CN115074286B - Bacillus pumilus for antagonizing tinea pedis pathogenic fungi and application thereof - Google Patents
Bacillus pumilus for antagonizing tinea pedis pathogenic fungi and application thereof Download PDFInfo
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Abstract
The invention discloses a strain X1 for antagonizing tinea pedis pathogenic fungi, belonging to bacillus pumilusBacillus pumilus) The preservation number is CGMCC NO.24605, and the strain X1 can simultaneously antagonize the strain K1 belonging to the alternaria alternate and trichophyton rubrum, reduce the use amount of antibiotics when antagonizing tinea pedis pathogenic fungi, and increase the cleanliness and the health level of feet.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to bacillus pumilus for antagonizing tinea pedis pathogenic fungi.
Background
Tinea pedis is a chronic skin disease with extremely high recurrence rate caused by fungal infection, and has extremely strong infectivity. Generally, the tinea pedis is treated by applying external medicines, but for intractable tinea pedis, oral medicines are also given, and the oral medicines possibly bring side effects such as liver toxicity to human bodies, and the external medicines are also carefully considered for pregnant women and children.
In recent years, research on tinea pedis is mostly focused on chemical drug development and clinical case statistics, and the occurrence, development and mechanism of tinea pedis are rarely researched from the microbiome level. Previous studies based on microbial culture did not fully demonstrate the microbiota associated with tinea pedis, which was considered only trichophyton rubrum and other cutaneous epidermophyton infections. However, its repeated infection process suggests that the microflora plays a role in the pathogenesis of tinea pedis. Current modulation of skin microorganisms generally includes the transplantation of antibiotics, probiotics and skin microbial complexes. The former has undoubted beneficial effects on eliminating pathogenic bacteria, but the long-term use of the former inevitably increases the probability of the body to generate dependence on the former, and even can cause drug resistance and drug resistance, which can lead the parasitic of pathogenic bacteria to be more rampant, and finally has no effect on the treatment and alleviation of diseases. And microbial agent therapy or alternative therapy using the relationship between microorganisms and hosts is becoming a hotspot in the field of microbial research.
Disclosure of Invention
The invention aims to provide bacillus pumilus X1 for antagonizing tinea pedis pathogenic fungi and application thereof, which can antagonize alternaria alternate and trichophyton rubrum simultaneously, reduce the use amount of antibiotics when antagonizing tinea pedis pathogenic fungi and increase the cleanliness and health level of feet.
The strain is a bacillus pumilus which is separated from foot samples and can antagonize tinea pedis pathogenic fungi, the bacillus pumilus grows well on YG and PDA culture mediums, and the bacillus pumilus is cultured for 24 hours to form 1-2mm, round, semitransparent, milky and smooth-edged bacterial colonies; the culture is continued for 48 hours, the colony is slightly enlarged and has the size of 2-3mm, and the colony is round, opaque and milky, the surface is wrinkled, and the edge is slightly irregular.
Genomic DNA (1440 bp) of Bacillus pumilus X1 was taken and sequenced. The obtained result is compared by BLAST to obtain the similarity of the strain X1 and the bacillus pumilus reaching 99.51 percent, and the strain is determined to be bacillus pumilus by combining the colony, the thallus characteristics and the molecular biology characteristicsBacillus pumilus) The gene sequence was submitted to GenBank under accession number MT798142. Combining morphological observation of bacterial colony and thallus, molecular biological identification and phylogenetic analysis to determine that the strain is bacillus pumilusBacillus pumilus) This was designated as X1.
The strain is preserved in China general microbiological culture Collection center (CGMCC) of China general microbiological culture Collection center (CGMCC) for 3 and 28 days of 2022. Address: beijing, chaoyang area, north Chen Xi Lu No. 1, 3, china academy of sciences microbiological institute, post code: 100101. the preservation number of the strain is CGMCC NO.24605.
The invention also discloses a method for culturing the bacillus pumilus X1, which comprises the steps of inoculating the strain X1 into a liquid culture medium and culturing at the temperature of 28 ℃ at 180 r/min.
The culture medium is YG culture medium (YCFA with glucose added as carbon source) with pH of 5.8-6.1, comprising: casein, yeast extract, cysteine, naHCO 3 、K 2 HPO 4 、KH 2 PO 4 、NaCl、MgSO 4 ·7H 2 O、CaCl 2 Glucose, folic acid, biotin, vitamin B12, potassium P-aminobenzoate (P-aminobenzoic), hemin, and vitamin B6.
Further, 10g casein, 2.5g yeast extract, 1g cysteine, 4g NaHCO are contained per liter of the culture medium 3 ，3g K 2 HPO 4 ，3g KH 2 PO 4 ，0.9g NaCl，1g MgSO 4 ·7H 2 O，1g CaCl 2 2.5g glucose, 10. Mu.L folic acid, 10. Mu.L biotin, 10. Mu.L vitamin B12, 10. Mu. L P-Potassium P-aminobenzoate (P-aminobenzoate), 10. Mu.L hemin, 10. Mu.L vitamin B6.
In addition, the invention uses Bacillus pumilus X1 for inhibiting tinea pedis pathogenic fungi.
Preferably, the Bacillus pumilus X1 may be used as a preparation for inhibiting alternaria alternata.
Preferably, the Bacillus pumilus X1 can be used as a preparation for inhibiting trichophyton rubrum.
At present, antibiotics or derivatives thereof are mostly adopted for treating tinea pedis, and long-term application can lead patients to have drug resistance or even drug resistance, and the bacillus pumilus X1 disclosed by the invention has obvious effect of inhibiting tinea pedis fungi and also has obvious effect on tinea pedis pathogenic bacteria extracted from tinea pedis samples in the laboratoryA. alternataK1 has obvious inhibition effect. In addition, the use of Bacillus pumilus X1 to purposefully antagonize the growth of tinea pedis pathogenic fungi is a technology with minimal risk, and is suitable for pregnant women, children, the elderly or people allergic to drugs, etc., and can be used in cases of inapplicable antibiotics and no application of drugsThe product or oral medicine can be used for treating tinea pedis, and is also suitable for long-term treatment without affecting daily production and life. Provides a new direction for the microbiological study of tinea pedis diseases, and provides basic reference and new direction and potential probiotic resources for the later microbiological preparation or probiotic study of tinea pedis diseases.
The bacterial strain X1 disclosed by the invention has higher antibacterial rate, and can inhibit newly discovered tinea pedis pathogenic fungi K1 and the existing more common trichophyton rubrum. Based on the high-throughput sequencing of the foot samples, the separation and identification of foot microorganisms are carried out, and the antagonism relationship among foot symbiotic microorganisms is deeply explored. The method utilizes the mutual antagonism relationship among microorganisms to screen out bacteria which have stronger antagonism on the suspected pathogenic fungi of tinea pedis, namely the alternaria alternate, namely the bacillus pumilus, provides a new direction for the microorganism research of tinea pedis, and also provides potential probiotic resources for probiotic preparations of tinea pedis.
Drawings
FIG. 1 is a diagram of strain morphology features of strain X1.
FIG. 2 is a phylogenetic tree of foot skin separation symbiotic bacteria.
FIG. 3 is an expanded phylogenetic tree of gene amplification of tinea pedis pathogenic fungi.
FIG. 4 shows the results of screening for the bacterial strain X1 against tinea pedis pathogenic bacteria K1, wherein A is the antibacterial activity of 12 strains of bacteria isolated from the skin of the foot against K1, E, F, G is the medium inoculated with X1 antagonistic K1 by the cross-plate method, the double-plate method and the single-plate method, respectively, and B, C, D is the control group inoculated with only E, F, G groups of K1, respectively.
FIG. 5 is the result of an experiment in which the strain X1 inhibits the growth of mycelium of the tinea pedis pathogenic bacterium K1, wherein the left graph of A shows that when no X1 is acting,A. alternata k1 hypha growth morphology, right panel shows after action of strain X1A. alternata K1 hypha growth morphology, B is left image corresponding to A, C is right image corresponding to A, D, E, H is control groupA. alternataOptical microscopy of the growth morphology of K1 mycelium, F, G, I is after X1 actionA. alternata Optical microscopy of growth status of K1 mycelium, wherein D-G pattern is 10 x under-view, H-I is a 100 x subplot; j is the number of mycelium in the control and experimental groups.
FIG. 6 is the result of an experiment for inhibiting proliferation of tinea pedis pathogenic bacteria K1 spore by strain X1, wherein A is a control groupA. alternataNumber and status under K1 sporoscope, B is X1 after actionA. alternataNumber and status under K1 sporoscope, C is X1 pairA. alternataEffect of K1 spore amount.
FIG. 7 shows the results of screening for Trichophyton rubrum against strain X1, wherein A is the antibacterial activity of 9 strains having an antagonistic effect on Trichophyton rubrum among 12 strains isolated from the skin of the foot, E, F, G is a medium for X1 antagonizing Trichophyton rubrum using the cross-plate counter method, the double-plate counter method and the single-plate counter method, and B, C, D is a control group of E, F, G groups inoculated with K1 alone, respectively.
Detailed Description
Specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples and comparative examples. The following examples and comparative examples are intended only to more clearly illustrate the technical aspects of the present invention so that those skilled in the art can better understand and utilize the present invention without limiting the scope of the present invention.
The experimental methods, processes, apparatuses and devices involved in the examples and comparative examples of the present invention, the names and abbreviations thereof are all conventional in the art, and are clearly understood in the relevant fields of use, and those skilled in the art can understand the conventional process steps and apply the corresponding devices according to the names, and perform the operations according to the conventional conditions or the conditions suggested by the manufacturer.
The various raw materials or reagents used in the examples and comparative examples of the present invention are not particularly limited in source, and are conventional products commercially available.
Trichophyton rubrum used in the following examplesTrichophyton rubrum) Purchased from the north nano cell collection management center.
The composition of the culture medium used was:
PDA solid medium: glucose 10g/L, potato 200g/L, agar 15g/L, distilled water to 1L. Washing potato, peeling, cutting into small pieces, boiling in water for 30min, filtering with eight layers of gauze for several times, cooling, adding glucose and agar, dissolving, adding distilled water to 1L, naturally adjusting pH, and sterilizing at 121deg.C for 20min.
PDA liquid medium: glucose 10g/L, potato 200g/L, distilled water to 1L. The preparation method is the same as that of the PDA solid medium.
YG solid medium: (YCFA added glucose as carbon source, g/L) Medium: agar 15, casein 10.0, yeast extract 2.5, cysteine 1.0, naHCO 3 4.0，K 2 HPO 4 3.0，KH 2 PO 4 3.0，NaCl 0.9，MgSO 4 ·7H 2 O 1.0，CaCl 2 1.0, 2.5g of glucose, 10.0 mu L of folic acid, 10.0 mu L of biotin, 10.0 mu L of vitamin B12, 10.0 mu L of p-potassium p-aminobenzoate (p-aminobenzoate), 10.0 mu L of hemin and 10.0 mu L of vitamin B6, after being uniformly dissolved, distilled water is added to 1.0L, the pH is adjusted to 5.8-6.1, and the mixture is sterilized at 121 ℃ for 20min.
YG liquid medium: (YCFA added glucose as carbon source, g/L) Medium: casein 10.0, yeast extract powder 2.5, cysteine 1.0, naHCO 3 4.0，K 2 HPO 4 3.0，KH 2 PO 4 3.0，NaCl 0.9，MgSO 4 ·7H 2 O 1.0，CaCl 2 1.0, 2.5g of glucose, 10.0 mu L of folic acid, 10.0 mu L of biotin, 10.0 mu L of vitamin B12, 10.0 mu L of p-potassium p-aminobenzoate (p-aminobenzoate), 10.0 mu L of hemin, 10.0 mu L of vitamin B6, 0 mu L of distilled water to a volume of 1.0L, adjusting the pH value to 5.8-6.1, and sterilizing at 121 ℃ for 20min.
Example 1 morphological and molecular biological characterization of strain X1.
Inoculating strain X1 into YG liquid culture medium, diluting with sterile PBS solution to 10 times -1 、10 -2 、…、10 -7 100 mu L of the gradient diluted antagonistic bacteria X1 are coated on YG solid culture medium and cultured for 48 hours at 37 ℃. The morphological characteristics of the strain were recorded by photographing, as in figure 1.
The genomic DNA of strain X1 was extracted and further studied and amplified using the following primers:
upstream primer 27F:5'-AAAGATGGCATCATCATTCAAC-3'
Downstream primer 1492R:5'-TACCGTCATTATCTTCCCCAAA-3'
The amplification system was prepared and amplified under the conditions shown in Table 1, and the amplified product was sent to Beijing sequencing department of biological engineering (Shanghai) Co., ltd for DNA sequencing. The results were submitted to GenBank after BLAST alignment, and accession number MT798142 was obtained. Drawing the phylogenetic tree shown in fig. 2 by using MEGA7.0 software, and determining that the strain X1 is bacillus pumilus @Bacillus pumilus) And the strain is stored at-80 ℃ for standby.
TABLE 1PCR reaction System and reaction conditions
Example 2 cultivation and isolation of tinea pedis pathogenic fungus K1.
6 fungi are separated from tinea pedis samples, the effective length of the PCR amplification of ITs rDNA genes is 280bp, and a phylogenetic tree shown in figure 3 is drawn after ITs sequencing and Blast sequence comparison. Wherein, the K1 bacteria are skin infectious disease related fungi which are easy to culture and grow faster, and are most likely to participate in the occurrence of tinea pedis, so the K1 bacteria are used as antagonistic subjects for related research. The K1 bacteria ITs rDNA sequence has been submitted to GenBank and accession numbers are obtained: MW686913, the closest relationship between the bacterium and the alternaria alternata, the homology is 99%, so the bacterium is named asA.alternataK1。
Example 3 screening, culturing and isolation of Bacillus pumilus X1 against tinea pedis pathogenic fungi.
The isolation and culture steps of the strain for antagonizing tinea pedis pathogenic fungi are as follows:
1) Autoclaved PDA, YG medium, PBS and 1.5mL, 4mL EP tube, syringe, gun head, etc., were placed in advance and sterilized by ultraviolet light for half an hour.
2) Taking a skin sample obtained from a focus of a patient suffering from tinea pedis, clamping the root of a cotton swab by using burnt forceps, and placing the head of the cotton swab into PBS to prepare a bacterial stock solution.
3) Taking 8 EP pipes of 1.5mL, respectively adding 900 mu L of PBS (phosphate buffer solution) into the EP pipes, numbering 1 and 2 …, extracting 100 mu L of bacterial stock solution, injecting the bacterial stock solution into the EP pipe of No. 1, extracting 100 mu L of bacterial stock solution from the EP pipe of No. 1, injecting the bacterial stock solution into the EP pipe of No.2, and so on, and mixing the bacterial stock solution according to the ratio of 10 -1 、10 -2 …10 -7 、10 -8 10-fold dilution was performed and 100. Mu.L of each was drawn therefrom and spread evenly on PDA solid medium and incubated upside down at 37℃for 24-48h.
4) Taking out the culture medium, picking single strain on the culture medium capable of obviously seeing single bacterial colony, recording the corresponding morphological characteristics, rinsing the strain in PBS (phosphate buffer solution) prepared in advance, preparing bacterial liquid, injecting the bacterial liquid into the corresponding YG liquid culture medium, recording, and shake culturing at 37 ℃/180rpm for 24-48 hours.
5) And (3) after the absorbance value of the bacterial liquid at the OD595 is greater than 1, preparing the bacterial liquid according to the dilution method of the multiple ratio in the step (3) and culturing.
6) Repeating the step 4 again to obtain purified bacterial liquid, and storing the bacterial liquid at 4 ℃ for standby after recording.
The isolated strain was deposited as follows:
1) Preparing seed-preserving glycerol: the proportion of glycerin to water is 1:4, after the preparation, the mixture is uniformly mixed, a plug is covered, a layer of gauze is covered, the gauze is tightly bound by rubber bands, and 1-2 syringe needles are inserted into the plug for discharging pressure in the high-pressure process.
2) The high-pressure matched seed-protecting glycerin, the EP pipe, the injector and the blue gun head are wrapped by newspapers, and all the articles are detached in a safety cabinet during use. And (5) immediately pulling out the syringe needle after the high pressure of the seed-retaining glycerol is finished, and placing the syringe needle into a safety cabinet for standby.
3) Irradiating with ultraviolet for 15-20min, and sterilizing the used equipment.
4) The mixture was stirred in a 4mL EP tube by sequentially extracting 2mL of glycerol and 2mL of the bacterial liquid, and 1mL of the mixture was extracted in a 1.5mL EP tube.
5) After the seed preservation sample is recorded, the seed preservation sample is preserved for 24 hours at 4 ℃ and then is placed at-20 ℃ for 24 hours, and finally is placed at-80 ℃ for long-term preservation.
The 12 strains isolated from the skin of the foot (containing coincident but non-recombinant) and liable to be cultured in the above-mentioned steps were used as screening targets, designated X1 to X12, and strains having antagonism against the K1 strain obtained in example 1 were preliminarily screened by the plate counter method, as follows:
1) The activated K1 bacteria were inoculated into PDA solid medium and cultured upside down in an environment of 28℃for 6d.
2) Taking the cultured K1 bacterial cake by using a 7mm sterile puncher, moving the bacterial cake to the center of a PDA culture medium, respectively placing a sterile filter paper sheet in four perpendicular directions of the bacterial cake, and respectively dripping the separated skin symbiotic bacterial liquid 6 mu L to ensure that the distance between the bacterial cake and the K1 bacterial cake is 30mm. Each treatment was repeated 3 times to inoculate only K1 bacteria as a control (CK group).
3) The treated medium was allowed to stand in a safety cabinet for several minutes and incubated upside down at 28℃for 6d. And (3) observing and recording bacteriostasis, measuring the growth diameter (mm) of each group of K1 bacterial cakes, and calculating the bacteriostasis rate according to the following formula.
Antibacterial ratio (%) = (average colony growth diameter of CK group-average colony growth diameter of treatment group)/average colony growth diameter of CK group×100
The culture results are shown in FIG. 4A, wherein 6 strains show different antagonistic activities on K1 bacteria, the antagonistic effect of the strain X1 is strongest, and the antibacterial rate reaches 60.31% after 6d culture at 28 ℃ and is far higher than that of other strains.
Then, re-screening the X1 strain with the maximum bacteriostasis rate of the fungus K1, wherein the re-screening culture medium is divided into three types:
1) Cross symmetry plate counter method:
taking activated K1 bacterial cake by using a 7mm sterile puncher, moving the activated K1 bacterial cake to the center of a PDA culture medium, placing a sterile filter paper sheet in each of four perpendicular directions of the activated K1 bacterial cake, dripping 6 mu L of X1 bacterial liquid to ensure that the distance between the activated K1 bacterial cake and the K1 bacterial cake is 30mm, placing the activated K1 bacterial cake in a safety cabinet for a plurality of minutes, and culturing the activated K1 bacterial cake upside down at 28 ℃ to only inoculate K1 bacteria as a control (CK group).
2) Double-sided plate counter method:
activated K1 bacterial cakes are picked up by using a 7mm sterile puncher, are moved to symmetrical positions on a PDA culture medium, sterile filter paper sheets are placed at a position 15mm away from the center, antagonistic bacteria liquid is added in a sample of 6 mu L, and after the antagonistic bacteria liquid is placed in a safety cabinet for a plurality of minutes, the culture is inverted at 28 ℃ to inoculate K1 bacteria only as a control (CK group).
3) Single-sided plate counter method:
activated K1 bacterial cake was picked up by a 7mm sterile punch, inoculated to one side of the center of the plate, a sterile filter paper sheet was placed at a position 30mm from the inoculation point, and 6. Mu.L of antagonistic bacteria liquid was applied and cultured upside down at 28℃for 6d to inoculate only K1 bacteria as a control (CK group).
As shown in FIGS. 4B-G, strain X1 significantly inhibited the growth of K1 by the cross-symmetric plate method, the double-sided plate method and the single-sided plate method, and it was found that strain X1 had a strong antagonism against K1 and possibly against Alternaria alternata homologous to K1.
Example 4 antagonism of X1 against tinea pedis pathogenic fungus K1.
The experiment of the effect of Bacillus pumilus X1 on the growth of mycelium of tinea pedis pathogenic fungi was performed as follows:
1) Taking mycelium on the activated K1 bacterial cake, preparing suspension, then injecting the suspension into a PDA liquid culture medium, and shake culturing for 24 hours at 28 ℃ at a speed of 180rpm for later use;
2) Centrifuging activated X1 at 12000rpm for 5min, washing with PBS solution for 3 times, suspending with PDA culture medium, injecting into the K1 mycelium suspension cultured in the previous step, culturing at 28deg.C and 180rpm for 48 hr without inoculating X1, and observing growth of K1 mycelium under microscope, wherein K1 mycelium treated with X1 bacterial liquid has different thickness, and serious distortion and even fracture appear;
3) 3mL of activated K1 bacteria are injected into PDA liquid culture medium, and 3mL of activated X1 bacteria liquid is injected into PDA culture medium containing K1 bacteria, each treatment is repeated for 3 times without inoculating X1 bacteria as a control, the culture is carried out at 28 ℃ and 180rpm for 48 hours, the whole mycelium number is counted in a solid culture dish, and the number of K1 mycelium is obviously reduced after the action of equal volume of X1 bacteria as shown in figure 5C.
The experiment of the effect of Bacillus pumilus X1 on the spore growth of tinea pedis pathogenic fungi was performed as follows:
1) Cleaning the surface of the activated K1 bacterial cake by using sterile water, preparing spore suspension, and adjusting the spore concentration to 106CFU/mL by using a blood cell counting plate;
2) According to spore suspension: mixing the X1 bacterial liquid=1:1 and the spore suspension in a ratio of sterile PDA culture medium=1:1, injecting into the PDA culture medium, and shake culturing at 28 ℃ and 180rpm for 24 hours;
3) The proliferation of spores was observed and recorded under a microscope, and the corresponding numbers of spores were counted, as shown in FIG. 6, the sporulation amount of K1 bacteria was significantly reduced after the action of X1 bacteria, 1.63×10 10 CFU, control group 6.08X10 10 CFU(P<0.01 The effect of strain X1 significantly reduced proliferation of K1 spores.
Example 5 antagonism of Bacillus pumilus X1 against Trichophyton rubrum.
Trichophyton rubrum (Fr.) karstTrichophyton rubrum) Is one of the most common pathogenic fungi of tinea pedis. The purchased Trichophyton rubrum strain was resuscitated and activated on PDA medium, and its purity was determined by DNA sequencing technique, and the pairs were selected as in example 3 using the foot 12 symbiotes (containing coincident but different batches of isolated bacteria) obtained in example 3 as screening subjects for antagonistic bacteriaT.rubrum9 strains of bacteria having antagonistic effect were used, as shown in FIG. 7, in which antagonistic activity was highest with the strain X1. After the culture is carried out for 12 days at 28 ℃, the bacteriostasis rate can reach 75.91 percent. The bacterial strain X1 has a wider bacteriostasis spectrum, and then the bacterial strain X1 is selected for carrying outT.rubrumIs a re-screening experiment. The regreening results are shown in fig. 7B-G: strain X1 pair was detected by 3 plate counter methodsT.rubrumHas antagonistic effect, wherein the antagonistic activity is highest by cross symmetry plate counter method, and even complete inhibition can be achievedT.rubrumGrowth and diffusion effects.
Wherein, the strain X3 is identified as bacillus safoci @Bacillus safensis) From the results of example 3, it can be seen that it has antagonism against Alternaria alternata K1, its efficacy is inferior to X1, but weak antagonism against Trichophyton rubrum, it can be seen that not allThe bacterial strain antagonizing the alternaria alternata K1 has antagonism on trichophyton rubrum, so that the bacterial strain X1 disclosed by the invention can be used for antagonizing the alternaria alternata K1 and trichophyton rubrum with high efficiency, and the antagonism range is wide and the inhibition effect is high.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced with equivalents; such modifications and substitutions do not depart from the spirit of the technical solutions according to the embodiments of the present invention.
Claims (6)
1. Bacillus pumilus X1 for antagonizing tinea pedis pathogenic fungi, belonging to the field of Bacillus pumilusBacillus pumilus) The preservation number is CGMCC NO.24605.
2. The method for culturing Bacillus pumilus X1 of claim 1, comprising the steps of: strain X1 was inoculated into a liquid medium and cultured at 180r/min and 28 ℃.
3. The culture method according to claim 2, wherein the culture medium is YG culture medium with pH of 5.8-6.1, and comprises casein, yeast extract, cysteine, naHCO 3 、K 2 HPO 4 、KH 2 PO 4 、NaCl、MgSO 4 ·7H 2 O、CaCl 2 Glucose, folic acid, biotin, vitamin B12, potassium P-para-aminobenzoate, hemin, vitamin B6 and distilled water.
4. A culture method according to claim 3, wherein the medium contains 10g casein, 2.5g yeast extract, 1g cysteine, 4g NaHCO per liter of the medium 3 ，3g K 2 HPO 4 ，3g KH 2 PO 4 ，0 .9g NaCl，1g MgSO 4 ·7H 2 O，1gCaCl 2 2.5g glucose, 10. Mu.L folic acid, 10. Mu.L biotin, 10. Mu.L vitamin B12, 10. Mu. L P potassium p-aminobenzoate, 10. Mu.L hemin, 10. Mu.L vitamin B6.
5. The preparation of Bacillus pumilus X1 as claimed in claim 1 for inhibiting alternaria alternataAlternaria alternata) The application in the preparation.
6. The preparation of Bacillus pumilus X1 as claimed in claim 1 for inhibiting Trichophyton rubrumTrichophyton rubrum) The application in the preparation.
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