CN115449501A - Application of Jujun grass juice as non-pathogenic fusarium oxysporum fermentation culture medium - Google Patents
Application of Jujun grass juice as non-pathogenic fusarium oxysporum fermentation culture medium Download PDFInfo
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- 239000001963 growth medium Substances 0.000 title claims abstract description 65
- 241000223221 Fusarium oxysporum Species 0.000 title claims abstract description 61
- 235000011389 fruit/vegetable juice Nutrition 0.000 title claims abstract description 42
- 238000000855 fermentation Methods 0.000 title claims abstract description 27
- 230000004151 fermentation Effects 0.000 title claims abstract description 27
- 230000001717 pathogenic effect Effects 0.000 title claims abstract description 25
- 244000025254 Cannabis sativa Species 0.000 title claims abstract description 22
- 239000007788 liquid Substances 0.000 claims abstract description 34
- 238000002360 preparation method Methods 0.000 claims abstract description 32
- 238000004519 manufacturing process Methods 0.000 claims abstract description 24
- 241000196324 Embryophyta Species 0.000 claims abstract description 23
- 238000009630 liquid culture Methods 0.000 claims abstract description 22
- 229960005486 vaccine Drugs 0.000 claims abstract description 18
- 230000001954 sterilising effect Effects 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 14
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 9
- 244000061456 Solanum tuberosum Species 0.000 claims abstract description 9
- 235000002595 Solanum tuberosum Nutrition 0.000 claims abstract description 9
- 239000008103 glucose Substances 0.000 claims abstract description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 239000002609 medium Substances 0.000 claims description 10
- 239000001888 Peptone Substances 0.000 claims description 7
- 108010080698 Peptones Proteins 0.000 claims description 7
- 235000019319 peptone Nutrition 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 239000000463 material Substances 0.000 claims description 6
- 239000002699 waste material Substances 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 244000130556 Pennisetum purpureum Species 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 11
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- JNPZQRQPIHJYNM-UHFFFAOYSA-N carbendazim Chemical compound C1=C[CH]C2=NC(NC(=O)OC)=NC2=C1 JNPZQRQPIHJYNM-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
-
- 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
- C12R2001/77—Fusarium
Abstract
The invention provides application of Jujun grass juice as a non-pathogenic fusarium oxysporum fermentation culture medium; the method comprises the following steps: s1, preparation of liquid strains for production: preparing a liquid strain for production by taking fusarium oxysporum FJAT-9290 as a strain and a potato glucose culture medium PDB as a culture medium for the strain; s2, preparation of a fusarium oxysporum plant vaccine preparation: and (2) taking the pennisetum hydridum juice as a liquid culture medium, sterilizing, inoculating the liquid strain prepared in the step (S1), and performing shaking culture at 26-30 ℃ and 160-200r/min for 6-8 days to obtain the non-pathogenic fusarium oxysporum plant vaccine preparation. The application method takes the giant-mycograss juice as the culture medium to ferment the nonpathogenic fusarium oxysporum, the fermentation method is simple, the cost is low, the content of the nonpathogenic fusarium oxysporum after fermentation is equivalent to the fermentation effect of the PDB culture medium commonly used by fungi, and the method has important significance for improving the high added-value utilization of the giant-mycograss.
Description
Technical Field
The invention relates to the technical field of bacterial culture media, in particular to application of pennisetum hydridum juice as a non-pathogenic fusarium oxysporum fermentation culture medium.
Background
Fusarium oxysporum (Fusarium oxysporum) nonpathogenic strains are used as natural plant vaccine strains because the non-pathogenic strains can be colonized in plants, do not cause plant diseases and have good antagonistic action on pathogenic strains, and are applied to control of various crop blight (Shishido et al, 2005 veloso et al, 2012, 2014. The nonpathogenic fusarium oxysporum strain FJAT-9290 disclosed in the prior art is a natural plant vaccine strain, and earlier researches show that the strain can not cause diseases of crops of solanaceae, cucurbitaceae, musaceae, liliaceae and the like, has good colonization ability on the crops of solanaceae, cucurbitaceae, musaceae and the like, and can reach 40-90 days. The method of applying in advance can promote the plant growth, and have better prevention and cure effect to tomato blight, wherein the prevention and cure effect of potted plant is 76.70%, the prevention and cure effect of field plot is 69.56%, the prevention and cure effect is 68.64% slightly higher than that of 50% carbendazim wettable powder treatment of chemical pesticide (xiao Rong Feng, etc., 2015. Colonization characteristic of nonpathogenic fusarium oxysporum FJAT-9290 and prevention and cure effect to tomato blight. Plant protection report, 2.
In the prior art, a PDB culture medium commonly used by fungi is generally used as a fermentation culture medium of nonpathogenic fusarium oxysporum.
Jujun grass is a perennial graminaceous upright fasciculate plant, has a developed root system and grows rapidly, and is known to be rich in organic acids, polyphenols, proteins, cellulose, sugars and other biological substances. The Jujun grass has strong adaptability and is widely planted in various regions of China. At present, the Jujun grass is mainly used in the traditional fields of feed, biogas fermentation production, combustion power generation, biomass ethanol production and the like.
At present, reports of using the pennisetum hydridum juice as a fermentation culture medium of non-pathogenic fusarium oxysporum are not found, and the pennisetum hydridum juice has important significance for high-added-value utilization of pennisetum hydridum.
Disclosure of Invention
The invention aims to solve the technical problem of providing the application of the pennisetum hydridum juice as a non-pathogenic fusarium oxysporum fermentation culture medium. The application method takes the giant-mycorrhizal grass juice as the culture medium to ferment the nonpathogenic fusarium oxysporum, the fermentation method is simple and low in cost, the content of the nonpathogenic fusarium oxysporum after fermentation is equivalent to the fermentation effect of the PDB culture medium commonly used by fungi, the giant-mycorrhizal grass juice waste can be changed into valuable, and the method has important significance for improving the high added value utilization of the giant-mycorrhizal grass.
In order to solve the technical problems, the invention adopts the following technical scheme:
the application of Jujun grass juice as a non-pathogenic fusarium oxysporum fermentation culture medium.
As an embodiment, the non-pathogenic fusarium oxysporum is fusarium oxysporum FJAT-9290.
Fusarium oxysporum FJAT-9290 in the application:
and (3) classification and naming: fusarium oxysporum (Fusarium oxysporum)
The preservation unit: china general microbiological culture Collection center
And (4) storage address: xilu No. 1 Hospital No. 3 of Beijing market facing Yang district
The preservation date is as follows: 3/29/2012
The preservation number is: CGMCC No.5910
As a preferred embodiment, the method steps of the application are as follows:
s1, preparation of liquid strains for production:
preparing a liquid strain for production by taking fusarium oxysporum FJAT-9290 as a strain and a potato dextrose agar PDB culture medium as a culture medium for the strain;
s2, preparation of a fusarium oxysporum plant vaccine preparation:
and (2) taking the megaterium juice as a liquid culture medium, sterilizing, inoculating the liquid strain prepared in the step (S1), and performing shaking culture at 26-30 ℃ and 160-200r/min for 6-8d to obtain the non-pathogenic fusarium oxysporum plant vaccine preparation.
In a preferred embodiment, in step S1, the preparation of the liquid seed culture for production comprises the following steps:
s11, activation of strains:
preparing PDA culture medium, sterilizing at 121 deg.C for 20min, cooling to 45 deg.C, and placing in a sterilizing plate; taking a nonpathogenic fusarium oxysporum FJAT-9290 strain stored at-70 ℃ to perform streak culture on a PDB culture medium plate, and performing constant-temperature culture at 28-30 ℃ for 5-7 days to obtain hyphae;
s12, preparing liquid strain for production
Preparing a PDB liquid culture medium, selecting the hypha obtained in the step S11, inoculating the hypha into the PDB liquid culture medium, and performing shake culture at the temperature of 28-30 ℃ and at the speed of 170-200r/min for 5-7 days to obtain the liquid strain for production.
In one embodiment, in step S11, the PDA medium includes: potato extract 5g/L, peptone 10.0g/L, glucose 15.0g/L, sodium chloride 5.0g/L, agar 20g/L, and pH =7.0.
In one embodiment, in step S12, the PDB liquid medium includes: potato extract 5g/L, peptone 10.0g/L, glucose 15.0g/L, sodium chloride 5.0g/L, and pH =7.0.
As a preferred embodiment, in step S2, the preparation of the fusarium oxysporum plant vaccine preparation comprises the following specific steps:
s21, squeezing fresh giant napier grass serving as a material by using a juicer to obtain juice, and filtering waste residues to obtain giant napier grass juice;
s22, taking pure megaterium juice as a liquid culture medium or a mixture of the megaterium juice and a PDB liquid culture solution as the culture medium, adjusting the pH value to 7.0, and sterilizing at 121 ℃ for 20min;
s23, inoculating the liquid strain for production obtained in the step S1 into the sterilized liquid culture medium according to the inoculation amount of 1%, and performing shaking culture at the constant temperature of 30 ℃ for 30h at the speed of 180r/min to obtain the fusarium oxysporum plant vaccine preparation.
Any range recited herein is intended to include the endpoints and any number between the endpoints and any subrange subsumed therein or defined therein.
The starting materials of the present invention are commercially available, unless otherwise specified, and the equipment used in the present invention may be any equipment conventionally used in the art or may be any equipment known in the art.
The invention has the advantages of
The application method of the invention takes the giant-fungus grass juice as the culture medium to ferment the nonpathogenic fusarium oxysporum, the fermentation method is simple and has low cost, the content of the nonpathogenic fusarium oxysporum after fermentation is equivalent to the fermentation effect of the PDB culture medium commonly used by fungi, and the giant-fungus grass juice waste can be changed into valuable, thus having important significance for improving the high added value utilization of the giant-fungus grass.
Detailed Description
In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
All percentages and ratios used herein are by weight of the total composition, unless otherwise specified. Unless otherwise indicated, all percentages, ratios, and levels of ingredients referred to herein are based on the actual amount of the ingredient, and do not include solvents, fillers, or other materials found in commercially available products with which the ingredient may be combined.
The term "comprising" as used herein means that other steps and ingredients which do not affect the end result can be added.
The term "preferably" and its variants herein refer to embodiments of the invention that are capable of providing specific benefits under specific circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the detailed description of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.
The method is carried out according to conventional conditions or conditions recommended by manufacturers if specific conditions are not indicated in the examples of the invention; the reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
As one aspect of the invention, the invention provides the use of Jujun grass juice as a non-pathogenic Fusarium oxysporum fermentation medium.
As an example, the non-pathogenic Fusarium oxysporum is Fusarium oxysporum FJAT-9290.
The fusarium oxysporum FJAT-9290 is preserved in China general microbiological culture Collection center of China general microbiological culture Collection management Committee in 3.29.2012, has the address of No. 3 Xilu No. 1 Hospital in North West Chen of the Korean district in Beijing and has the preservation number of CGMCC No.5910.
As a preferred embodiment, the method steps of the application are as follows:
s1, preparation of liquid strains for production:
preparing a liquid strain for production by taking fusarium oxysporum FJAT-9290 as a strain and a potato dextrose agar PDB culture medium as a culture medium for the strain;
s2, preparation of a fusarium oxysporum plant vaccine preparation:
and (2) taking the megaterium juice as a liquid culture medium, sterilizing, inoculating the liquid strain prepared in the step (S1), and performing shaking culture at 26-30 ℃ and 160-200r/min for 6-8d to obtain the non-pathogenic fusarium oxysporum plant vaccine preparation.
As a preferred embodiment, in step S1, the preparation of the liquid seed culture for production comprises the following specific steps:
s11, activation of strains:
preparing PDA culture medium, sterilizing at 121 deg.C for 20min, cooling to 45 deg.C, and placing in a sterilizing plate; taking a nonpathogenic fusarium oxysporum FJAT-9290 strain stored at-70 ℃ to perform streak culture on a PDB culture medium plate, and performing constant-temperature culture at 28-30 ℃ for 5-7 days to obtain hyphae;
s12, preparing liquid strain for production
Preparing a PDB liquid culture medium, selecting the hyphae in the step S11, inoculating the hyphae into the PDB liquid culture medium, and performing shake culture at the temperature of 28-30 ℃ and at the speed of 170-200r/min for 5-7 days to obtain the liquid strain for production.
As an example, in step S11, the PDA medium includes: potato extract 5g/L, peptone 10.0g/L, glucose 15.0g/L, sodium chloride 5.0g/L, agar 20g/L, and pH =7.0.
As an example, in step S12, the PDB liquid medium includes: potato extract 5g/L, peptone 10.0g/L, glucose 15.0g/L, sodium chloride 5.0g/L, and pH =7.0.
As a preferred embodiment, in step S2, the preparation of the fusarium oxysporum plant vaccine preparation comprises the following specific steps:
s21, taking fresh Jujun grass as a material, squeezing by a juicer to obtain juice, and filtering waste residues to obtain Jujun grass juice;
s22, taking pure megaterium juice as a liquid culture medium or a mixture of the megaterium juice and PDB liquid culture solution as a culture medium, adjusting the pH value to 7.0, and sterilizing at 121 ℃ for 20min;
s23, inoculating the liquid strain for production obtained in the step S1 into the sterilized liquid culture medium according to the inoculation amount of 1%, and performing shaking culture at the constant temperature of 30 ℃ for 30h at the speed of 180r/min to obtain the fusarium oxysporum plant vaccine preparation.
Examples 1-5, control
The application of the pennisetum hydridum juice as a non-pathogenic fusarium oxysporum fermentation culture medium comprises the following steps:
1) Preparation of test materials
Fungus grass juice: fresh giant-fungus grass juice is used as a material, and a juicer is used for squeezing and filtering waste residues to obtain the giant-fungus grass juice.
The main strains are as follows: the non-pathogenic fusarium oxysporum FJAT-9290 is preserved by a strain bank of biological resources of agricultural academy of sciences of Fujian province; 3.29 days 2012, is preserved in China general microbiological culture Collection center, no. 3 of Xilu No. 1 of Beijing province of rising area, and the preservation number is CGMCC No.5910.
PDB liquid medium: 5g/L of potato extract powder and 10.0g/L of peptone; 15.0g/L of glucose; sodium chloride 5.0g/L, pH =7.0; subpackaging in 250mL triangular flasks, each flask containing 100mL of culture medium; autoclaving at 121 deg.C for 20min, and cooling for use.
2) Test method
2.1 Activation of nonpathogenic Fusarium oxysporum): preparing PDA culture medium, sterilizing at 121 deg.C for 20min, cooling to 45 deg.C, and placing in a sterilizing plate; taking a nonpathogenic fusarium oxysporum FJAT-9290 strain stored at the temperature of-70 ℃ to perform streak culture on a PDB culture medium plate, and performing constant-temperature culture at the temperature of 28-30 ℃ for 5-7 days to obtain activated hyphae;
2.2 Preparation of non-pathogenic liquid strains for Fusarium oxysporum production: inoculating the activated hypha into a PDB culture medium, and performing shake culture at the temperature of between 28 and 30 ℃ and at 180r/min for 5 to 7 days to obtain a liquid strain for production;
2.3 Preparation of a Meconopsis megateri liquid medium: mixing the megalophora juice with a PDB liquid culture medium according to the volume ratio of 0%, 20%, 40%, 60%, 80% and 100%, adjusting the pH to =7.0, subpackaging in 250mL triangular bottles, wherein the liquid filling amount of each triangular bottle is 100mL, and sterilizing at 121 ℃ for 20min; 3 replicates per treatment;
2.4 Preparation of a non-pathogenic Fusarium oxysporum plant vaccine formulation: inoculating the prepared non-pathogenic fusarium oxysporum seed liquid into a pennisetum hydridum liquid culture medium at the inoculation amount of 1%, and carrying out shaking culture at the constant temperature of 28-30 ℃ and at the speed of 180r/min for 7d, namely obtaining a plant vaccine liquid fermentation product; inoculating to PDB liquid culture medium as control;
2.5 Detection of spore number of fermentation product: double-layer sterile yarn for fermentation productFiltering with cloth to obtain spore suspension, and filtering with a filter cloth according to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 After serial dilution, coating on a PDA culture medium plate, culturing at 28 ℃ for 5d, and counting the number of spores;
3) Test results
See table 1 for the following: the non-pathogenic Fusarium oxysporum FJAT-9290 can be fermented and grown in culture medium containing grass juice, and the content of viable bacteria fermented in each treatment group is (4.33-7.23) × 10 7 cuf/ml, all compared to pure PDB medium control (4.28X 10) 7 cuf/ml), and the content of the pennisetum hydridum juice has no significant adverse effect on the growth of the bacteria, and can be used for fermentation of the bacteria.
Table 1: growth effects of Jujun grass juice on non-pathogenic Fusarium oxysporum
| Treatment group (Jujun juice content%) | Number of spores (cfu/ml) |
| Control group (0%) | (4.28±0.17)×10 7a |
| Example 1 (20%) | (5.50±0.51)×10 7a |
| Example 2 (40%) | (5.08±0.45)×10 7a |
| Example 3 (60%) | (7.23±1.36)×10 7a |
| Example 4 (80%) | (6.53±1.84)×10 7a |
| Example 5 (100%) | (4.33±0.47)×10 7a |
The invention unexpectedly discovers that the content of the non-pathogenic fusarium oxysporum after fermentation is equivalent to or slightly superior to the fermentation effect of the common PDB culture medium by taking the megaterium juice as the culture medium or taking the mixed solution of the megaterium juice and the PDB culture medium in any proportion as the culture medium.
It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. Not all embodiments are exhaustive. All obvious changes and modifications which are obvious to the technical scheme of the invention are covered by the protection scope of the invention.
Claims (7)
1. The application of Jujun grass juice as a non-pathogenic fusarium oxysporum fermentation culture medium.
2. Use according to claim 1, characterized in that: the nonpathogenic fusarium oxysporum is fusarium oxysporum FJAT-9290.
3. The application according to claim 1, characterized in that the method steps of the application are as follows:
s1, preparation of liquid strains for production:
preparing a liquid strain for production by taking fusarium oxysporum FJAT-9290 as a strain and a potato glucose culture medium PDB as a culture medium for the strain;
s2, preparation of a fusarium oxysporum plant vaccine preparation:
and (2) taking the megaterium juice as a liquid culture medium, sterilizing, inoculating the liquid strain prepared in the step (S1), and performing shaking culture at 26-30 ℃ and 160-200r/min for 6-8d to obtain the non-pathogenic fusarium oxysporum plant vaccine preparation.
4. Use according to claim 3, characterized in that: in step S1, the preparation of the liquid strain for production comprises the following specific steps:
s11, activation of strains:
preparing PDA culture medium, sterilizing at 121 deg.C for 20min, cooling to 45 deg.C, and placing in a sterilizing plate; taking a nonpathogenic fusarium oxysporum FJAT-9290 strain stored at-70 ℃ to perform streak culture on a PDB culture medium plate, and performing constant-temperature culture at 28-30 ℃ for 5-7 days to obtain hyphae;
s12, preparing liquid strain for production
Preparing a PDB liquid culture medium, selecting the hyphae in the step S11, inoculating into the PDB liquid culture medium, and carrying out shake culture at the temperature of 28-30 ℃ and at the speed of 170-200r/min for 5-7 days to obtain the liquid strain for production.
5. Use according to claim 4, characterized in that: in step S11, the PDA medium includes: potato extract 5g/L, peptone 10.0g/L, glucose 15.0g/L, sodium chloride 5.0g/L, agar 20g/L, and pH =7.0.
6. Use according to claim 4, characterized in that: in step S12, the PDB liquid medium includes: potato extract 5g/L, peptone 10.0g/L, glucose 15.0g/L, sodium chloride 5.0g/L, and pH =7.0.
7. Use according to claim 3, characterized in that: in step S2, the preparation of the fusarium oxysporum plant vaccine preparation comprises the following specific steps:
s21, squeezing fresh giant napier grass serving as a material by using a juicer to obtain juice, and filtering waste residues to obtain giant napier grass juice;
s22, taking pure megaterium juice as a liquid culture medium or a mixture of the megaterium juice and a PDB liquid culture medium as a culture medium, adjusting the pH value to 7.0, and sterilizing at 121 ℃ for 20min;
s23, inoculating the liquid strain for production obtained in the step S1 into the sterilized liquid culture medium according to the inoculation amount of 1%, and performing shaking culture at the constant temperature of 30 ℃ for 30h at the speed of 180r/min to obtain the fusarium oxysporum plant vaccine preparation.
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