CN110628625A - Separation and screening method of patchouli endophytic fungi - Google Patents
Separation and screening method of patchouli endophytic fungi Download PDFInfo
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Abstract
The invention relates to the technical field of microorganisms, in particular to a separation and screening method of patchouli endophytic fungi. Comprises a culture medium preparation step, an endophytic fungi separation and purification step, an endophytic fungi solid fermentation cultivation and crude extract preparation step and an endophytic fungi activity screening step. A tissue mass separation method is adopted to separate a plurality of endophytic fungi strains from patchouli plants, and then a filter paper sheet method is used to screen out the endophytic fungi with ralstonia solanacearum resistance. The obtained various bacterial strains with antagonistic action on the ralstonia solanacearum are subjected to strain identification of antagonistic bacteria by morphological observation, and the biological prevention and control effect of endophytic fungi on the ralstonia solanacearum is measured by a pot experiment, so that a theoretical basis is provided for resource utilization and bacterial wilt prevention and control of the pogostemon cablin endophytic fungi, and a new bacterial strain resource for reference is provided for biological prevention and control of the tomato bacterial wilt.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a separation and screening method of patchouli endophytic fungi.
Technical Field
Herba Agastaches (Pogostemon cablin (Blanco) Benth.) is a dry aerial part of plants of genus Pogostemon of family Labiatae, and is one of the famous-region herbs and the ten-large Guangdong herbs. The endophytic fungi of the medicinal plants widely exist in the bodies of the medicinal plants, have rich biological diversity and have positive effects on host plants, and secondary metabolites have activities of resisting tumors, oxidation, bacteria and the like, so the endophytic fungi of the medicinal plants have great application prospects in the aspects of medicines, agriculture and the like.
According to investigation, the bacterial wilt disease is obtained in the tomato planting process, the bacterial wilt disease can be rapidly spread in a short time, and the control work in the later period is very difficult, so that large-area withering death and even complete harvest of tomato crops are caused, the tomato yield is reduced finally, and the economic benefit of a grower is seriously damaged. The endophytic fungi separated from roots, stems and leaves of patchouli plants has positive effect on host plants to inhibit bacterial wilt.
Disclosure of Invention
The invention aims to provide a separation and screening method of patchouli endophytic fungi, and the technical scheme provided by the invention provides a theoretical basis for resource utilization and bacterial wilt prevention and control of the patchouli endophytic fungi.
In order to achieve the above object, the present invention provides a method for separating and screening an endophytic fungus of Pogostemon cablin, comprising the following steps:
a culture medium preparation step, which is used for preparing a culture medium required by the endophytic fungi separation and screening method;
the separation and purification step of the endophytic fungi is used for separating the endophytic fungi from the patchouli plants and purifying to obtain the strain of the endophytic fungi;
solid fermentation cultivation and crude extract preparation steps of the endophytic fungi, which are used for obtaining crude extracts of the endophytic fungi by fermenting and concentrating strains obtained by completing purification;
and an endophytic fungi activity screening step, which is used for screening the endophytic fungi with ralstonia solanacearum resistance to the crude extract of the endophytic fungi.
Preferably, in the medium preparing step, the medium includes:
PDA culture medium formed by culturing potato, glucose and agar;
is prepared from potato, glucose and KH2PO4、MgSO4·7H2O, vitamin B1 and water;
NA culture medium formed by culturing peptone, beef extract, sodium chloride and agar; and
NA liquid medium formed by culturing peptone, beef extract and sodium chloride.
Preferably, the step of isolating and purifying the endophytic fungi comprises the following steps:
a100, collecting healthy patchouli plants, cleaning and cutting the patchouli plants into small pieces to prepare samples;
a200, sequentially soaking a sample in ethanol, a NaClO solution and ethanol to finish disinfection and sterilization, and then washing with sterile water;
a300, placing the washed sample in a PDA culture medium for constant-temperature culture; and (3) timely picking out a single hypha in the PDA culture medium to a new PDA culture medium for constant-temperature culture after the hypha grows out of the sample, and repeatedly operating A300 until a plurality of purified endophytic fungi strains are obtained.
Preferably, the solid fermentation cultivation and crude extract preparation steps of the endophytic fungi comprise the following steps: b100, inoculating the endophytic fungi strain obtained by purifying in the step A300 into a PDA culture medium;
b200, fermenting for several days, and then adding ethyl acetate to soak;
and B300, mashing the soaked PDA culture medium, soaking again, sequentially filtering, and concentrating under reduced pressure to obtain a crude extract of the endophytic fungi.
Preferably, the step of screening for endophytic fungi activity comprises the steps of:
c100, taking a proper amount of the crude extract obtained in the step B300, and preparing a sample solution by using a DMSO solution;
c200, respectively culturing the ralstonia solanacearum in a plurality of NA liquid culture media, and preparing a bacterial liquid by shaking culture of a constant-temperature culture shaker;
c300, dipping a proper amount of bacteria liquid, respectively and uniformly coating the bacteria liquid on a plurality of NA culture media, dropwise adding a sample solution on a sterilized filter paper sheet, and placing the filter paper sheet on the NA culture media containing the bacteria liquid;
and C400, carrying out constant-temperature culture on the NA culture medium, measuring the diameter of the inhibition zone of the NA culture medium, and screening out the endophytic fungi with ralstonia solanacearum resistance.
Preferably, the preparation method of the PDA culture medium comprises the following steps of: 10-7The potato, the glucose, the agar, the kanamycin sulfate and the ampicillin are cultured by sterilizing for 40-45 min at the temperature of 121 ℃ in a high-temperature high-pressure environment at the natural pH value;
the preparation method of the PDA liquid culture medium comprises the following steps of mixing the components in a mass concentration ratio of 200-220: 20-25: 2-5: 0.1-0.2: 0.01:1 of potato, glucose and KH2PO4、MgSO4·7H2O, vitamin B1 and water, the natural pH value, and the sterilization is carried out for 25-35 min in the high-temperature high-pressure environment of 121 ℃ to form the culture medium;
the preparation method of the NA culture medium comprises the steps of sterilizing peptone, beef extract, sodium chloride and agar at the mass concentration ratio of 8-11: 2-4: 4-6: 15-20 for 25-35 min at 121 ℃ in a high-temperature high-pressure environment, and culturing;
the preparation method of the NA liquid culture medium comprises the steps of sterilizing peptone, beef extract and sodium chloride at the mass concentration ratio of 8-12: 2-3: 5-7 for 25-35 min at 121 ℃ in a high-temperature high-pressure environment, and culturing.
Preferably, in step a300, the obtained endophytic fungal strain is subjected to molecular biological identification comprising:
a301, primarily screening 2 high-efficiency bacteria from the obtained endophytic fungi strains, culturing for 72h, and extracting DNA according to a TSP101 kit;
a302, observing through agarose electrophoresis, and performing genome PCR amplification on the strain by using a universal primer;
and A303, carrying out gene ITS sequencing on the amplified product, and comparing the sequencing result in Gen Bank by BLAST.
Preferably, in the step B200, after fermenting for 30-40 days at 28 ℃, adding ethyl acetate to soak for 2-3 hours;
in the step B300, the soaked PDA culture medium is smashed and soaked again for 12-13 h, and after filtration, the crude extract of the endophytic fungi is obtained by decompression and concentration at 40-50 ℃ and 60 r/min.
Preferably, in step C200, the NA liquid medium is subjected to shake culture for 36h in a30 ℃ constant temperature culture shaker;
in step C400, the NA medium is incubated at 30 ℃ for 24 h.
From the above, the following beneficial effects can be obtained by applying the invention: the method comprises the steps of separating endophytic fungi from pogostemon cablin plants by adopting a tissue block separation method so as to obtain various strains with antagonistic action on ralstonia solanacearum, identifying the strains of the antagonistic bacteria by morphological observation, measuring the biological prevention and control effect of the biocontrol microbial inoculum on the ralstonia solanacearum by using a pot experiment, researching the related application of the biocontrol microbial inoculum in the biological prevention and control of the ralstonia solanacearum, providing a theoretical basis for the resource utilization of the endophytic fungi of pogostemon cablin and the prevention and control of the ralstonia solanacearum, and providing a new strain resource for reference in the biological.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments of the present invention or the prior art will be briefly described below. It is obvious that the drawings in the following description are only some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive effort.
FIG. 1 is a flow chart of a method for separating and screening endophytic fungi of Pogostemon cablin according to an embodiment of the present invention;
FIG. 2 is a flow chart of the steps of separating and purifying endophytic fungi according to the embodiment of the invention;
FIG. 3 is a block diagram of the solid fermentation cultivation and crude extract preparation steps of the endophytic fungi of the embodiment of the invention;
FIG. 4 is a block diagram of the steps of molecular biological identification of endophytic fungi according to an embodiment of the present invention;
FIG. 5 is a flow chart of the steps of screening for endophytic fungi activity according to an embodiment of the present invention;
FIG. 6 is a colony chart of GY14 strain against Ralstonia solanacearum in example 6;
FIG. 7 is a colony chart of GY06 strain against Ralstonia solanacearum in example 7;
FIG. 8 is a diagram of the inhibition zone of GY14 strain against Staphylococcus aureus in the example of the present invention;
FIG. 9 is the bacteriostatic circle diagram of GY06 strain against E.coli in this example.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
As shown in fig. 1, in order to solve the technical problem that ralstonia solanacearum affects crop harvest, the present embodiment provides a separation and screening method for pogostemon cablin endophytic fungi, which can separate and form endophytic fungi with high resistance to ralstonia solanacearum. The roots, stems and leaves of the patchouli have endophytic fungi, but the types and the number of the endophytic fungi contained in different parts are different. The separation and screening method provided in this embodiment adopts a tissue mass separation method to separate and purify endophytic fungi with high bacterial wilt resistance, as shown in fig. 2, and includes the following steps:
a100, collecting healthy patchouli plants, cleaning and cutting the patchouli plants into small pieces to prepare samples;
collecting healthy herba Agastaches, cleaning with distilled water, and cutting root, stem and leaf into small pieces of different specifications to obtain samples.
A200, sequentially soaking a sample in ethanol, a NaClO solution and ethanol to finish disinfection and sterilization, and then washing with sterile water;
sequentially placing the sample into 75% ethanol for soaking for 1-2min, soaking in 3% NaClO solution for 2-3min, soaking in 75% ethanol for 1-2min, washing with sterile water, and culturing in PDA solid culture medium at constant temperature of 26 deg.C.
A300, placing the washed sample in a PDA culture medium for constant-temperature culture; and (3) timely picking out a single hypha in the PDA culture medium to a new PDA culture medium for constant-temperature culture after the hypha grows out of the sample, and repeatedly operating A300 until a plurality of purified endophytic fungi strains are obtained.
The preparation method of the PDA culture medium comprises the following steps of: 10-7The potato, the glucose, the agar, the kanamycin sulfate and the ampicillin are cultured by sterilizing for 40-45 min at the temperature of 121 ℃ in a high-temperature high-pressure environment at the natural pH value. In step A300, PDA medium was placed in a constant temperature environment at 26 ℃ for the purpose of growing hyphae in the medium.
In order to ensure that the obtained bacterial strain is the endophytic fungus of the cablin potchouli herb, the surface-sterilized materials are directly inoculated on the same PDA culture medium without any shearing in the separation process of the endophytic fungus to be cultured, whether the fungi grow is observed, the blank control is used, and if no bacterial strain grows on the materials, the step is indicated to separate the endophytic fungus of the cablin potchouli herb.
And (4) carrying out solid fermentation culture on the separated and purified strain, and preparing a crude extract of the strain. Referring to fig. 3, the solid fermentation cultivation and crude extract preparation steps of the endophytic fungi comprise the following steps:
b100, inoculating the endophytic fungi strain obtained by purifying in the step A300 into a PDA culture medium;
in this step, the purified endophytic fungal strain was inoculated into PDA medium and subjected to solid fermentation at 28 ℃ for 30 days.
B200, after fermenting for 30 days, adding ethyl acetate to soak for 2-3 h;
and B300, mashing the soaked PDA culture medium, soaking again, sequentially filtering, and concentrating under reduced pressure to obtain a crude extract of the endophytic fungi.
In the step B300, the soaked PDA culture medium is smashed and soaked again for 12-13 h, and after filtration, the crude extract of the endophytic fungi is obtained by decompression and concentration at 40-50 ℃ and 60 r/min.
Further, the crude extract of the obtained endophytic fungi is subjected to activity screening so as to obtain the endophytic fungi with ralstonia solanacearum resistance. Several NA liquid media and several NA media were prepared prior to screening.
Wherein the NA liquid culture medium comprises 200g/L potato, 20g/L glucose and KH2PO43g/L，MgSO4·7H2O0.15 g/L, vitamin B110mg/L, 1000mL of water, natural pH, and autoclaving at 121 ℃ for 30 min.
The NA culture medium is formed by culturing 10g/L of peptone, 3g/L of beef extract, 5g/L of sodium chloride and 18g/L of agar for 30min under high pressure sterilization at 121 ℃.
As shown in fig. 4, in this embodiment, a filter paper sheet method is used to perform the screening of the activity of the endophytic fungi, and the screening step of the activity of the endophytic fungi comprises the following steps:
c100, taking a proper amount of the crude extract obtained in the step B300, and preparing a sample solution with the concentration of 50mg/mL by using a DMSO solution;
c200, respectively culturing the ralstonia solanacearum in a plurality of NA liquid culture media, and preparing a bacterial liquid by shaking culture of a constant-temperature culture shaker;
wherein the NA liquid culture medium is placed in a30 ℃ constant temperature culture oscillator for shaking culture for 36h, and then diluted with normal saline to prepare 105-107cfu/mL of bacterial liquid.
C300, dipping a proper amount of bacteria liquid, respectively and uniformly coating the bacteria liquid on a plurality of NA culture media, dropwise adding a sample solution on a sterilized filter paper sheet, and placing the filter paper sheet on the NA culture media containing the bacteria liquid;
and C400, carrying out constant-temperature culture on the NA culture medium, measuring the diameter of the inhibition zone of the NA culture medium, and screening out the endophytic fungi with ralstonia solanacearum resistance.
And (3) placing the NA culture medium in a 37 ℃ incubator for culturing for 24h, measuring the diameter of the inhibition zone, judging the effect of the inhibition effect of the endophytic fungi according to the diameter of the inhibition zone, and further selecting the endophytic fungi resisting ralstonia solanacearum.
In this example, 42 endophytic fungi were co-isolated from a sample by tissue mass isolation, 10 of which were isolated from roots, 16 of which were isolated from stems, and 16 of which were isolated from leaves, accounting for 23.8%, 38.1%, and 38.1% of the number of isolated strains, respectively. As can be seen from the number of strains isolated from different parts, the root separation rate is the lowest, while the leaf and stem separation rates are equal and higher than the root.
And (3) screening the antibacterial activity of the 42 separated endophytic fungi cablin potchouli herb endophytic fungi to obtain 6 bacterial strains with ralstonia solanacearum activity, wherein the bacterial strains are named as GJ06, GG05, GY06, GJ13, GG03 and GY 14.
TABLE 1 Pogostemon cablin endophytic fungi having ralstonia solanacearum antagonistic activity
The results are shown in Table 1, wherein the radius of inhibition of GY06 is 6.6mm, the radius of inhibition of GY14 is 7.1mm, the radius of inhibition of GG05 is 4.5mm, and the radius of inhibition of the rest of GJ06, GJ13 and GG03 is smaller. As shown in fig. 6-7, the inhibition effect of the GY06 strain and the GY14 strain against ralstonia solanacearum is more significant for the GY14 strain and the GY06 strain, respectively.
Example 2
This example performed bacteriostatic activity tests on the isolated endophytic fungi and provided methods for identifying endophytic fungi.
The endophytic fungi separated in the example 1 is subjected to an antibacterial activity test, staphylococcus aureus and escherichia coli are prepared into bacterial liquid, a cotton swab is used for dipping a proper amount of the staphylococcus aureus bacterial liquid and the escherichia coli liquid, the bacterial liquid and the escherichia coli liquid are respectively and uniformly coated on an NA culture medium, and a plurality of plate culture media are respectively arranged on each bacterial.
Specifically, Staphylococcus aureus and Escherichia coli are respectively cultured in NA liquid medium, shake-cultured in constant temperature 37 deg.C shaking table for 24h, diluted with normal saline, and formulated into 105-107cfu/mL concentration of bacterial liquid. Wherein, the NA liquid culture medium is prepared by adopting the following raw materials in percentage by mass: 10g/L of peptone, 3g/L of beef extract and 5g/L of sodium chlorideg/L, autoclaving at 121 deg.C for 30min, and setting several plate culture media for each strain for testing activity of the separated multiple endophytic fungi strains.
Dipping a cotton swab with a proper amount of bacterial liquid, uniformly coating the bacterial liquid on an NA culture medium, respectively dropwise adding 4-5 uL of endophytic fungi sample solution on a sterilized filter paper, clamping the filter paper by using sterile forceps, placing the filter paper on a plate culture medium containing golden yellow staphylococcus liquid and escherichia coli liquid, placing the plate on a 37 ℃ incubator for culturing for 24 hours, measuring the diameter of a bacteriostatic circle, and calculating the average diameter of the bacteriostatic circle.
The DMSO solution was used as a blank control, and the Staphylococcus aureus and Escherichia coli were respectively used as positive controls with ampicillin at a concentration of 200. mu.g/mL and kanamycin sulfate at a concentration of 200. mu.g/mL. The active patchouli endophytic fungus strain has the effect of inhibiting the growth of staphylococcus aureus and escherichia coli, and the effect of the inhibition can be judged according to the diameter of a bacteriostatic zone.
TABLE 2 inhibitory Effect of the strains on Staphylococcus aureus
TABLE 3 inhibition of E.coli by the strains
The test results are shown in table 2, 5 strains of the strain capable of inhibiting staphylococcus aureus exist, as shown in fig. 8, wherein the bacterial strain GY14 has the best antibacterial effect, the antibacterial radius reaches 7.5mm, the antibacterial effects of the strains GG05, GY04 and GJ06 are the second order, and the antibacterial effect of the strain GY03 is relatively poor.
The test results are shown in Table 3, and there are 4 strains of the strain which can inhibit Escherichia coli, as shown in FIG. 9, among which, the inhibition effect of strain GY06 is the best, the inhibition radius reaches 13mm, the inhibition effect of strain GY14 and strain GJ06 is the second best, and the inhibition effect of strain GY04 is relatively poor.
The plant endophytic fungi have various types, and different plants can be separated to obtain different types and quantities of endophytic fungi. The endophytes isolated from the same plant also vary under different experimental circumstances and isolation methods. In the embodiment of the invention, 42 endophytic fungi are separated from roots, stems and leaves of patchouli by adopting a tissue mass separation method, 5 strains with antagonistic activity to staphylococcus aureus and 4 strains with antagonistic activity to escherichia coli are screened out by a bacteriostatic test, and the test result can draw a conclusion that the antagonistic activity of the strain GY14 and the strain GJ06 is particularly obvious.
In order to perform molecular biological identification on the obtained and screened 2 high-efficiency endophytic fungi, namely strain GY14 and strain GJ06, the embodiment provides a molecular biological identification method for the obtained endophytic fungi strain, which comprises the following steps:
a301, primarily screening 2 high-efficiency bacteria from the obtained endophytic fungi strains, culturing for 72h, and extracting DNA according to a TSP101 kit;
a302, observing through agarose electrophoresis, and performing genome PCR amplification on the strain by using a universal primer;
and A303, carrying out gene ITS sequencing on the amplified product, and comparing the sequencing result in Gen Bank by BLAST.
The genome detection electrophoresis result shows that the DNA separation effect of 2 samples is good, no breakage exists, the concentration is considerable, and the next analysis can be carried out. As can be seen from the electrophoretogram detected after PCR amplification, 5 samples can separate bands which are clear, the separation effect is good, and the concentration is high.
The genome ITS sequence of the endophytic fungus GY06 is determined by biotechnology as follows:
TTCGGAGGTGCGAGCTCGCGGCTCACCTCCCACCCTTGTCTCTCTACACCCTGTTGCTTTGGCGGGCCCACCGGGGCCACCCGGTCGCCGGGGGACGCACGTCCCCGGGCCCGCGCCCGCCGAAGCGCTCTGTGAACCCTGATGAAGATGGGCTGTCTGAGTACTATGAAAATTGTCAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCCTGGCATTCCGGGGGGCATGCCTGTCCGAGCGTCATTTCTGCCCTCAAGCACGGCTTGTGTGTTGGGTGCGGTCCCCCCGGGGACCCGCCCGAAAGGCAGCGGCGACGTCCGTCTGGTCCTCGAGCGTATGGGGCTTTGTCACTCGCTCGGGAAGGACCTGCGGGGGTTGGTCACCACCATATTTTTACCACGGTTGACCTCGGATCAGGTAGGAGTTACCCGCTGAACTTAAGCATATCAATAAGCCGGAGGAAAAAAACGCCCCC
the endophytic fungus GY06 was found to cover 99% similar to Aspergillus flavus (Talaromyces sp 12 MGS-2017) by Blast alignment analysis. The genus was initially determined to be Aspergillus flavus.
Sequencing by biotechnology, the genome ITS sequence of the endophytic fungus GY14 is as follows:
AATGGATTGATGACGGGTTGTTGCTGGCCCTTACTGGGCATGTGCACGCTTTGTACATTCCAATTCTTATACCTCTGTGCACTTTTCATAGATTTAGTGTGGAAGAGATCTTTTTGATCTTGGAAATGCTGGATCTGTGTTTTTACAAACGCTTTAGTATTAGAATGTCATCTGCGAATAACGCAATAAATACAACTTTCAGCAACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCCCCTTGGTATTCCGAGGGGCATGCCTGTTTGAGTGTCATGGTATTCTCAATGCCCTACATCTTTGCGGATGAAGGTGTATTGGATTTGGAGGTTTATGTTGGCTCTCTTGAGAGTCAGCTCCTCTTAAATGTATTAGCAGAGATGTTACTGCTACTCTCCAGTGTGATAATTGTCTACACTGTTAGTAGTGCAGTAAAAATTAAGTCTATGCTTCTAATCGTCTCCGGACAGTTTTTTGACATCTGACCTCAAATCAGGTAGGACTACCCGCTGAACTTAAGCATATCATAAGCGGAGGAAAAGAAAAGGAGAT
the comparison analysis by Blast shows that the coverage of the endophytic fungus GY14 is 99% similar to that of the Pogostemon cablin endophytic fungus (Cerrena sp MG 295). The genus was preliminarily determined to be odontobutis.
Example 3
This example also provides a potting test to test the disease resistance of the strain of Pogostemon cablin GY14 and GY06 against Ralstonia solanacearum, based on the isolated and screened Pogostemon cablin endophytic fungi.
Bacterial wilt of tomato, pepper, patchouli and other plants can be caused by bacterial wilt infection. The healthy tomato seedlings are inoculated by adopting a root injury soaking method and a root irrigation method. The test pot culture was divided into 4 groups, which were a blank group inoculated with only clear water, a control group inoculated with ralstonia solanacearum, an experimental group 1 inoculated with the GY14 strain and ralstonia solanacearum, and an experimental group 2 inoculated with the GY06 strain and ralstonia solanacearum, respectively.
Specifically, tomato seedlings which are identical in growth condition, size and development and are clean in sterile water are selected, wounds of the same degree are cut on the cutting seedlings by using a sterile scalpel, and the cutting seedlings are soaked in the ralstonia solanacearum suspension for 15 minutes. Placing the soaked cutting seedlings into a seedling recovery pot, adding 25mL of ralstonia solanacearum suspension into a control group, adding 25mL of ralstonia solanacearum-resistant suspension into an experimental group 1 and an experimental group 2, and inoculating clear water only into a blank group, namely:
experiment group 1, tomato seedlings were inoculated with ralstonia solanacearum, while 25mL of endophyte suspension of strain GY14 was added;
experiment group 2, tomato seedlings were inoculated with ralstonia solanacearum, while 25mL of endophyte suspension of strain GY06 was added;
in a control group, young tomato seedlings are inoculated with ralstonia solanacearum, and 25mL of ralstonia solanacearum suspension is added;
in the blank group, the tomato seedlings are not inoculated with ralstonia solanacearum, and only clear water is added.
And observing and recording the disease condition of tomato seedling bacterial wilt after 7 days of culture.
Dividing the disease condition of tomato seedling bacterial wilt into 5 grades: grade 0 is no obvious symptoms of the leaf; 1-25% of leaves with grade 1 show wilting symptom; 2, 26 to 50 percent of leaves have wilting symptoms; the leaf with grade 3 of 51-75% shows wilting symptom; the 4 grade is 76-100% of leaves with wilting symptom. The calculation method of disease index and prevention and treatment effect is as follows:
disease index (%) × Σ (number of diseased plants × representative value)/(total number of plants × representative value of the most serious level of disease) × 100;
the disease prevention effect (%) is (control disease index-treatment disease index)/control disease index × 100.
After 7 days of treatment, the disease condition of the tomato seedlings is recorded according to the potted plant result display, and the disease condition index results of the experimental group 1, the experimental group 2, the control group and the blank group are calculated by the disease condition index calculation method as follows:
experiment group 1, tomato seedlings are treated by the strain GY14, simultaneously ralstonia solanacearum is inoculated, and the disease index of the tomato seedlings to the bacterial wilt is 20%;
experiment group 2, tomato seedlings are treated by the strain GY06, simultaneously ralstonia solanacearum is inoculated, and the disease index of the tomato seedlings to the bacterial wilt is 20%;
in the control group, only ralstonia solanacearum is inoculated, and the bacterial wilt disease index of the tomato seedlings is 40%, and the strains GY14 and GY06 are not inoculated;
and (3) a blank group, wherein only clear water is inoculated, the strain GY14, the strain GY06 and ralstonia solanacearum are not inoculated, and the disease index of the tomato seedlings to the bacterial wilt is 0.
According to the disease indexes of the experimental group 1, the experimental group 2 and the control group, the disease indexes of the experimental group 1 and the experimental group 2 are both 20%, the disease index of the control group is 40%, the disease prevention effect of the strain GY14 can be calculated to be 50% and the disease prevention effect of the strain GY06 can be calculated to be 50% through the prevention and treatment effect calculation method, and the results show that the strain GY14 and the strain GY06 have good disease prevention effects.
According to the tomato pot experiment provided by the embodiment of the invention, the control effects of GY14 and GY06 reach 50%, which shows that the strain GY14 and the strain GY06 have good control effects on bacterial wilt in practical application.
The above-described embodiments do not limit the scope of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the above-described embodiments should be included in the protection scope of the technical solution.
Claims (9)
1. A separation and screening method of patchouli endophytic fungi is characterized in that: the method comprises the following steps:
a culture medium preparation step, which is used for preparing a culture medium required by the endophytic fungi separation and screening method;
the separation and purification step of the endophytic fungi is used for separating the endophytic fungi from the patchouli plants and purifying to obtain the strain of the endophytic fungi;
solid fermentation cultivation and crude extract preparation steps of the endophytic fungi, which are used for obtaining crude extracts of the endophytic fungi by fermenting and concentrating strains obtained by completing purification;
and an endophytic fungi activity screening step, which is used for screening the endophytic fungi with ralstonia solanacearum resistance to the crude extract of the endophytic fungi.
2. The separation and screening method according to claim 1, wherein: in the medium preparing step, the medium includes:
PDA culture medium formed by culturing potato, glucose and agar;
is prepared from potato, glucose and KH2PO4、MgSO4·7H2O, vitamin B1 and water;
NA culture medium formed by culturing peptone, beef extract, sodium chloride and agar; and
NA liquid medium formed by culturing peptone, beef extract and sodium chloride.
3. The separation and screening method according to claim 2, wherein: the separation and purification steps of the endophytic fungi comprise the following steps:
a100, collecting healthy patchouli plants, cleaning and cutting the patchouli plants into small pieces to prepare samples;
a200, sequentially soaking a sample in ethanol, a NaClO solution and ethanol to finish disinfection and sterilization, and then washing with sterile water;
a300, placing the washed sample in a PDA culture medium for constant-temperature culture; and (3) timely picking out a single hypha in the PDA culture medium to a new PDA culture medium for constant-temperature culture after the hypha grows out of the sample, and repeatedly operating A300 until a plurality of purified endophytic fungi strains are obtained.
4. The separation and screening method according to claim 3, wherein: the solid fermentation cultivation and crude extract preparation steps of the endophytic fungi comprise the following steps:
b100, inoculating the endophytic fungi strain obtained by purifying in the step A300 into a PDA culture medium;
b200, fermenting for several days, and then adding ethyl acetate to soak;
and B300, mashing the soaked PDA culture medium, soaking again, sequentially filtering, and concentrating under reduced pressure to obtain a crude extract of the endophytic fungi.
5. The separation and screening method according to claim 4, wherein: the activity screening step of the endophytic fungi comprises the following steps:
c100, taking a proper amount of the crude extract obtained in the step B300, and preparing a sample solution by using a DMSO solution;
c200, respectively culturing the ralstonia solanacearum in a plurality of NA liquid culture media, and preparing a bacterial liquid by shaking culture of a constant-temperature culture shaker;
c300, dipping a proper amount of bacteria liquid, respectively and uniformly coating the bacteria liquid on a plurality of NA culture media, dropwise adding a sample solution on a sterilized filter paper sheet, and placing the filter paper sheet on the NA culture media containing the bacteria liquid;
and C400, carrying out constant-temperature culture on the NA culture medium, measuring the diameter of the inhibition zone of the NA culture medium, and screening out the endophytic fungi with ralstonia solanacearum resistance.
6. The separation and screening method according to claim 2, wherein:
the preparation method of the PDA culture medium comprises the following steps of mixing the PDA culture medium with a mixture of the components in a mass concentration ratio of 200-220: 20-25: 15-20: 10-7The potato, the glucose, the agar, the kanamycin sulfate and the ampicillin are cultured by sterilizing for 40-45 min at the temperature of 121 ℃ in a high-temperature high-pressure environment at the natural pH value;
the preparation method of the PDA liquid culture medium comprises the following steps of mixing the components in a mass concentration ratio of 200-220: 20-25: 2-5: 0.1-0.2: 0.01:1 of potato, glucose and KH2PO4、MgSO4·7H2O, vitamin B1 and water, the natural pH value, and the sterilization is carried out for 25-35 min in the high-temperature high-pressure environment of 121 ℃ to form the culture medium;
the preparation method of the NA culture medium comprises the steps of sterilizing peptone, beef extract, sodium chloride and agar at the mass concentration ratio of 8-11: 2-4: 4-6: 15-20 for 25-35 min at 121 ℃ in a high-temperature high-pressure environment, and culturing;
the preparation method of the NA liquid culture medium comprises the steps of sterilizing peptone, beef extract and sodium chloride at the mass concentration ratio of 8-12: 2-3: 5-7 for 25-35 min at 121 ℃ in a high-temperature high-pressure environment, and culturing.
7. The separation and screening method according to claim 3, wherein: in step a300, the obtained endophytic fungal strain is subjected to molecular biological identification comprising:
a301, primarily screening 2 high-efficiency bacteria from the obtained endophytic fungi strains, culturing for 72h, and extracting DNA according to a TSP101 kit;
a302, observing through agarose electrophoresis, and performing genome PCR amplification on the strain by using a universal primer;
and A303, carrying out gene ITS sequencing on the amplified product, and comparing the sequencing result in Gen Bank by BLAST.
8. The separation and screening method according to claim 4, wherein:
in the step B200, after fermenting for 30-40 days at 28 ℃, adding ethyl acetate to soak for 2-3 h;
in the step B300, the soaked PDA culture medium is smashed and soaked again for 12-13 h, and after filtration, the crude extract of the endophytic fungi is obtained by decompression and concentration at 40-50 ℃ and 60 r/min.
9. The separation and screening method according to claim 5, wherein:
in step C200, the NA liquid culture medium is subjected to shake culture for 36h in a30 ℃ constant temperature culture shaker;
in step C400, the NA medium is incubated at 30 ℃ for 24 h.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112226384A (en) * | 2020-09-29 | 2021-01-15 | 广州中医药大学(广州中医药研究院) | Pathogenic strain causing patchouli bacterial wilt and application thereof |
| CN112300975A (en) * | 2020-09-29 | 2021-02-02 | 广州中医药大学(广州中医药研究院) | Low-pathogenicity mutant strain of pogostemon cablin ralstonia solanacearum and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828665A (en) * | 2017-11-01 | 2018-03-23 | 华南理工大学 | A kind of isolation and purification method of purpleback murdannia herb endogenetic fungus and its application |
-
2019
- 2019-09-26 CN CN201910914405.9A patent/CN110628625A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828665A (en) * | 2017-11-01 | 2018-03-23 | 华南理工大学 | A kind of isolation and purification method of purpleback murdannia herb endogenetic fungus and its application |
Non-Patent Citations (9)
| Title |
|---|
| NASRUN等: "《Bacterial wilt disease on patchouli and its control strategy》", 《JURNAL PENELITIAN DAN PENGEMBANGAN PERTANIAN 》 * |
| 丁奇等: "《青枯菌侵染广藿香的致病因素及寄主植物相关酶活性分析》", 《安徽农业科学》 * |
| 佘小漫等: "《广东广藿香青枯病病原菌鉴定》", 《植物病理学报》 * |
| 张 健等: "《毛泡桐内生真菌的分离、拮抗细菌菌株的筛选和鉴定》", 《微生物学杂志》 * |
| 易天凤等: "《海南广藿香内生真菌分离鉴定及拮抗菌株筛选》", 《热带作物学报》 * |
| 杨洁彬等编著: "《乳酸菌 生物学基础及应用》", 30 April 1996, 中国轻工业出版社 * |
| 王营等: "《广藿香内生真菌类群分析及其抗菌活性研究》", 《中国中药杂志》 * |
| 福建农学院主编: "《害虫生物防治》", 31 October 1981, 农业出版社 * |
| 谢华蓉等: "《广藿香内生真菌多样性及其对青枯菌的拮抗活性》", 《微生物学通报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112226384A (en) * | 2020-09-29 | 2021-01-15 | 广州中医药大学(广州中医药研究院) | Pathogenic strain causing patchouli bacterial wilt and application thereof |
| CN112300975A (en) * | 2020-09-29 | 2021-02-02 | 广州中医药大学(广州中医药研究院) | Low-pathogenicity mutant strain of pogostemon cablin ralstonia solanacearum and application thereof |
| CN112226384B (en) * | 2020-09-29 | 2022-05-10 | 广州中医药大学(广州中医药研究院) | Pathogenic strain causing patchouli bacterial wilt and application thereof |
| CN112300975B (en) * | 2020-09-29 | 2022-06-14 | 广州中医药大学(广州中医药研究院) | Low-pathogenicity mutant strain of pogostemon cablin ralstonia solanacearum and application thereof |
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