium wilt
Example 11
Determination of control effect of fermentation liquor of Paenibacillus roseus SYP-1-1 strain on root-knot nematode on cucumber
Soaking cucumber seeds in 2% NaClO solution for 5min, washing with tap water for several times, placing in a culture dish, adding a little sterile water, and accelerating germination at 28deg.C. The soil adopted in the test is farmland soil with serious disease of the root-knot nematodes of the gallery test base, the density of the nematodes is measured to be 53 per 100g of soil, the nematode soil is subpackaged into flowerpots with the diameter of 12cm, and the soil loading amount of each pot is 600g. The seeds with germination acceleration are directly planted in diseased soil, 2 seedlings are reserved in each pot after emergence, 6 pots are treated each, and 3 times are repeated. After 2-3 true leaves grow out of the cucumber, 50mL of SYP-1-1 fermentation liquor prepared in example 7 is added into each pot, and root irrigation treatment is carried out. Cucumber with 50mL of tap water was used as control. After 45 days, the disease index of cucumber root knot nematode and the disease prevention and control effect of the fermentation liquor of the trichosporon roseum SYP-1-1 on diseases are counted.
The disease degree of the root-knot nematode is divided into 5 grades, and 0 grade: the root system is completely normal, and the symptoms of root-knot nematodes are avoided; stage 1: the total length of the root knot is 1% -15% of the total length of the root system; 2 stages: the total length of the root knot is 16% -25% of the total length of the root system; 3 stages: the total length of the root knot is 51% -75% of the total length of the root system; 4 stages: the total length of root knot is more than 75% of the total length of root system, and the degree grade of the root knot nematode disease of each plant treated is investigated. Disease index = [ Σ (number of disease plants at each stage x corresponding number of stages)/total number of investigation x highest number of stages value ] ×100; disease preventing effect (%) = (control disease index-treatment disease index)/control disease index x 100%.
The result shows that the fermentation liquor of the Saprolegnia rosea SYP-1-1 has better control effect on cucumber root knot nematode disease, and the control effect reaches 53.5%.
Although the invention has been described by way of examples, it will be appreciated by those skilled in the art that modifications and variations may be made thereto without departing from the spirit and scope of the invention.
Claims (10)
1. A strain of Paenibacillus roseus is characterized in that the preservation registration number is CGMCC No.18153.
2. The microbial agent obtained by using the strain of the Paenibacillus roseus according to claim 1, wherein the microbial agent is in the form of liquid, powder or granule.
3. The microbial agent according to claim 2, characterized by being prepared by the following method:
1) Spore suspension preparation: inoculating the strain of the Paenispira rosea on a Potato Dextrose Agar (PDA) culture medium, culturing for 6-8 days at 25-28 ℃, adding sterile water to elute spores, and preparing spore suspension;
2) Seed culture: adding a seed culture medium into a triangular flask, sterilizing at high temperature and moist heat, inoculating spore suspension into the triangular flask when cooling to room temperature, and placing the triangular flask on a shaking table for shaking and dark culture at 25-28 ℃ for 48-84 hours to obtain seed liquid;
3) Liquid fermentation culture: preparing a fermentation medium, sterilizing, cooling, inoculating seed liquid, and culturing at 25-28 ℃ for 48-96 hours to obtain fermentation liquor.
4. A microbial agent according to claim 3, wherein in step 2) the seed medium is Potato Dextrose (PD) medium or a complex medium comprising, in 1000ml volume: 15-30 g glucose, 5-15 g yeast extract powder and K 2 HPO 4 0.5-2 g of MgSO 4 ·7H 2 O0.5-1.0 g, water to 1000ml, and HCl solution of 1M to adjust pH to 5.5-6.5.
5. The microbial agent according to claim 3, which isCharacterized in that in step 3), the fermentation medium comprises the following components in volume of 1000 milliliters: 10-25 g of glucose, 10-25 g of corn meal, 5-15 g of yeast extract powder, 5-15 g of bean cake powder and K 2 HPO 4 0.5-2 g of MgSO 4 0.2-1 g, adding water to 1000ml, adjusting pH to 5.5-6.5, sterilizing to obtain the fermentation medium.
6. The microbial agent of claim 3, wherein the liquid fermentation culture conditions of step 3) are: the inoculation amount is 0.5-5%, the rotating speed is 150-220 r/min, the temperature is 25-28 ℃, and the culture time is 48-96 hours.
7. The microbial agent according to claim 2, wherein the strain of polyporus roseus is subjected to liquid fermentation to obtain a fermentation broth, and a stabilizer is added or not added to obtain a liquid agent; or directly adding a carrier into the fermentation broth according to the mass ratio of the fermentation broth to the carrier of 1-3:1, or adding the carrier after centrifugally concentrating the fermentation broth, or adding a combination of the carrier and an auxiliary agent, wherein the adding amount of the carrier is 10-30 percent (mass percent) of the fermentation broth, mixing, granulating and drying at 35-45 ℃; or pulverizing to obtain powder; the carrier is one or more of diatomite, light calcium carbonate, talcum powder, turf, rice hull powder and straw powder.
8. The microbial agent according to claim 2, wherein the microbial agent has a bacterial content of (1 to 10) ×10 8 CFU/g。
9. Use of the strain of trichosporon roseum according to claim 1 for controlling crop nematodes, sclerotinia, wilt, root rot and verticillium wilt.
10. Use of a microbial agent according to any one of claims 2 to 8 for controlling crop nematodes, sclerotinia, wilt, root rot and verticillium wilt.
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