CN112239736A - Bacillus amyloliquefaciens, microbial inoculum, screening method and application - Google Patents
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Abstract
The invention relates to a Bacillus amyloliquefaciens strain, which is named as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) A23 and is preserved in China center for type culture collection with the preservation number of CCTCC NO: M2020381. The invention also discloses a microbial inoculum prepared from the strain, a screening method and application. The strain is separated from the intestinal tract of the procambarus clarkii, can improve the intestinal flora of the procambarus clarkii and can obviously improve the disease resistance of the procambarus clarkii to WSSV. The strain has the advantages of safety, high temperature resistance, acid resistance and convenient storage. The strain has broad-spectrum antibacterial activity, and has antagonistic effect on various aquatic common pathogenic bacteria.
Description
Technical Field
The invention relates to the technical field of strain cultivation, in particular to bacillus amyloliquefaciens, a microbial inoculum, a screening method and application.
Background
White spot syndrome virus (wssv) is a pathogen that causes White Spot Syndrome (WSS). Under natural conditions, WSSV breaks through the first defense line of the prawn, enters the prawn body and is usually in a latent state, and the behavior and the body surface characteristics of the prawn carried by the virus are normal; under the coordination of certain environmental factors, the virus in a latent state can be activated to cause diseases of prawns, and particularly, the breeding of procambarus clarkia is seriously damaged. The prawn culture pond is a relatively independent ecological system for high-density stocking, the biological species are limited, the complexity of a food net is low, the self-withering energy-saving capability is low, and the stability is poor; in addition, many ecological environmental stressors (stressors) can induce the development of viral diseases.
In the actual production, the ecological environment can be improved and optimized by adopting physical, chemical and biological methods, a multi-colony biological community which is beneficial to keeping ecological stability is constructed, the ecological niche of the shrimp pond is utilized in a three-dimensional manner, and the health level of the prawns and the capability of resisting diseases are improved. However, the existing various methods for improving the disease resistance of prawns are limited in the effect of improving the self resistance of prawns and poor in regulating capability.
Disclosure of Invention
In view of the above, the invention provides a Bacillus amyloliquefaciens strain, wherein the Bacillus amyloliquefaciens strain is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) A23 which is preserved in China center for type culture collection with the preservation number of CCTCC NO: M2020381.
Specifically, the 16S rDNA sequence of the bacillus amyloliquefaciens A23 is shown as SEQ ID No. 1.
The invention also provides a screening method of the bacillus amyloliquefaciens, which comprises the following steps:
s1, separating and purifying the procambarus clarkii intestinal tract to obtain bacillus;
s2, screening the bacillus obtained in the step S1 through pathogen antagonistic screening to screen out a strain with a bacteriostatic action on the indicator bacterium;
s3, screening the strain obtained in the step S2 through extracellular amylase to obtain a strain which simultaneously produces the extracellular amylase and the protease, namely the bacillus amyloliquefaciens A23.
Specifically, the indicator bacteria comprise aeromonas hydrophila, edwardsiella catarrhalis, vibrio alginolyticus, vibrio harveyi and vibrio parahaemolyticus.
Specifically, the S2 specifically includes the following steps:
s21, sucking the indicator bacterium liquid, adding the indicator bacterium liquid into the unset BHI culture medium, uniformly mixing, and pouring the mixture into a flat plate for solidification;
s22, selecting the strains screened in the step S1, activating for 24 hours, dibbling the strains on a flat plate, culturing for 24 hours at 28 ℃, and selecting the colony with the largest inhibition zone for further screening.
Specifically, the S3 specifically includes the following steps:
s31, inoculating the strain obtained in the step S2 on a protease-producing culture medium, culturing at 37 ℃, selecting a single colony capable of producing a hydrolysis loop, measuring the diameter of the hydrolysis loop and the diameter of the colony, and selecting the strain with the largest difference between the diameter of the hydrolysis loop and the diameter of the colony for further screening;
s32, inoculating the strain obtained in S31 on an amylase-producing medium, and culturing at 37 ℃. And selecting a single bacterial colony capable of generating a hydrolysis ring, dyeing, measuring the diameter of the hydrolysis ring and the diameter of the bacterial colony, and selecting a bacterial strain with the largest difference between the diameter of the hydrolysis ring and the diameter of the bacterial colony as the bacillus amyloliquefaciens A23.
The invention also provides a bacillus amyloliquefaciens microbial inoculum which is prepared by activating, fermenting and culturing the bacillus amyloliquefaciens strain.
The invention also provides application of the bacillus amyloliquefaciens and the microbial inoculum thereof in the cultivation of procambarus clarkii.
The invention has at least the following beneficial effects:
the bacillus amyloliquefaciens strain is separated from the intestinal tract of the procambarus clarkii, can improve the intestinal flora of the procambarus clarkii and can obviously improve the disease resistance of the procambarus clarkii to WSSV. The strain has the advantages of safety, high temperature resistance, acid resistance and convenient storage. The strain has broad-spectrum antibacterial activity, and has antagonistic effect on various aquatic common pathogenic bacteria.
Drawings
FIG. 1 is a graph showing the antagonistic effect of Bacillus amyloliquefaciens A23 on pathogenic bacteria.
FIG. 2 is a graph showing the effect of the extracellular enzyme-producing plate of Bacillus amyloliquefaciens A23 according to the present invention.
FIG. 3 is a phylogenetic diagram of Bacillus amyloliquefaciens A23 according to an embodiment of the present invention.
FIG. 4 is a graph showing the survival effect of Bacillus amyloliquefaciens A23 on the white spot syndrome virus of procambarus clarkii.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Screening of Bacillus amyloliquefaciens A23
In one embodiment of the invention, the Bacillus amyloliquefaciens is named as Bacillus amyloliquefaciens A23 and is preserved in Wuhan Chinese type culture collection center in 7-30 months in 2020, with the preservation number of CCTCC NO: M2020381 and the preservation address of No. 299 in eight paths in Wuchang region in Wuhan city, Hubei province. The 16S rDNA sequence of the bacillus amyloliquefaciens A23 is shown as SEQ ID No. 1.
1. Screening of relevant materials
Screening sources: the healthy procambarus clarkii is selected from ponds of professional cooperation society for feeding Wuhan Wowo soil wish shrimps in ChuanDian district, Wuhan City, Hubei province and aquiculture bases of Huazhong agricultural university, the weight of the pond is 25-30 g, the pond is used as an experimental shrimp, and intestinal contents are used as screening samples.
And (3) indication bacteria: aeromonas hydrophila (A. hydrophila), Edwardsiella catarrhalis (E.ictaluri), Vibrio alginolyticus (V.alginoticus), Vibrio harveyi (V.harveyi) and Vibrio parahaemolyticus (V.parahaemolyticus)
2. Screening method
S1, separation and purification of bacillus
Adding 0.1g of screening sample into 1mL of sterile PBS, treating for 20min in a water bath at 80 ℃ for pasteurization, inoculating into an LB culture medium according to the inoculum size of 3%, and performing shake culture at 37 ℃ and 180r/min for 24 h. The suspension was diluted in gradients, 0.1mL of each gradient was spread evenly on sterile solid LB plates and incubated in an incubator at 37 ℃ for 24 h. And (5) randomly selecting colonies after the colonies grow out, purifying and numbering.
S2 screening pathogenic antagonistic bacteria
S21, adjusting the concentration of the indicator bacterium liquid to 10 by adopting a double-layer agar dibbling method7CFU/mL, sucking 0.1mL, adding into a slightly-heated (about 40 ℃) 3.9mL BHI agar culture medium, inverting up and down, mixing uniformly, pouring into a BHI flat plate immediately after mixing uniformly, and waiting for solidification;
s22, adjusting the concentration to 10 after the candidate strain is activated for 24 hours7CFU/mL suspension, 2. mu.L of the suspension was spotted on the plate; and then putting the plate into a constant temperature box at 28 ℃ for culturing for 24h, measuring the size of the inhibition zone, and selecting the colony with the largest inhibition zone for further screening.
S3 screening of extracellular enzyme-producing strains
S31, inoculating the strain obtained in S2 into an LB liquid culture medium, culturing at 37 ℃ and 180r/min for 24 hours, inoculating a bacterial liquid on a protease-producing culture medium, culturing at 37 ℃ for 48 hours, selecting a single bacterial colony capable of producing a hydrolysis ring, taking out a flat plate, placing the flat plate into a sterile operating platform, measuring the diameter (D) of the hydrolysis ring and the diameter (D) of the bacterial colony, and selecting the strain with the largest difference between the diameter of the hydrolysis ring and the diameter of the bacterial colony as a strain for re-screening;
s32, culturing the strain obtained in S31 at 37 ℃ and 180r/min for 24h, inoculating the bacterial liquid on an amylase production culture medium, and culturing at 37 ℃ for 48 h. And (3) selecting a single bacterial colony capable of generating a hydrolysis ring, taking out the flat plate, putting the flat plate into a sterile operating platform, measuring the diameter (H) of the hydrolysis ring and the diameter (C) of the bacterial colony by a Lugol iodine solution staining method, and selecting a bacterial strain with the largest difference between the diameter of the hydrolysis ring and the diameter of the bacterial colony, namely the bacillus amyloliquefaciens A23 obtained by final screening. The results are shown in table 1 and fig. 2.
TABLE 1 extracellular enzyme-producing ability of Strain A23
Culture medium for culturing the strain obtained in S2:
protease-producing medium (400 mL): weighing 4.0g of casein, 0.8g of dipotassium phosphate, 1.2g of beef extract, 2.0g of sodium chloride, 6.0g of agar, 400mL of distilled water, 2mL of 1% bromothymol blue solution, pH7.2, and sterilizing at 121 ℃ for 20 min.
Amylase-producing medium (400 mL): weighing peptone 2.0g, yeast extract 4.0g, NaCl2.0g, soluble starch 4.0g, agar 6.0g, distilled water 400mL, pH5.6, sterilizing at 121 deg.C for 20 min.
The embodiment of the invention provides understanding of bacillus amyloliquefaciens a23 antagonistic bacteria, please refer to fig. 1. As can be seen from the figure 1, when pathogenic bacteria all grow on a vessel, an obvious inhibition zone appears on a culture medium inoculated with the bacillus amyloliquefaciens A23 isolate, the diameter of the inhibition zone is more than 2.0cm, and the appearance of the inhibition zone can show that the bacillus amyloliquefaciens A23 provided by the embodiment of the invention has an obvious inhibition effect on the pathogenic bacteria, so that the bacillus amyloliquefaciens A23 can be well applied to the aquaculture for preventing and treating microbial diseases.
The preparation method of the fermentation liquor of the bacillus amyloliquefaciens A23 provided by the embodiment of the invention comprises the following steps: inoculating the bacillus amyloliquefaciens A23 in a liquid fermentation culture medium, carrying out fermentation culture at 25-30 ℃, and shaking in a shaking table for 2-4 days to obtain a fermentation liquid of the bacillus amyloliquefaciens A23.
Wherein the shaking table is oscillated at a rotating speed of 180 r/min.
The liquid fermentation medium comprises the following components in concentration: 10g/L of tryptone, 5g/L of yeast extract, 20g/L of sucrose and 5g/L of NaCl. The pH value of the liquid fermentation medium during preparation is 7.2-7.4, and the liquid fermentation medium is sterilized for 20min at the temperature of 121 ℃.
Identification of strains
1. Physiological and biochemical identification
According to Bergey's Manual of bacteria identification (ninth edition), A23 was identified based on morphological, physiological and biochemical characteristics. The results are shown in Table 2.
TABLE 2
2. 16S rDNA sequence analysis
Extracting DNA of A23 with kit, and using primer
27F：5′-AGAGTTTGATCCTGGCTCAG-3′，
1492R: 5 '-TACGGYTACCTTGTTACGACTT-3' to carry out PCR amplification, wherein the PCR reaction system comprises: 2 XTaq PCR Master Mix 12.5. mu.L, primers 27F and 1492R each 1. mu.L, template DNA 1. mu.L, and finally 25. mu.L was supplemented with sterile double distilled water. The reaction procedure is as follows: 5min at 94 ℃; at 94 ℃ for 40s, at 55 ℃ for 40s, and at 72 ℃ for 2.5min, for 35 cycles; finally, the reaction is finished at 72 ℃ for 10min and 4 ℃. And (3) carrying out 1% (w/v) agarose gel electrophoresis on the PCR amplification product, cutting and recovering the product, and then sending the product to Shanghai biological engineering technical service company Limited for sequencing. The 16S rDNA sequence of the bacillus amyloliquefaciens A23 is shown IN SEQ IN No. 1. The sequences were Blast aligned on Genbank and strains with high similarity to the aligned sequences were selected. And a phylogenetic tree was constructed using MEGA6.06 software as shown in FIG. 3.
The results of physiological and biochemical analysis and 16S rDNA identification show that the strain is highly homologous with bacillus amyloliquefaciens (B. amyloliquefaciens), and is determined and named as bacillus amyloliquefaciens A23.
The bacillus amyloliquefaciens A23 and the fermentation liquid thereof provided by the embodiment of the invention can be used for preventing common diseases such as fish, shrimp or crab caused by common pathogenic bacteria of aquatic products, wherein the pathogenic bacteria comprise aeromonas hydrophila (A. hydrophila), edwardsiella catarrhalis (E.ictaluri), vibrio alginolyticus (V.alginoticus), vibrio harveyi (V.harveyi) and vibrio parahaemolyticus (V.parahaemolyticus).
Meanwhile, the bacillus amyloliquefaciens A23 provided by the embodiment of the invention has stronger capability of producing amylase and protease extracellularly. When the bacillus amyloliquefaciens A23 provided by the embodiment of the invention is fed with bait, the activity of intestinal digestive enzymes of procambarus clarkii can be obviously improved, the intestinal flora is improved, and the disease resistance to White Spot Syndrome Virus (WSSV) of the procambarus clarkii is improved. The result of the invention shows that the strain has broad-spectrum bacteriostatic ability, provides good biocontrol resources for preventing and treating diseases in the procambarus clarkia cultivation, and lays a foundation for the application of probiotics of the procambarus clarkia.
3. High temperature resistance
The activated bacterial suspension (72 h of culture) was adjusted to a concentration of about 1X 106CFU/mL, firstly sucking diluted 0.1mL bacterial liquid plate, coating, culturing and counting as a control group; the bacterial solutions were then boiled in water (100 ℃) for 5 and 15min, and then cooled and diluted at 90 ℃ for 5 and 15min, and the plate counts were performed, and the survival rates of the bacteria in the test groups (survival rate (%): bacterial solution concentration after high-temperature treatment/initial bacterial solution concentration × 100%) were calculated, respectively, as compared with the control group. The results are shown in table 3, which indicates that the survival rate of the A23 strain provided by the invention reaches more than 90% in 5min at 90 ℃, the survival rate of the A23 strain in 15min is close to 80%, and the A23 strain has good high temperature resistance.
TABLE 3 survival (%) -of A23 treated at different bath temperatures and times
4. Low pH tolerance
Activating probiotic bacteria, and adjusting bacteria concentration to 1 × 108And (3) inoculating the CFU/mL into BHI culture media with pH values of 2.0, 3.0, 4.0 and 5.0 according to the inoculation amount of 5%, performing shake culture at 37 ℃ at 200r/min, taking bacterial liquid after 1h and 2h, performing plate coating counting, and calculating the survival rate. Table 4 shows that the survival rate of the A23 strain provided by the invention within 1h under the condition of pH3.0 is close to 70%, which has excellent low pH tolerance.
TABLE 4 survival rate (%) after 1 hour treatment under various acidic conditions
Applications of
The embodiment of the invention researches the fungus diseases of bacillus amyloliquefaciens A23 and the fermentation liquid thereof, and the research contents are as follows.
1. Preparation of bacillus amyloliquefaciens A23 bacterial powder
Bacillus amyloliquefaciens a23 was inoculated from a slant into the liquid medium provided in the above example, the liquid fermentation medium comprising the following components in concentrations: 10g/L of tryptone, 5g/L of yeast extract, 20g/L of sucrose and 5g/L of NaCl. The pH value of the liquid fermentation medium during preparation is 7.2-7.4, and the liquid fermentation medium is sterilized for 20min at the temperature of 121 ℃.
Fermenting to final concentration of 1010And after CFU/mL, adding a protective agent and the like, and freeze-drying to prepare bacterial powder for later use.
2. Design of specific experiment
The experiments were divided into 3 groups:
healthy procambarus clarkii was divided into CON group, a23-1 group and a23-2 group, and adult shrimps (about 20g) were fed 4w, each group was set in triplicate, each of 30 replicates. The correlation index was determined at the 28 th sample.
And (2) CON group: adding sterile water to the feed; group A23-1: adding Bacillus amyloliquefaciens A23 powder into feed until the final concentration of bacterial liquid is 107CFU/g; group A23-2: adding Bacillus amyloliquefaciens A23 powder into feed until the final concentration of bacterial liquid is 108CFU/g。
2.1 digestive enzyme Activity
After feeding 4w, 6 adult procambarus clarkii are randomly selected from each group, and the water of the procambarus clarkii is absorbed by filter paper to take the whole intestines of the procambarus clarkii. Weighing intestinal tract, placing in a glass homogenizer, taking 9 times of precooled homogenate medium (pH 7.4, 0.01mol/L Tris-HCl, 0.0001mol/L EDTA-2Na, 0.01mol/L sucrose and 0.8% sodium chloride solution) of a tissue block by using a pipette, fully homogenizing, centrifuging for 10min at 2000r/min by using a low-temperature low-speed centrifuge, taking supernatant fluid which is crude enzyme solution, and storing at-80 ℃ to be detected.
The activities of trypsin, amylase and lipase were all determined using a kit (Nanjing institute of bioengineering). The OD value was read with an ultraviolet spectrophotometer in a water bath at 37 ℃. OD was read in the trypsin reaction at 253 nm. The trypsin activity is defined as the change in absorbance per minute of trypsin contained in a protein of 0.003 per mg at 37 ℃ as one unit of enzyme activity (U). The OD value in the amylase reaction system was read at a wavelength of 660 nm. Amylase activity is defined as: at 37 deg.C, 10mg of starch was hydrolyzed as one amylase activity unit (U) by allowing the substrate to react with each mg of tissue protein for 30 min. And reading the OD value in the lipase reaction system at the wavelength of 420 nm. Lipase activity is defined as: at 37 ℃, the substrate acts for 1min per mg of tissue protein, and one enzyme activity unit (U) is consumed per 1 mu mol of substrate.
2.2 Effect on serum non-specific immune enzymes
After the 4w feeding is finished, 6 adult procambarus clarkii are randomly selected from each group, the water of the bodies of the procambarus clarkii is sucked dry by using filter paper, and the hemolymph, the hepatopancreata and the whole intestine of the procambarus clarkii are taken. After the hemolymph is static overnight, the hemolymph is centrifuged at 800g for 10min at 4 ℃, and the supernatant is taken and stored at-80 ℃ for testing.
Serum superoxide dismutase, phenol oxidase, acid phosphatase, alkaline phosphatase, lysozyme and catalase were all measured using a kit (Nanjing institute of bioengineering).
2.3 disease resistance to WSSV
After the 4w feeding is finished, a white spot syndrome virus is used for carrying out a virus challenge experiment. Each treatment group had 30 tail crayfish procambarus clarkii. Before challenge experiments, the semi-lethal concentration of white spot syndrome virus on procambarus clarkii was first determined by 8d preliminary experiments (LD50, 8 d). The pathogen was injected into the 1, 2 abdominal muscle of procambarus clarkii using a syringe. The survival rate was counted at 0, 12, 24, 48, 72, 96h after injection of white spot syndrome virus. Here, the survival rate (%) ═ number of surviving shrimps/number of infected shrimps × 100%. The results are shown in table 5 and fig. 4.
TABLE 5 relevant Biochemical indicators of Procambrus clarkii after feeding with probiotic feed
From the results, it can be seen that:
1. amylase, trypsin and lipase from groups a23-1 and a23-2, respectively, were significantly elevated relative to CON group and increased significantly with increasing concentrations of a23 feed. The A23 bacteria provided by the invention can promote the secretion of digestive enzymes in the intestinal tract and enhance the digestive function of the intestinal tract, thereby improving the microbial environment of the intestinal tract and being beneficial to the absorption of nutrition in the intestinal tract.
2. The levels of SOD, LSZ and ACP in each of groups a23-1 and a23-2 were significantly elevated relative to CON and increased significantly with increasing concentrations of a23 feed. It is known that non-specific immune enzymes in bloodlymph such as superoxide dismutase (SOD), Lysozyme (LSZ) and acid phosphatase (ACP) are all important immune indexes, and unlike mammals, procambarus clarkia does not have an acquired immune system, and the non-specific immunity is mainly relied on to enhance the resistance to diseases and maintain the normal physiological state of the body. LSZ is the material basis for phagocyte sterilization and can hydrolyze the cell wall of gram-positive bacteria, leading to cell rupture and death. SOD can remove excessive oxygen free radicals in vivo and prevent biomolecule damage. ACP is a marker enzyme of macrophage lysosome, has close relation with organism substance metabolism, plays an important role in immune response and can be induced by exogenous substances; an increase in ACP activity corresponds to an increase in anabolism of the substance to protect against oxidative damage. This indicates that the A23 strain provided by the invention can enhance the immunoregulation function of the procambarus clarkia and enhance the resistance of the procambarus clarkia to attack of foreign pathogenic microorganisms.
In addition, as can be seen from fig. 4, the survival rates of procambarus clarkia in the 8 th days after the challenge and 1-8 th days after the challenge of the groups a23-1 and a23-2 are both significantly higher than those of the CON group, which indicates that the bacillus amyloliquefaciens provided by the invention has the effect of resisting white spot syndrome virus, has a protective effect on the intestinal tract of procambarus clarkia and can significantly improve the WSSV disease resistance of the procambarus clarkia.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.
Claims (8)
1. The Bacillus amyloliquefaciens is characterized in that the Bacillus amyloliquefaciens strain is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) A23, is preserved in China Center for Type Culture Collection (CCTCC) NO: M2020381.
2. The bacillus amyloliquefaciens according to claim 1, wherein the 16S rDNA sequence of the bacillus amyloliquefaciens a23 is shown as SEQ ID No. 1.
3. The method for screening bacillus amyloliquefaciens according to claim 1 or 2, comprising the steps of:
s1, separating and purifying the procambarus clarkii intestinal tract to obtain bacillus;
s2, screening the bacillus obtained in the step S1 through pathogen antagonistic screening to screen out a strain with a bacteriostatic action on the indicator bacterium;
s3, screening the strain obtained in the step S2 through extracellular amylase to obtain a strain which simultaneously produces the extracellular amylase and the protease, namely the bacillus amyloliquefaciens A23.
4. The screening method according to claim 3, wherein the indicator bacteria include Aeromonas hydrophila, Edwardsiella ictarda, Vibrio alginolyticus, Vibrio harveyi and Vibrio parahaemolyticus.
5. The screening method according to claim 3, wherein the S2 specifically comprises the steps of:
s21, sucking the indicator bacterium liquid, adding the indicator bacterium liquid into the unset BHI culture medium, uniformly mixing, and pouring the mixture into a flat plate for solidification;
s22, selecting the strains screened in the step S1, activating for 24 hours, dibbling the strains on a flat plate, culturing for 24 hours at 28 ℃, and selecting the colony with the largest inhibition zone for further screening.
6. The screening method according to claim 3, wherein the S3 specifically comprises the steps of:
s31, inoculating the strain obtained in the step S2 on a protease-producing culture medium, culturing at 37 ℃, selecting a single colony capable of producing a hydrolysis loop, measuring the diameter of the hydrolysis loop and the diameter of the colony, and selecting the strain with the largest difference between the diameter of the hydrolysis loop and the diameter of the colony for further screening;
s32, inoculating the strain obtained in S31 on an amylase-producing medium, and culturing at 37 ℃. And selecting a single bacterial colony capable of generating a hydrolysis ring, dyeing, measuring the diameter of the hydrolysis ring and the diameter of the bacterial colony, and selecting a bacterial strain with the largest difference between the diameter of the hydrolysis ring and the diameter of the bacterial colony as the bacillus amyloliquefaciens A23.
7. A Bacillus amyloliquefaciens bacterial agent, which is prepared by activating, fermenting and culturing the Bacillus amyloliquefaciens strain of claim 1.
8. Use of the bacillus amyloliquefaciens according to claim 1 or 2 or the bacillus amyloliquefaciens microbial agent according to claim 7 for cultivating procambarus clarkia.
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