CN115247134A - Application of Jujun grass juice as fermentation medium of paecilomyces lilacinus for nematode biocontrol bacteria - Google Patents
Application of Jujun grass juice as fermentation medium of paecilomyces lilacinus for nematode biocontrol bacteria Download PDFInfo
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- 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
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Abstract
The invention provides application of Jujun grass juice as a fermentation culture medium of nematode biocontrol bacteria paecilomyces lilacinus. The method for applying comprises the following steps: s1, preparation of liquid strains for production: preparing a liquid strain for production by using Paecilomyces lilacinus FJAT-9041 as a strain and a potato glucose PDB culture medium as a culture medium for the strain; s2, preparing a paecilomyces lilacinus preparation for nematode biocontrol bacteria: taking the pennisetum hydridum juice as a liquid culture medium, sterilizing, inoculating the liquid strain prepared by the S1, and performing shaking culture for 6-8 days at the temperature of 26-30 ℃ and at the speed of 160-200r/min to obtain a liquid fermentation product of the nematode biocontrol bacterium paecilomyces lilacinus. The application method of the invention takes the pennisetum hydridum juice as the culture medium, and the number of the cultured paecilomyces lilacinus spores can reach (7.15 +/-0.84) multiplied by 10 7 cfu/ml. The invention has simple application method, low cost and fermentationThe content of the obtained paecilomyces lilacinus for the nematode biocontrol bacteria is obviously higher than the fermentation effect of a PDB culture medium commonly used by fungi.
Description
Technical Field
The invention relates to the technical field of bacterial culture media, in particular to application of Jujun grass juice as a paecilomyces lilacinus fermentation culture medium for nematode biocontrol bacteria.
Background
Paecilomyces lilacinus (thorn.) Samson is a known soil filamentous fungus with extensive activity against plant parasitic nematodes (kutscher a et al, 2012 mani et al, 2013, khan et al, 2014, larexa et al, 2016), potentially parasitic fungi to nematode eggs, larvae and females, thereby reducing the plant nematode population in the soil. Strains of this fungus have been used by many countries to control nematodes (Larec et al, 2009, yu et al, 2015, sharma et al, 2014. Paecilomyces lilacinus FJAT-9041 is a natural antagonistic bacterium and is publicly sold in the market (such as Anhui Hede agricultural biotechnology, inc., shandong Changtai biotechnology, inc., and the like), and early researches show that the strain has high poisoning effect on the sweet potato stem nematode (Ditylenchus destructor) and has a corrected lethality rate of 57.1% (Li Fang, and the like, 2005. Toxicity of the Paecilomyces lilacinus to the sweet potato stem nematode. Chinese agronomy report, 21 (3): 255-258'); research also proves that the bacillus has strong bacteriostatic action on fusarium oxysporum which is pathogenic bacterium of fusarium wilt, the produced beta-glucosidase can digest the cell wall of the pathogenic bacterium, and produce polysaccharide to inhibit the growth and reproduction of fusarium oxysporum, and the inhibition rate on the generation of fusarium oxysporum spores is more than 95% (Li Fang and the like, 2005. The antagonistic action and mechanism of paecilomyces lilacinus on fusarium oxysporum are divided intoAnalytical plant protection journal, 12.32 (4): 373-378); the pesticide residue can be degraded while preventing and treating the nematode and wilt of crops, the degradation effect on phoxim pesticide is obvious, the degradation rate of phoxim can reach more than 98 percent after the mixed culture of the bactericides for 5 days (Li Fang and the like, 2006. Degradation effect of paecilomyces lilacinus on phoxim. Application and environmental biology report, 12 (01): 104-107.). In view of this, the biological culture of Paecilomyces lilacinus is of great significance. At present, the common PDB culture medium is mainly used for culturing the paecilomyces lilacinus. However, the paecilomyces lilacinus cultured in the medium can only reach 1.00X 10 4 5363 and about cuf/ml, and the cost is high.
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. Currently, the Jujun grass is mainly used in the traditional fields of feed, biogas fermentation production, combustion power generation, biomass ethanol production and the like. In addition, jujun grass has been reported as a medium for producing wood-rotting fungi such as shiitake mushroom, for example: chinese patent application CN 108633622A discloses a method for producing mushroom by taking pennisetum giganteum as a culture medium, wherein the culture medium comprises the following components in percentage by weight: 54.6% of broad-leaf wood chips, 23.4% of Jujun grass, 20% of wheat bran, 1% of gypsum, 1% of sugar and 55-60% of water content. The invention takes Jujun grass and broad-leaved wood dust as mushroom culture medium.
At present, reports of using the pennisetum hydridum juice as a culture medium of paecilomyces lilacinus are not found, and the pennisetum hydridum culture medium has important significance for high-added-value utilization of pennisetum hydridum.
Disclosure of Invention
The invention aims to solve the technical problem of providing an application of Jujun grass juice as a fermentation culture medium of nematode biocontrol bacteria paecilomyces lilacinus. The application method uses Jujun grass juice as culture medium, and can make cultured Paecilomyces lilacinus spore number ≧ 7.15 + -0.84 x 10 7 cfu/ml. The application method is simple and low in cost, and the content of the nematode biocontrol bacteria paecilomyces lilacinus obtained after fermentation is obviously higher than the fermentation effect of the PDB culture medium commonly used by fungi.
In order to solve the technical problem, the invention adopts the following technical scheme:
the application of the pennisetum hydridum juice as a fermentation culture medium of the nematode biocontrol bacterium paecilomyces lilacinus.
In one embodiment, the Paecilomyces lilacinus is Paecilomyces lilacinus FJAT-9041.
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 using Paecilomyces lilacinus (Thorn.) Samson) FJAT-9041 as a strain and a potato dextrose PDB (dextrose) culture medium as a culture medium for the strain;
s2, preparing a paecilomyces lilacinus preparation for nematode biocontrol bacteria:
taking the pennisetum hydridum juice as a liquid culture medium, sterilizing, inoculating the liquid strain prepared by the S1, and performing shaking culture for 6-8 days at the temperature of 26-30 ℃ and at the speed of 160-200r/min to obtain a liquid fermentation product of the nematode biocontrol bacterium paecilomyces lilacinus.
As a preferred embodiment, in step S1, the specific steps for preparing the liquid seed culture for production are as follows:
s11, activation of strains:
preparing potato glucose agar PDA culture medium, sterilizing at 121 deg.C for 20min, cooling to 45 deg.C, and placing into a sterilized plate; taking Paecilomyces lilacinus FJAT-9041 strain stored at-70 ℃ to perform streak culture on a PDA culture medium plate, and culturing at the constant temperature of 28-30 ℃ for 6-8 days until Paecilomyces lilacinus FJAT-9041 hyphae appears in the culture medium;
s12, preparing liquid strain for production
Preparing a potato glucose PDB culture medium, selecting the hyphae in the step S11, inoculating into a PDB culture solution medium, and performing shake culture at the temperature of 28-30 ℃ and the rpm of 170-200 for 5-7 days to obtain the liquid strain for production.
As an embodiment, in step S11, the potato dextrose agar PDA medium includes: potato extract 5g/L, peptone 10g/L, glucose 15g/L, sodium chloride 5g/L, agar 5g/L, and pH =7.0.
As an embodiment, in step S12, the potato dextrose PDB medium includes: potato extract 5g/L, peptone 10g/L, glucose 15g/L, sodium chloride 5g/L, and pH =7.0.
In a preferred embodiment, in step S2, the preparation of the paecilomyces lilacinus preparation as the nematode biocontrol bacterium 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 grass juice as a liquid 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 S12 into the sterilized liquid culture medium according to the inoculation amount of 1%, and carrying out shaking culture at the constant temperature of 28-30 ℃ and 180r/min for 7d, namely obtaining a liquid fermentation product of the nematode biocontrol bacterium paecilomyces lilacinus.
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 can be obtained commercially, unless otherwise specified, and the equipment used in the present invention can be carried out by conventional equipment in the art or by referring to the prior art in the art.
The invention has the advantages of
The application method of the invention uses the megaterium juice as the culture medium, and the number of the directly cultured paecilomyces lilacinus spores is ≧ (7.15 +/-0.84) x 10 7 cfu/ml. The application method is simple and low in cost, and the content of the nematode biocontrol bacterium paecilomyces lilacinus obtained after fermentation is obviously higher than the fermentation effect of a PDB culture medium commonly used by fungi.
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 Jujun grass juice is used as a fermentation culture medium of the nematode biocontrol bacterium paecilomyces lilacinus.
As a preferred example, the Paecilomyces lilacinus is Paecilomyces lilacinus FJAT-9041.
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 using Paecilomyces lilacinus (Thorn.) Samson) FJAT-9041 as a strain and a potato dextrose PDB (dextrose) culture medium as a culture medium for the strain;
s2, preparing a paecilomyces lilacinus preparation for nematode biocontrol bacteria:
taking the pennisetum hydridum juice as a liquid culture medium, sterilizing, inoculating the liquid strain prepared by the S1, and performing shaking culture for 6-8 days at the temperature of 26-30 ℃ and at the speed of 160-200r/min to obtain a liquid fermentation product of the nematode biocontrol bacterium paecilomyces lilacinus.
As a preferred embodiment, in step S1, the specific steps for preparing the liquid strain for production are as follows:
s11, activation of strains:
preparing potato glucose agar PDA culture medium, sterilizing at 121 deg.C for 20min, cooling to 45 deg.C, and placing into a sterilized plate; taking Paecilomyces lilacinus FJAT-9041 strain stored at-70 ℃ to perform streak culture on a PDA culture medium plate, and culturing at the constant temperature of 28-30 ℃ for 6-8 days until Paecilomyces lilacinus FJAT-9041 hyphae appears in the culture medium;
s12, preparing liquid strain for production
Preparing a potato glucose PDB culture medium, selecting the hyphae in the step S11, inoculating into a PDB culture solution medium, and performing shake culture at the temperature of 28-30 ℃ and the rpm of 170-200 for 5-7 days to obtain the liquid strain for production.
As an example, in step S11, the potato dextrose agar PDA medium includes: potato extract 5g/L, peptone 10g/L, glucose 15g/L, sodium chloride 5g/L, agar 5g/L, and pH =7.0.
As an example, in step S12, the potato dextrose PDB medium includes: potato extract 5g/L, peptone 10g/L, glucose 15g/L, sodium chloride 5g/L, and pH =7.0.
As a preferred embodiment, in step S2, the preparation of the paecilomyces lilacinus preparation as the nematode biocontrol bacterium 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 grass juice as a liquid 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 S12 into the sterilized liquid culture medium according to the inoculation amount of 1%, and carrying out shaking culture at the constant temperature of 28-30 ℃ and 180r/min for 7d, namely obtaining a liquid fermentation product of the nematode biocontrol bacterium paecilomyces lilacinus.
Example 1
The application of the pennisetum hydridum juice as the fermentation medium of the nematode biocontrol bacterium paecilomyces lilacinus comprises the following steps:
1) Preparation of test materials
Fungus grass juice: fresh Jujun grass juice is taken as a material, a juicer is adopted to squeeze, and then waste residue is filtered to obtain the Jujun grass juice;
the main strains are as follows: paecilomyces lilacinus FJAT-9041, preserved by strain stock of biological resources of agricultural academy of sciences of Fujian province;
PDB medium (g/L): 5g/L of potato extract powder, 10g/L of peptone, 15g/L of glucose, 5g/L of sodium chloride and pH7.0, and subpackaging in 250mL triangular flasks with 100mL of culture medium per flask; sterilizing at 121 deg.C under high pressure for 20min, and cooling;
2) Test method
2.1 Activation of paecilomyces lilacinus): preparing PDA culture medium, sterilizing at 121 deg.C for 20min, cooling to 45 deg.C, and placing in a sterilizing plate; taking Paecilomyces lilacinus FJAT-9041 strain stored at-70 ℃ to perform streak culture on a PDA culture medium plate, and performing constant-temperature culture at 28-30 ℃ for 5-7 days;
2.2 Preparation of Paecilomyces lilacinus seed solution: inoculating the activated hypha into PDB culture solution, and performing shake culture at 28-30 ℃ and 170-200rpm for 5-7 days to obtain liquid strain for production;
2.3 Preparation of liquid medium: adjusting the pH of 100% Jujun grass juice to be =7.0, subpackaging in 250mL triangular bottles, filling liquid in each triangular bottle to be 100mL, and sterilizing at 121 ℃ for 20min; 3 replicates per treatment;
2.4 Preparation of paecilomyces lilacinus nematode biocontrol agent: inoculating the prepared paecilomyces lilacinus seed liquid into a pennisetum liquid culture medium with the inoculation amount of 1%, and carrying out shaking culture at the constant temperature of 28-30 ℃ and 180r/min for 7d, thus obtaining the liquid fermentation product of the nematode biocontrol preparation. Inoculating in PDB liquid culture medium as control;
2.5 Detection of spore number of fermentation product: filtering the fermentation product with double-layer sterile gauze to obtain spore suspension, and filtering by 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10-6、10 -7 After serial dilution, the suspension was spread on PDB medium plates and cultured at 28 ℃ for 5 daysCounting the number of spores;
3) Test results
See table 1 for the following: the Paecilomyces lilacinus FJAT-9041 can ferment and grow in a culture medium containing grass juice, and the content of the viable bacteria fermented in the treatment group of the embodiment is (7.15 +/-0.84) multiplied by 10 7 cuf/ml, significantly higher than the pure PDB medium control, 100% pennisetum giganteum juice is suitable as the fermentation medium for Paecilomyces lilacinus FJAT-9041.
Comparative example 1
Example 1 was repeated with the only difference that:
in the step 2.3), PDB liquid culture medium is used as a culture medium instead of 100 percent of Jujun grass juice.
The number of spores tested in this comparative example is shown in Table 1.
Comparative example 2
Example 1 was repeated with the only difference that:
in the step 2.3), a mixed solution of a PDB liquid culture medium accounting for 80% by weight and megalophora juice accounting for 20% by weight is used as a culture medium.
The number of spores tested in this comparative example is shown in Table 1.
Comparative example 3
Example 1 was repeated with the only difference that:
in the step 2.3), a mixed solution of 60% by weight of PDB liquid culture medium and 40% by weight of pennisetum hydridum juice is used as a culture medium.
The number of spores tested in this comparative example is shown in Table 1.
Comparative example 4
Example 1 was repeated with the only difference that:
in the step 2.3), a mixed solution of 40% by weight of PDB liquid culture medium and 60% by weight of megalophora juice is used as a culture medium.
The number of spores tested in this comparative example is shown in Table 1.
Comparative example 5
Example 1 was repeated with the only difference that:
in the step 2.3), a mixed solution of 20% by weight of PDB liquid culture medium and 80% by weight of pennisetum hydridum juice is used as a culture medium.
The number of spores tested in this comparative example is shown in Table 1.
Table 1:
| treatment group | Number of spores (cfu/mL) |
| Comparative example 1 | (1.00±0.00)×10 4d |
| Comparative example 2 | (6.77±0.39)×10 6cd |
| Comparative example 3 | (2.45±0.35)×10 7bc |
| Comparative example 4 | (4.10±0.58)×10 7b |
| Comparative example 5 | (4.37±0.27)×10 7b |
| Example 1 | (7.15±0.84)×10 7a |
The inventionSurprisingly, it was found that the number of directly cultured Paecilomyces lilacinus spores can reach (7.15 +/-0.84) multiplied by 10 by using 100% pennisetum giganteum juice as a culture medium 7 cfu/mL. The application method is simple and low in cost, and the content of the nematode biocontrol bacteria paecilomyces lilacinus obtained after fermentation is obviously higher than the fermentation effect of the PDB culture medium commonly used by fungi
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. Obvious changes and modifications of the technical scheme of the invention are included in the protection scope of the invention.
Claims (7)
1. The application of the pennisetum hydridum juice as a fermentation culture medium of the nematode biocontrol bacterium paecilomyces lilacinus.
2. Use according to claim 1, characterized in that: the paecilomyces lilacinus is paecilomyces lilacinus FJAT-9041.
3. Use according to claim 1, characterized in that: the method for applying comprises the following steps:
s1, preparation of liquid strains for production:
preparing a liquid strain for production by using Paecilomyces lilacinus FJAT-9041 as a strain and a potato glucose PDB culture medium as a culture medium for the strain;
s2, preparing a paecilomyces lilacinus preparation for nematode biocontrol bacteria:
taking the pennisetum hydridum juice as a liquid culture medium, sterilizing, inoculating the liquid strain prepared by the S1, and performing shaking culture for 6-8 days at the temperature of 26-30 ℃ and at the speed of 160-200r/min to obtain a liquid fermentation product of the nematode biocontrol bacterium paecilomyces lilacinus.
4. Use according to claim 3, characterized in that: in step S1, the specific steps for preparing the liquid strain for production are as follows:
s11, activation of strains:
preparing potato glucose agar PDA culture medium, sterilizing at 121 deg.C for 20min, cooling to 45 deg.C, and placing into a sterilized plate; taking Paecilomyces lilacinus FJAT-9041 strain stored at-70 ℃ to perform streak culture on a PDA culture medium plate, and culturing at the constant temperature of 28-30 ℃ for 6-8 days until Paecilomyces lilacinus FJAT-9041 hyphae appears in the culture medium;
s12, preparing liquid strain for production
Preparing a potato glucose PDB culture medium, selecting the hyphae in the step S11, inoculating into a PDB culture solution medium, and performing shake culture at the temperature of 28-30 ℃ and the rpm of 170-200 for 5-7 days to obtain the liquid strain for production.
5. Use according to claim 4, characterized in that: in step S11, the potato dextrose agar PDA medium includes: potato extract 5g/L, peptone 10g/L, glucose 15g/L, sodium chloride 5g/L, agar 5g/L, and pH =7.0.
6. Use according to claim 4, characterized in that: in step S12, the potato dextrose PDB medium includes: potato extract 5g/L, peptone 10g/L, glucose 15g/L, sodium chloride 5g/L, and pH =7.0.
7. Use according to claim 1, characterized in that: in the step S2, the preparation of the nematode biocontrol bacterium paecilomyces lilacinus 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 grass juice as a liquid 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 S12 into the sterilized liquid culture medium according to the inoculation amount of 1%, and carrying out shaking culture at the constant temperature of 28-30 ℃ and 180r/min for 7d, namely obtaining a liquid fermentation product of the nematode biocontrol bacterium paecilomyces lilacinus.
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