CN114854665B - Production method and application of paecilomyces lilacinus microsclerotia - Google Patents
Production method and application of paecilomyces lilacinus microsclerotia Download PDFInfo
- Publication number
- CN114854665B CN114854665B CN202210807022.3A CN202210807022A CN114854665B CN 114854665 B CN114854665 B CN 114854665B CN 202210807022 A CN202210807022 A CN 202210807022A CN 114854665 B CN114854665 B CN 114854665B
- Authority
- CN
- China
- Prior art keywords
- microsclerotia
- paecilomyces lilacinus
- culture
- fermentation
- seed
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
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
- C12N3/00—Spore forming or isolating processes
-
- 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
-
- 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
-
- 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
- C12R2001/79—Paecilomyces
Abstract
The invention provides a production method and application of paecilomyces lilacinus microsclerotia, wherein the production method comprises the following steps: 1) culturing Paecilomyces lilacinus strain on PDA plate culture medium for 1 week, eluting spore, or scraping hypha and spore on the plate with inoculating ring, inoculating into seed culture medium in shake flask; 2) shake-flask shake culture with Paecilomyces lilacinus, and performing primary or secondary shake-flask culture to obtain seed solution; 3) inoculating the seed liquid into a fermentation tank to prepare the micro-sclerotium fermentation liquid. 4) Adding carrier filler into the fermentation liquid, drying, and sieving to obtain the microsclerotia preparation. The invention provides a preparation technology and production process parameters for pilot-scale fermentation production of paecilomyces lilacinus microsclerotia, and realizes large-scale production of paecilomyces lilacinus microsclerotia.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a pilot-scale production method of biocontrol fungi.
Background
Plant nematodes are a disease of the world that infests 3000 crops, including most food and commercial crops, and are a major cause of continuous cropping obstacles. After nematode infection, crops are hindered in growth, the yield and the quality are seriously reduced, and the annual loss caused by plant nematodes is up to 1570 hundred million dollars. Because the nematodes can survive in soil and disease residues in winter and are difficult to remove, once the nematodes have suitable environment and hosts, the nematodes can be infected again, and the nematodes are easy to spread and spread in a large area after infection, so that great pressure is brought to prevention and control of diseases. The plant parasitic nematodes are seriously harmed by root-knot nematodes and cyst nematodes, wherein 15 common species of the root-knot nematodes can infect most of food crops, oil crops, fiber crops, vegetables, fruit trees, tobacco, tea leaves, Chinese herbal medicines, flowers and the like, and the annual loss of the plant parasitic nematodes exceeds 700 million yuan in China. After infected, a large number of root knots are formed on the roots of the plants, so that the absorption and transmission of nutrients and water are blocked, meanwhile, wounds caused by the root knots are easy to infect other fungi and bacterial diseases, and crop failure can be caused when the root knot nematodes are serious. Cyst nematodes mainly damage wheat, rice, potatoes and soybeans, and can reduce yield by 70-80% in severe cases.
In production, the generation of nematode diseases can be controlled by methods such as crop rotation, planting of disease-resistant varieties, reasonable fertilization, high-temperature greenhouse closing and the like, but the methods have certain limitations and are difficult to implement in a large area. At present, the prevention and the treatment of the nematodes are mainly realized by relying on chemical pesticides and through soil treatment and seed and seedling treatment. However, the long-term application of a large amount of chemical pesticides inevitably causes the environmental pollution of farmland soil and the pesticide residue of agricultural products exceeds the standard, and meanwhile, the problems of pesticide damage at the seedling stage, drug resistance of pathogenic substances and the like are easy to occur. Plant nematodes are found to have numerous natural enemies, such as fungi, bacteria, predatory nematodes. Paecilomyces lilacinus (A)Paecilomyces lilacinusAlso known as lilac violet sporePurpureocillium lilacinum) Is a kind of filamentous fungi, also an important nematophagous fungi, belonging to the class of hyphomycetes, the order of arbuscular sporophytes, the family of arbuscular sporophyceae of the subdivision Deuteromycotina, and is extremely widely distributed. Paecilomyces lilacinus can parasitize eggs of plant nematodes such as root-knot nematodes, cyst nematodes and the like,Female and cyst produce cell wall hydrolase of chitinase, glucanase, etc. and may secrete nematocidal compound and bacteriostasis active matter. In the early stage, the investigation on the root-knot nematode parasitic bacteria on over 80 crops in farmland soil of 11 provinces and cities in China finds that the paecilomyces lilacinus has the highest separation frequency and is a dominant species in many places. Paecilomyces lilacinus has shown great biocontrol potential since the first discovery by Jatala in 1979 of its parasitic action on nematodes, and some commercial preparations have been registered and used. At present, the main active components of the paecilomyces lilacinus preparation are conidium and hypha, the stress resistance is poor, the shelf life is short, the requirement of agricultural production is difficult to meet, and the large-scale production and application of the paecilomyces lilacinus preparation are also limited to a great extent.
Paecilomyces lilacinus has been found to form a specific resistant structure, namely Microsclerotia (Microclerotia), under certain conditions. The microsclerotia is formed by winding and specializing a large number of hyphae and is a resistant dormancy body of filamentous fungi for resisting adverse environments such as high temperature, low temperature, drought and the like. Unlike those sclerotia which are large in size and have remarkable hypha variation differentiation and tissue structure, there are sometimes no specific tissue differentiation in microsclera, and there is also a great difference in the degree of compactness of hypha aggregation. Some plant pathogenic fungi, such as verticillium dahliae, soybean anthracnose, etc., can generate microsclerotia in plant aging tissues to help the plant to survive overwintering, and when the temperature and humidity conditions are appropriate, the microsclerotia germinate again. Recent studies have found that entomogenous fungi such as Metarrhizium anisopliae, Nomuraea japonica and Beauveria bassiana can also produce microsclerotia in a specific liquid medium. The resistance structure of the microsclerotia has important significance for prolonging the shelf life of the biocontrol fungus preparation and promoting the commercialization of the fungus biocontrol fungus agent.
Disclosure of Invention
The invention mainly aims at the problems of short shelf life and unstable field control effect of paecilomyces lilacinus spore preparations in the current production, provides a pilot-scale production method for liquid fermentation of paecilomyces lilacinus microsclerotia and preparation processing, prepares a paecilomyces lilacinus microsclerotia preparation with strong stress resistance and remarkable biological control effect, and is applied to control of important plant nematodes such as root-knot nematodes, cyst nematodes and the like. Therefore, the first purpose of the invention is to provide a production method of paecilomyces lilacinus microsclerotia.
A second object of the invention is to provide the use of said production method.
The technical scheme for realizing the above purpose of the invention is as follows:
a production method of paecilomyces lilacinus microsclerotia comprises the following steps:
1) culturing Paecilomyces lilacinus strain on PDA plate culture medium for 1 week, eluting spore, or scraping hypha and spore on the plate with inoculating loop, and inoculating in shake flask seed culture medium;
2) shake-flask shaking culture inoculated with Paecilomyces lilacinus, and performing primary or secondary shake-flask culture to obtain seed solution;
3) inoculating the seed liquid into a fermentation tank to prepare a micro-sclerotium fermentation liquid; the volume ratio of the seed liquid to the liquid fermentation medium is 1: (100-200), wherein the fermentation culture temperature is 25-28 ℃, the stirring speed is 150-200 r/min, the ventilation volume is 0.3-1, and the culture time in a fermentation tank is 3-4 days;
the fermentation medium contained in the fermenter per unit volume contained the following dry matter weights of the various raw materials: 30-50 g/L of sucrose, 7.5-15 g/L of yeast extract powder, 5-8 g/L of soybean meal powder and 800.01-0.05 mL/L, KH of Tween 2 PO 4 4.0-5.0 g/L of anhydrous CaCl 2 0.7~1.2 g/L、MgSO 4 •7H 2 O 0.5~1.0 g/L、FeSO 4 •7H 2 O 0.05~0.1 g/L、ZnSO 4 •7H 2 O 15~30 mg/L、MnSO 4 15~30 mg/L、Na 2 MoO 4 •2H 2 10-30 mg/L of O, 0.5-1 mg/L of thiamine, and adjusting the pH value to 5.0-6.5.
4) Adding carrier filler into the fermentation liquor, drying, sieving, and preparing the microsclerotia preparation.
Wherein, the weight of dry matters of various raw materials in the unit volume of the seed culture medium in the step 1) is as follows: 20-40 g/L of cane sugar and 7.5-15 g/L, K of yeast extract powder 2 HPO 4 •3H 2 O 0.5~1 g/L、MgSO 4 •7H 2 O 0.5~1g/L、NaCl 0.5~1 g/L、ZnSO 4 •7H 2 O15-30 mg/L, and adjusting the pH value to 6.0-6.5.
Wherein, the seed culture conditions in the step 2) are 25-28 ℃ and 150-200 r/min, and the inoculation amount of the 2 nd-level seed liquid cultured by the second-level shake flask is 2-3%.
Further preferably, in the step 2), a seed solution is prepared by first-stage shake flask culture, and the culture time is 3-4 days; or preparing a seed solution by two-stage shake flask culture, wherein the 1 st seed solution is subjected to shake culture for 2-3 days, and the 2 nd seed solution is subjected to shake culture for 2-4 days.
Based on the shake flask culture condition, the concentration of the seed liquid obtained by shake flask culture is 2-10 multiplied by 10 8 spores/mL.
In the step 3), the ventilation amount is 0.3-1, the initial ventilation amount is 0.3-0.4, and the adjustment is needed in the fermentation process, wherein the adjustment mode is a known mode in the field.
The liquid filling coefficient of the fermentation tank in the step 3) can be 0.5-0.7.
Preferably, the filler in the step 4) is one or more of diatomite, starch and rice hull powder; the weight ratio of the micro-bacterium core fermentation liquid to the filler is 1: (0.7-1.5).
Because the diatomite has good adsorbability and strong water absorption capacity, and the rice hull powder is easy to disperse after adsorption, the filler in the step 4) is preferably a combination of the diatomite and the rice hull powder, and the weight ratio of the diatomite to the rice hull powder is (2-4): 1.
by adopting the fermentation conditions of the invention, the fermentation liquor in the step 3) contains 1.0-5.0 multiplied by 10 4 each/mL of microsclerotia, wherein the size of the microsclerotia is 200-600 mu m; the formation process of the microsclerotia is that spores are gathered and germinate firstly, and then hyphae are wound continuously to form a compact stress-resistant structure.
Wherein the drying temperature of the microsclerotia preparation in the step 4) is 30-35 ℃, and the microsclerotia preparation is dried until the water content is below 13%. The drying time can be 6-24 h.
The production method of paecilomyces lilacinus microsclerotia provided by the invention is applied to the prevention and treatment of root-knot nematodes and cyst nematodes.
The invention has the beneficial effects that:
the invention firstly provides a preparation technology and production process parameters of paecilomyces lilacinus microsclerotia pilot-scale fermentation production, and realizes the large-scale production of paecilomyces lilacinus microsclerotia.
The inventor finds that under the culture condition limited by the invention, the inoculated paecilomyces lilacinus spores start to massively gather and germinate, then hyphae are interwoven to form microsclerotia prototypes, and finally compact mature microsclerotia are formed, but not formed by massively dispersed hyphae gathering. Through seed and fermentation culture, a large amount of paecilomyces lilacinus microsclerotia with regular shapes and uniform sizes can be stably produced, and the fermentation time can be shortened to 3-4 days. Compared with conidiospores and mycelia, the microsclerotia cells have thicker cell walls, more carbohydrate substances and lipid substances, stronger dryness resistance and high-salt stress resistance, stronger tolerance to common bactericides and insecticides in agricultural production, the shelf life of the researched and developed paecilomyces lilacinus microsclerotia preparation can reach 1-2 years, and the preparation can adapt to a high-temperature environment above 40 ℃, so that the problems of short shelf life of the fungal preparation and poor product stability are effectively solved. In addition, the microsclerotia preparation has high content, small dosage, long lasting period, stable effect and difficult drifting, and has important significance for developing novel paecilomyces lilacinus biopesticide and improving product stability and field effect.
Drawings
FIG. 1 shows the sclerotium of Paecilomyces lilacinus formed in the fermentation broth. The left side of FIG. 1 is a photograph of shake flask culture, and the right side of FIG. 1 is the microsclerotia morphology observed under a stereomicroscope, at 4-fold magnification.
FIG. 2 shows the formation of sclerotium of Paecilomyces lilacinus in the fermentation broth. A: 4 h; b: 8 h; c: 14 h; d: 96 h; scale size in fig. 2: a: 10 mu m; b: 20 mu m; c: 50 μm; d: 200 μm.
FIG. 3 is a photograph of the morphological structure of the microsclerotia of Paecilomyces lilacinus cultured for 4 days in a fermentation broth. A. B: microsclerotia internal structure (semi-thin section); C. d: microsclerotia surface structure (scanning electron microscope).
FIG. 4 is a photograph (transmission electron microscope) of a sclerotium cell structure of Paecilomyces lilacinus cultured for 4 days in a fermentation broth. A. B: conventionally cultured conidia and hyphae; C. d: spores and hyphae in microsclerotia.
FIG. 5 shows the accumulation of carbohydrates in the sclerotium of Paecilomyces lilacinus. A: spores in microsclerotia; b: conventionally cultured conidia; c: microsclerotia hyphae; d: hyphae were cultured routinely.
FIG. 6 shows the accumulation of lipid substances in the sclerotium of Paecilomyces lilacinus. A: spores and hyphae in the microsclerotia; b: conidia and hyphae cultured conventionally.
FIG. 7 is a graph showing the comparison of the inhibitory effect of a fermentation filtrate of a microsclerotia of Paecilomyces lilacinus on the growth of pathogenic bacteria and the formation of sclerotia.
FIG. 8 is a photograph showing the parasitism of the Microbacterium lilacinum to the root knot nematode egg.
FIG. 9 is a photograph of a paecilomyces lilacinus microsclerotia preparation adsorbed with diatomaceous earth and rice hull powder.
FIG. 10 is a graph showing the effect of the application of a P.lilacinus microsclerotia preparation on root knot formation in cucumber roots. A: comparison; b: the microsclerotia preparation is administered.
FIG. 11 shows germination of Scutellaria lilacinus microsclerotia after standing at room temperature for 1 year. In the figure, A is newly cultured microsclerotia, and B is microsclerotia left for 1 year.
Detailed Description
The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention.
Unless otherwise specified, the test materials and test instruments used in the specification are commercially available.
Unless otherwise specified, the parts in the examples are parts by mass, and the percentages are percentages by mass.
The paecilomyces lilacinus strain used in the invention is separated from a root-knot nematode sample by the research team, and the classification of the strain is named as lilacinus (A)Purpureocillium lilacinum) The preservation number of the strain is CGMCC No.9344, the preservation time is 2014, 7 and 4 days, and the preservation place is Chinese microorganism culture preservation management Committee, namely common microorganism center, zip code 100101.
Example 1 Observation of the morphology and Structure of the Microsclerotium of Paecilomyces lilacinus
Culturing paecilomyces lilacinus microsclerotia in a shake flask byThe culture medium is as follows: sucrose: 40 g/L, yeast extract powder: 10 g/L, soybean meal powder: 6 g/L, Tween-80: 0.05mL/L, KH 2 PO 4 : 5g/L anhydrous CaCl 2 ：1 g/L、MgSO 4 •7H 2 O：0.5 g/L、FeSO 4 •7H 2 O：0.05 g/L、ZnSO 4 •7H 2 O：30 mg/L、MnSO 4 ：15 mg/L、Na 2 MoO 4 •2H 2 O: 10 mg/L, thiamine: 0.5mg/L, and the pH value is adjusted to 5.5.
Sampling at different time points, and observing the form and the formation process of the microsclerotia in the fermentation broth, wherein the results are shown in figures 1 and 2. Observing under a microscope, inoculating for 4-8 h, enabling paecilomyces lilacinus conidia to gather and start to germinate, interweaving hyphae after 14 h, gradually forming a microsclerotia embryonic form, continuously gathering and winding the hyphae, continuously growing microsclerotia, forming microsclerotia with consistent size and compact structure on day 4, enabling fermentation liquor to be reddish brown, and enabling the number of the microsclerotia not to change any more, thereby determining that the shake flask culture time is optimal for 4 days.
Microsclerotium was collected with a 150 μm-sized screen, and a semi-thin section was prepared, and the internal structure of the microsclerum was observed, and the results are shown in A-B of FIG. 3. The paecilomyces lilacinus microsclerotia is divided into an inner layer and an outer layer, the outer part is compact, the inner part is mainly hypha, the inner part is loose, and a certain amount of spores are contained. The microsclerotia were fixed and the surface structure of the microsclerotia was observed under a scanning electron microscope, and the results are shown in C-D of FIG. 3. The mature microsclerotia are found to be spherical or ellipsoidal, are compact and are formed by interweaving and winding a large number of hyphae. It can also be seen under transmission microscope that the cell walls of the sclerotinia sclerotiorum hyphae and spores are significantly thicker than those of the conventional vegetative growth, and the cells contain melanin granules, see fig. 4. A, B in FIG. 4 are conidia and hyphae obtained by conventional culture. The 1L culture solution for conventional culture contains sucrose 20 g, yeast extract 15 g, and K 2 HPO 4 •3H 2 O 1 g、MgSO 4 •7H 2 0.5 g of O and 0.5 g of NaCl, and the fermentation temperature is 28 ℃.
The results of staining carbohydrate substances in microsclerotia with periodic acid-Schiff reagent are shown in FIG. 5. The carbohydrate in the spores and hyphae of the paecilomyces lilacinus sclerotium is found to be obviously more than that in the conidia and hyphae formed by conventional culture.
The results of staining lipid material in microsclerotia with sudan iii are shown in fig. 6. The lipid material in the sclerotium hyphae of paecilomyces lilacinus is found to be obviously more than that in the hyphae formed by conventional culture.
Experimental results show that the culture medium is a suitable culture medium for culturing paecilomyces lilacinus microsclerotia, and the cultured microsclerotia has better characteristics. The fermentation condition also provides a process condition reference for large-scale production of the fermentation tank.
Example 2: paecilomyces lilacinus microsclerotia pilot scale fermentation culture (1T fermentation tank)
1. Preparation of Paecilomyces lilacinus inoculum
The paecilomyces lilacinus strain preserved in glycerol at the temperature of-80 ℃ is connected to a PDA plate for activation, and is transferred to a fresh PDA plate after sporulation, and is cultured in an incubator at the temperature of 28 ℃ for 1 week. And (3) sucking 10 mL of 0.05% sterile Tween-80 solution by a pipette, adding the solution into the plate, gently scraping spores by using a sterile coating rod, transferring the eluent into a 50mL sterile centrifuge tube, and oscillating and mixing the eluent uniformly by using an oscillator to prepare the paecilomyces lilacinus inoculum.
2. Preparation of Paecilomyces lilacinus seed liquid
Preparing a seed culture medium, wherein the seed culture medium comprises the following components: sucrose: 20 g/L, yeast extract powder: 15 g/L, K 2 HPO 4 •3H 2 O：1 g/L、MgSO 4 •7H 2 O：0.5 g/L、NaCl：0.5 g/L、ZnSO 4 •7H 2 O: 15 mg/L and pH 6.0. And (3) subpackaging the seed culture medium into 2000 mL triangular flasks, filling the triangular flasks with liquid in an amount of 600 mL, sterilizing at 121 ℃ for 30 min, and cooling to room temperature for later use.
10 mL of spore suspension is sucked and inoculated into a seed culture medium, and the seed solution is prepared by shaking culture for 90h at the temperature of 28 ℃ and at the speed of 180 r/min.
3. Paecilomyces lilacinus micro-sclerotium fermentation culture
The pilot fermentation was performed by Highbio-technology Co., Ltd, Beijing, using a 1T automatic fermentation tank (HND-BI 06000 type intelligent control system for fermentation tank). Feeding after high-temperature steam of fermentation tank disappearsThe material comprises the following components: sucrose: 40 g/L, yeast extract powder: 10 g/L, soybean meal powder: 6 g/L, Tween-80: 0.05mL/L, KH 2 PO 4 : 5g/L anhydrous CaCl 2 ：1 g/L、MgSO 4 •7H 2 O：0.5 g/L、FeSO 4 •7H 2 O：0.05 g/L、ZnSO 4 •7H 2 O：30 mg/L、MnSO 4 ：15 mg/L、Na 2 MoO 4 •2H 2 O: 10 mg/L, thiamine: 0.5mg/L, the pH value is adjusted to 5.5, and the liquid filling coefficient is 0.6.
Sealing the cover, sterilizing at 121 deg.C for 30 min, and performing flame inoculation with 0.6% inoculation amount when the temperature of the fermenter is up to 30 deg.C. The fermentation conditions are that the temperature is 28 ℃, the rotating speed is 180 r/min, the ventilation volume is 0.3-0.8, the tank pressure is 0.03-0.05 MPa, the fermentation period is 84h, and the number of the microbial cores in the fermentation liquor is 2.8 multiplied by 10 4 one/mL.
Example 3 Michelia lilacina Mikrothrix pilot scale fermentation culture (1T fermenter)
1. Paecilomyces lilacinus first-order seed culture
Preparing a seed culture medium, wherein the formula is as follows: 20 g/L of cane sugar and 15 g/L, K of yeast extract powder 2 HPO 4 •3H 2 O 1 g/L、MgSO 4 •7H 2 O 0.5 g/L、NaCl 0.5 g/L、ZnSO 4 •7H 2 O15 mg/L, pH 6.0. And (3) subpackaging the seed culture medium into 500 mL triangular flasks, filling the flask with 100 mL of liquid, sterilizing at 121 ℃ for 30 min, and cooling to room temperature for later use.
Inoculating the paecilomyces lilacinus strain on a PDA (personal digital assistant) plate, culturing for 1 week until sporulation occurs, scraping hypha and spores by using an inoculating loop, inoculating the hypha and the spores in a seed culture medium, and performing shake-flask culture at 28 ℃ and 180 r/min for 48 hours to prepare a first-stage seed solution.
2. Paecilomyces lilacinus secondary seed culture
The seed medium was prepared as described above, and the resulting mixture was dispensed into 2000 mL triangular flasks, and the liquid loading was 650 mL. Inoculating the first-stage seed solution at an inoculum size of 2%, culturing at 28 deg.C and 200 r/min for 72h to obtain second-stage seed solution with a strain content of 1 × 10 9 spores/mL.
3. Paecilomyces lilacinus micro-sclerotium fermentation culture
Performing light purple simulation in a 1T fermentation tankFermenting and culturing penicillium microsclerotia. The medium composition was as follows: 50 g/L of sucrose, 10 g/L of yeast extract powder, 5g/L of soybean meal powder and 800.05 mL/L, KH of Tween 2 PO 4 4 g/L of anhydrous CaCl 2 1 g/L、MgSO 4 •7H 2 O 0.5 g/L、FeSO 4 •7H 2 O0.1 g/L、ZnSO 4 •7H 2 O30 mg/L、MnSO 4 15 mg/L、Na 2 MoO 4 •2H 2 O10 mg/L and thiamine 0.5mg/L, the pH value is adjusted to 5.2, and the liquid filling coefficient is 0.7.
After sterilization, flame inoculation is carried out, and the inoculation amount is 0.5 percent (volume ratio). The fermentation conditions are 29 ℃, the rotating speed is 180 r/min, the ventilation volume is 0.3-1, the tank pressure is 0.03-0.05 MPa, and the fermentation period is 76 h. Measuring the number of microsclerotia in the fermentation broth to be 4.2 multiplied by 10 4 one/mL.
EXAMPLE 4 preparation of a Paecilomyces lilacinus microsclerotia preparation
Taking 100L of paecilomyces lilacinus fermentation liquor obtained in example 3, and mixing the fermentation liquor according to the weight ratio of 1: 0.9, adding diatomite and rice hull powder in a weight ratio of 7: and 3, fully stirring in the mixer, uniformly mixing, then flatly paving in a tray, and performing air drying by adopting an air drying oven, wherein the drying temperature is 30-35 ℃, and drying until the water content is less than 13%. Sieving with 18 mesh sieve to obtain paecilomyces lilacinus microsclerotia fine granule. The product is shown in the photograph of FIG. 9.
Example 5 bacteriostatic Effect of Paecilomyces lilacinus Microsclerotium fermentation solution
The bacteriostatic activity of the paecilomyces lilacinus micro-sclerotium fermentation solution prepared in example 1 was measured. Centrifuging at 4000r/min for 10 min, filtering with 0.22 μm microporous membrane to remove thallus, mixing the obtained fermentation filtrate with PDA culture medium at volume ratio of 1: 4, fully and uniformly mixing to prepare the PDA flat plate containing the fermentation filtrate of the microsclerotia. Inoculating Rhizoctonia solani and Sclerotinia sclerotiorum cake with diameter of 0.5 cm to the center of the above plate, culturing at 28 deg.C, and measuring the growth of pathogenic bacteria after 1 week. As a result, as shown in FIG. 7, the inhibitory rates of the micronucleus fermentation filtrate against Rhizoctonia solani and Sclerotinia sclerotiorum were 53.5% and 70.5%, respectively, and the formation of sclerotia could be effectively inhibited.
Example 6 parasitism of Paecilomyces lilacinus Microbacterium Corvum on root knot nematode eggs
The microsclerotia of paecilomyces lilacinus prepared in example 1 was collected, the culture solution was washed with sterile water to remove the culture solution, 300 microsclerotia and 200 eggs of root knot nematode were added into 24-well tissue culture plates, 1 mL of water was added, the mixture was mixed uniformly, the mixture was cultured at 25 ℃ for 7 days, and the parasitism of the eggs of root knot nematode by the microsclerotia was observed under an inverted microscope, and the results are shown in FIG. 8. The paecilomyces lilacinus microsclerotia germinates to generate hyphae, parasitizes nematodes and grows and breeds in nematode bodies, and finally the hyphae of the paecilomyces lilacinus penetrates through the nematode egg body wall to digest the nematodes.
Example 7 Effect of Paecilomyces lilacinus Microsclerotium preparation on control of cucumber root-knot nematodes
The paecilomyces lilacinus microsclerotia preparation prepared in example 4 is tested for controlling cucumber root knot nematode disease. Picking root-knot nematode oocysts to prepare an egg suspension, and mixing sterilized soil with the egg suspension to ensure that the concentration of the nematodes is 300 eggs/100 g soil. The diseased soil is filled into plastic flowerpots with the diameter of 7 cm, and each flowerpot is 150 g.
And (3) disinfecting the surface of the Zhongnong No. 6 cucumber seeds by using 2% NaClO for 5 min, washing, accelerating germination and then sowing in a seedling tray. And (3) transplanting the cucumber seedlings into nematode disease soil when the cucumber seedlings grow to 2-3 true leaves, wherein 1 plant is planted in each pot, and meanwhile, the paecilomyces lilacinus microsclerotia preparation is subjected to hole application and inoculated in a concentration of 300/100 g soil. The disease condition of the roots is investigated after 30 days of transplanting, the result is shown in figure 10, the number of root knots on the cucumber roots is obviously reduced after the paecilomyces lilacinus microsclerotia preparation is applied, and the control effect on the root-knot nematodes reaches 62.5%.
Example 8 Heat resistance of Penicillium lilacinum microsclerotia preparations
The paecilomyces lilacinus microsclerotia preparation prepared in example 4 was subjected to heat resistance measurement. Weighing 1 g of microsclerotia preparation and conidia preparation, putting into a 50mL centrifuge tube, covering tightly, and heating in drying ovens of 40 ℃ and 60 ℃ respectively. And adding sterile water for dilution after treatment, sucking 0.5 mL of the solution on an anti-PDA (personal digital assistant) plate to uniformly distribute the microsclerotia in the plate, culturing at 28 ℃, and measuring the germination rate of the microsclerotia after 24 hours under a microscope. The result shows that the germination rate of the microsclerotia preparation is kept at 100% after being treated for 24 hours at 40 ℃, the germination rate of the microsclerotia can still reach more than 60% after being heated for 8 hours at 60 ℃, and the activity of the spore preparation is reduced to 0.
Example 9 determination of the storage Properties of the Microsclerotium preparations of Paecilomyces lilacinus
The prepared paecilomyces lilacinus microsclerotia preparation is placed in a ventilated and dry place, and the germination rate of the paecilomyces lilacinus microsclerotia preparation is measured after the paecilomyces lilacinus microsclerotia preparation is stored for 6 months and 12 months at room temperature. Weighing 1 g of microsclerotia preparation, adding water for dilution, and placing on an anti-PDA plate, wherein the number of microsclerotia on each plate is more than 100. Culturing at 28 deg.C for 24 hr, and determining germination rate. The results showed that the germination rate of microsclerotia was 100% at 12 months (see FIG. 11).
Although the present invention has been described in the foregoing by way of examples, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (6)
1. A production method of paecilomyces lilacinus microsclerotia is characterized by comprising the following steps:
1) culturing Paecilomyces lilacinus strain on PDA plate culture medium for 1 week, eluting spore, scraping hypha and spore on the plate with sterilizing coating rod or inoculating ring, and inoculating in seed culture medium in shake flask; the paecilomyces lilacinus strain is a paecilomyces lilacinusPurpureocillium lilacinumThe preservation number of the strain is CGMCC No.9344, the preservation time is 2014, 7 and 4 days, and the preservation place is China general microbiological culture Collection center;
2) shaking culture the inoculated paecilomyces lilacinus in a shaking flask, and preparing a seed solution through primary or secondary shaking culture;
3) inoculating the seed solution into a fermentation tank to prepare a micro-sclerotium fermentation solution; the volume ratio of the seed liquid to the liquid fermentation medium is 1: (100-200), wherein the fermentation culture temperature is 25-28 ℃, the stirring speed is 150-200 r/min, the ventilation quantity is 0.3-1, and the culture time in a fermentation tank is 3-4 days;
the weight of dry matter of each raw material contained in a unit volume of liquid fermentation medium in the fermentation tank is as follows: 30-50 g/L of sucrose, 7.5-15 g/L of yeast extract powder, 5-8 g/L of soybean meal powder and 800.01-0.05 mL/L, KH of Tween 2 PO 4 4.0-5.0 g/L of anhydrous CaCl 2 0.7~1.2 g/L、MgSO 4 •7H 2 O 0.5~1.0 g/L、FeSO 4 •7H 2 O 0.05~0.1 g/L、ZnSO 4 •7H 2 O 15~30 mg/L、MnSO 4 15~30 mg/L、Na 2 MoO 4 •2H 2 10-30 mg/L of O and 0.5-1 mg/L of thiamine, and adjusting the pH value to 5.0-6.5;
4) adding a carrier filler into the microsclerotia fermentation liquid, drying and sieving to prepare a microsclerotia preparation, wherein the weight ratio of the microsclerotia fermentation liquid to the filler is 1: (0.7-1.5), wherein the filler is diatomite and rice hull powder in a weight ratio of (2-4): 1, and mixing.
2. The method for producing Paecilomyces lilacinus sclerotium according to claim 1, wherein the weight of each dry matter contained in the seed medium of step 1) per unit volume is as follows: 20-40 g/L of cane sugar and 7.5-15 g/L, K of yeast extract powder 2 HPO 4 •3H 2 O 0.5~1 g/L、MgSO 4 •7H 2 0.5-1 g/L, NaCl 0.5.5-1 g/L of O and ZnSO 4 •7H 2 O15-30 mg/L, and adjusting the pH value to 6.0-6.5.
3. The method for producing paecilomyces lilacinus microsclerotia according to claim 1, wherein the shake flask culture conditions in step 2) are 25-28 ℃ and 150-200 r/min, and the inoculation amount of the 2 nd-stage seed liquid by the second-stage shake flask culture is 2-3%.
4. The method for producing paecilomyces lilacinus microsclerotia according to claim 1, wherein in the step 2), a seed solution is prepared by first-stage shake flask culture, and the culture time is 3-4 days; or preparing a seed solution by two-stage shake flask culture, wherein the 1 st seed solution is subjected to shake culture for 2-3 days, and the 2 nd seed solution is subjected to shake culture for 2-4 days.
5. The method for producing sclerotium microsclerotia in paecilomyces lilacinus according to claim 1, wherein the drying temperature of the sclerotium microsclerotia preparation in step 4) is 30-35 ℃, and the sclerotium microsclerotia preparation is dried until the water content is below 13%.
6. Use of a process for the production of paecilomyces lilacinus sclerotium according to any one of claims 1 to 5 for the control of root-knot and cyst nematodes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210807022.3A CN114854665B (en) | 2022-07-11 | 2022-07-11 | Production method and application of paecilomyces lilacinus microsclerotia |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210807022.3A CN114854665B (en) | 2022-07-11 | 2022-07-11 | Production method and application of paecilomyces lilacinus microsclerotia |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114854665A CN114854665A (en) | 2022-08-05 |
| CN114854665B true CN114854665B (en) | 2022-09-23 |
Family
ID=82626946
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210807022.3A Active CN114854665B (en) | 2022-07-11 | 2022-07-11 | Production method and application of paecilomyces lilacinus microsclerotia |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114854665B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102337221A (en) * | 2011-10-10 | 2012-02-01 | 重庆大学 | Inductive production method of Nomuraea rileyi microsclerotia |
| CN102925405A (en) * | 2012-05-29 | 2013-02-13 | 中国林业科学研究院森林生态环境与保护研究所 | Preparation method of paecilomyces lilacinus spore powder |
| CN104357338A (en) * | 2014-11-21 | 2015-02-18 | 重庆大学 | Fermentation method and applications of paecilomyce lilacinus microsclerotia |
| CN108949588A (en) * | 2018-08-23 | 2018-12-07 | 重庆大学 | A kind of application of the preparation method and preparation of beauveria bassiana Microsclerotia and its preparation |
| CN113999778A (en) * | 2021-12-28 | 2022-02-01 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | Trichoderma atroviride microsclerotia and preparation method and application of preparation thereof |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3939771A1 (en) * | 1989-12-01 | 1991-06-06 | Behringwerke Ag | PROCESS FOR THE BIOLOGICAL INACTIVATION OF NUCLEIC ACIDS |
| CN103320327B (en) * | 2012-03-20 | 2015-08-19 | 华中农业大学 | A kind of Paecilomyces lilacinus and cultural method thereof and application |
| CN104212722B (en) * | 2013-12-12 | 2015-11-25 | 谢明 | One strain Paecilomyces lilacinus and cultural method thereof and the application on crop insect pest preventing and controlling |
| EP3684185A1 (en) * | 2017-09-20 | 2020-07-29 | Bayer CropScience Biologics GmbH | Method of improving storage stability and fitness of fungal spores |
| AR118276A1 (en) * | 2019-03-08 | 2021-09-22 | Ypf Tecnologia Sa | BIOINSECTICIATED FORMULATION INCLUDING HIGH RESISTANCE MICROSCLEROTICS FROM BEAUVERIA BASSIANA AND PREPARATION METHOD OF THE SAME |
| CN112111411B (en) * | 2020-09-27 | 2021-10-26 | 江西华威科技有限公司 | Environment-responsive paecilomyces lilacinus micro-bead and preparation method thereof |
-
2022
- 2022-07-11 CN CN202210807022.3A patent/CN114854665B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102337221A (en) * | 2011-10-10 | 2012-02-01 | 重庆大学 | Inductive production method of Nomuraea rileyi microsclerotia |
| CN102925405A (en) * | 2012-05-29 | 2013-02-13 | 中国林业科学研究院森林生态环境与保护研究所 | Preparation method of paecilomyces lilacinus spore powder |
| CN104357338A (en) * | 2014-11-21 | 2015-02-18 | 重庆大学 | Fermentation method and applications of paecilomyce lilacinus microsclerotia |
| CN108949588A (en) * | 2018-08-23 | 2018-12-07 | 重庆大学 | A kind of application of the preparation method and preparation of beauveria bassiana Microsclerotia and its preparation |
| CN113999778A (en) * | 2021-12-28 | 2022-02-01 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | Trichoderma atroviride microsclerotia and preparation method and application of preparation thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114854665A (en) | 2022-08-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR100331125B1 (en) | How to Inhibit Phytopathogens Using Streptomyces WYEC108 | |
| CN100549158C (en) | A kind of campelyco and preparation method thereof | |
| CN106754426B (en) | Trichoderma asperellum and application thereof | |
| CN104686196B (en) | Method for preserving and separating toadstool strain through sporocarp dried in shade | |
| CN111733081A (en) | Biological agent formula suitable for preventing and treating tomato root-knot nematode diseases | |
| CN113980877B (en) | Compound microbial agent and preparation method thereof | |
| CN110896782A (en) | Artificial cultivation method of fossa orchioides | |
| CN112280692B (en) | Crop growth-promoting disease-resistant DSE strain and application thereof | |
| JP3601928B2 (en) | A new microorganism showing anthracnose control effect | |
| CN102960369B (en) | Preparation method of granular paecilomyces lilacinus biological nematocide | |
| CN109661972B (en) | Breeding method of high-temperature-resistant shiitake mushrooms | |
| CN114854665B (en) | Production method and application of paecilomyces lilacinus microsclerotia | |
| CN114507626B (en) | Bacillus subtilis 2JQ3 with poisoning activity on root-knot nematode and application thereof | |
| CN113999778B (en) | Trichoderma atroviride microsclerotia and preparation method and application of preparation thereof | |
| CN110129242B (en) | Continuous cropping resistant composite microbial preparation and preparation method thereof | |
| CN111172064B (en) | Bacillus laterosporus and application thereof | |
| CN110724640B (en) | Tomato root knot nematode biocontrol bacteria, preparation and application thereof | |
| CN114107070A (en) | Piriformospora indica and preparation method thereof | |
| CN110760450A (en) | Culture method of trichoderma longibrachiatum spores, trichoderma longibrachiatum water dispersible granule and preparation method thereof | |
| CN110079478A (en) | Bateriostatics of bacillus amyloliquefaciens and preparation method thereof and the application in prevention and treatment Fusarium moniliforme peanut root rot | |
| CN109355207B (en) | Liquid fermentation culture medium for producing spores from anthrax | |
| CN107964523A (en) | Methylotrophic bacillus and its application | |
| CN111808778B (en) | Bacillus wegener for preventing and treating verticillium wilt and culture method thereof, microbial inoculum and preparation method and application thereof | |
| JPH10506288A (en) | Fungi for biological control of plant diseases Gliocladium catenulatum | |
| CN113817613B (en) | Trichoderma asperellum 6S-2 and application thereof in relieving apple continuous cropping obstacle |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |