CN111748488B - Application of marine actinomyces HN60 in preparation of antifungal active substances - Google Patents

Application of marine actinomyces HN60 in preparation of antifungal active substances Download PDF

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CN111748488B
CN111748488B CN202010492998.7A CN202010492998A CN111748488B CN 111748488 B CN111748488 B CN 111748488B CN 202010492998 A CN202010492998 A CN 202010492998A CN 111748488 B CN111748488 B CN 111748488B
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杨革
孙萍
车程川
刘金锋
巩志金
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Abstract

The invention belongs to the technical field of agriculture, and particularly relates to application of a marine actinomycete strain HN60 in preparation of antifungal active substances. Through 16S rDNA sequence analysis and physiological and biochemical experiments, the strain is streptomyces griseofulensis, and the preservation number of the strain is CGMCC No. 18948. The antibacterial active substance is derived from fermentation liquor obtained by liquid fermentation of a strain HN60, and the liquid fermentation conditions of the strain HN60 are as follows: performing shake culture in culture medium at 30 deg.C and 180r/min for 6 d; wherein the culture medium comprises the following components: 4g of yeast extract powder, 4g of glucose, 10g of malt extract powder, 1L of seawater and pH 7.2-7.4. The antibiotic produced by the strain has good inhibition effect on a plurality of pathogenic bacteria such as aspergillus flavus, rice blast, soybean anthrax, asparagus wilt and the like, and can be used for preparing microbial agents for preventing and treating crop diseases.

Description

Application of marine actinomyces HN60 in preparation of antifungal active substances
Technical Field
The invention belongs to the technical field of agriculture, relates to microorganisms and application thereof, and particularly relates to application of marine actinomyces HN60 in preparation of antibacterial true active substances.
Technical Field
Biological control is one of the important contents for crop disease control. The long-term continuous use of chemical pesticides brings a series of serious problems to crop diseases and the environment, and mainly shows that the control effect is reduced, the pesticide residue exceeds the standard, the natural control force is weakened and the environment is seriously polluted. Particularly, the concepts of ecological agriculture, organic agriculture, environmental protection and the like are emphasized since 90 years, and the biological control development of China is rapid.
Actinomycetes have obvious antagonistic effect on a plurality of pathogenic bacteria as biocontrol bacteria, and a plurality of forming preparations such as jinggangmycin are applied to production. With the application of agricultural antibiotics, actinomycetes as an antibiotic-producing bacterium has been widely noted. Streptomyces is a higher actinomycete, is one of actinomycete with more varieties and quantities, and plays an important role in preventing and treating plant diseases.
Disclosure of Invention
The invention aims to provide application of marine actinomycetes HN60 in preparation of antifungal active substances, and fermentation liquor of the strain HN60 has a good inhibition effect on pathogenic bacteria of aspergillus flavus, rice blast, soybean anthrax and asparagus wilt.
A marine actinomycete strain HN60 is an actinomycete separated from soil collected in Haikou mangrove area by the inventor. The preservation information of the strain is as follows:
name:Streptomyces griseoplanus HN60, hereinafter abbreviated as HN 60;
the preservation number is: CGMCC No. 18948;
preservation time: 11/13/2019.
The method for separating and culturing the marine actinomycete HN60 comprises the following steps:
the soil collected in the Haikou mangrove area is placed for 14-21 days at room temperature for air drying, and then 4 different culture media are used for screening the marine actinomycetes on the sample, and the formula of the screening culture medium is shown in table 1. Serial dilution of soil sample with sterile physiological saline solution to concentration of 10-1~10-3. And (3) respectively taking 100 mu L of the diluents with different concentrations, coating the diluents with different concentrations on different screening culture media (potassium dichromate and nalidixic acid are respectively added into all the screening culture media, the final concentrations are respectively 75 mu g/mL and 25 mu g/mL, culturing at 28 ℃ for 14-28 d, separating and purifying the screened actinomycetes by adopting a Gauss first culture medium, and storing the purified actinomycetes at-80 ℃ for later use.
TABLE 1 Marine Actinomycetes screening Medium formulation
Figure DEST_PATH_IMAGE001
The invention also classifies the strain HN60 according to morphological characteristics. Strain HN60 was inoculated on a medium and exhibited morphological characteristics as: the bacterial colony is round, light yellow, has dry surface and is convex, and hyphae are rich, branched and have no fracture.
The invention classifies and identifies the strain HN60 by a 16S rDNA sequence method. The DNA of the strain was extracted and the 16S rDNA sequence was amplified. It can be known from phylogenetic trees at the 16S rDNA sequence construction position in the prior publications of the strain HN60 and streptomycete, and the strain HN60 belongs to Streptomyces griseoplanus.
Furthermore, the antibacterial active substance is derived from a fermentation liquor obtained by liquid fermentation of a strain HN60, and the fermentation conditions are as follows: performing shake culture in a culture medium at 28 deg.C and 180r/min for 6 d; wherein the culture medium comprises the following components: 4g/L of yeast extract powder, 4g/L of glucose, 10g/L of malt extract powder and pH of 7.2-7.4.
Furthermore, the fermentation liquor of the strain HN60 is used for inhibiting the biological activity of Aspergillus flavus, Magnaporthe grisea, soybean anthrax or asparagus fusarium wilt pathogenic bacteria.
Further, the fermentation liquor of the strain HN60 is used for preparing microbial agents for preventing and treating crop diseases.
The strain preservation information of the marine actinomycetes is as follows:
preservation time: 11/13/2019, and 11/month,
the preservation unit: china general microbiological culture Collection center,
the preservation number is: the CGMCC No.18948,
the address of the depository: the microbiological research institute of western road 1, 3, national academy of sciences, north-kyo, chaoyang, the postal code: 100101
And (3) classification and naming:Streptomyces griseoplanus。
advantageous effects
The invention provides application of marine actinomyces HN60 in preparation of antibacterial active substances. The strain can be used as a biological material for researching and developing a high-efficiency broad-spectrum microbial bactericide, the fermentation liquor of the strain HN60 can be directly used as a bactericidal substance for inhibiting various crop pathogenic bacteria, the operation process is simple, and the stability is good.
Drawings
FIG. 1(a) is a colony morphology of strain HN 60; FIG. 1 (b) is the scanning electron microscope result of strain HN 60;
FIG. 2 is a phylogenetic analysis of strain HN 60.
Detailed description of the invention
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and 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.
Example 1: isolation and characterization of Strain HN60
(1) The marine actinomycete strain HN60 is an actinomycete separated from the soil of Haikou mangrove.
1 g of the sample was weighed, dissolved in 9 mL of sterile water, and shaken in a shaker at 28 ℃ for 30 min. Taking the upper layer soil suspension for gradient dilution, and respectively diluting to 10-1~10-3100 uL of each gradient is taken and respectively coated on a Gao's first culture medium, a YMG culture medium, an HVA culture medium and a sodium propionate culture medium (all the culture media are added with 25 ug/mL nalidixic acid to inhibit fast-growing bacteria, particularly gram-negative bacteria and 75 ug/mL potassium dichromate to inhibit the growth of bacteria and fungi before being poured into a flat plate), a single colony is selected for further purification, and the culture medium used for separating the single colony is the Gao's first culture medium. Streaking was performed 3 times in succession to purify a single colony, yielding strain HN 60.
The seed culture conditions of the strain HN60 are as follows: performing shake culture at 28 deg.C and 180r/min for 48 h; the culture medium comprises the following components: 4g of yeast extract powder, 4g of glucose, 10g of malt extract powder and pH 7.2-7.4.
(2) Morphological observation and physiological and biochemical experiment of actinomycete strain HN60
The bacterial colony morphology of the strain is observed, a scanning electron microscope is used for observing the strain by adopting an insert method, and the experimental result is shown in figure 1. The reference strain HN60 refers to the Streptomyces identification handbook for physiological and biochemical analysis, and the experimental results are shown in Table 2.
TABLE 2 physiological and biochemical experiments with strain HN60
Figure DEST_PATH_IMAGE002
Note: "+" indicates positive, and "-" indicates negative.
As can be seen from FIG. 1, the strain grows well on the solid culture medium of Gao's I, and when the strain is cultured for 7 days at 28 ℃, the colony is round and light yellow, and the surface is dry and convex. The strain is rich in hypha and free of rupture phenomenon when observed under a scanning electron microscope.
Table 2 shows the results of physiological and biochemical experiments with strain HN 60. As a result of experiments, the bacterial strain HN60 can liquefy gelatin, does not use cellulose, hydrolyzes starch, can reduce nitrate and cannot produce H2S, milk can not be coagulated or peptonized, and tyrosinase and melanoid can not be generated. Glucose, lactose, maltose, arabinose can be used, sucrose, inositol, mannitol, fructose can not be used.
(3) 16S rDNA gene sequencing method is adopted to classify and identify the actinomycete strain HN 60.
The 16S rDNA sequencing was performed by Jinan Leigo Co. The specific sequencing method is as follows: extracting genome DNA from thallus by liquid nitrogen grinding method, amplifying 16S rDNA by universal primer, detecting PCR product and sequencing.
27F：5’-AGAGTTTGATCCTGGCTCAG-3’
1492R：5’-AAGGAGGTGATCCAGCCGCA-3’
The 16S rDNA sequence of the strain HN60 (see appendix for sequence) was determined as follows:
it can be seen from the phylogenetic tree (fig. 2) constructed by the strain HN60 and streptomycete effective promiscuous 16S rDNA sequence: the similarity of the 16S rDNA sequence of the strain HN60 and the reference strain is between 98% and 99%, andStreptomyces griseoplanusthe phylogenetic relationship of (a) is the most closely, and the sequence similarity between them is 100%.
Example 2: inhibitory activity of bacterial strain HN60 fermentation liquor on pathogenic bacteria
The inhibitory activity of the fermentation liquor on aspergillus flavus, rice blast, soybean anthrax and reed is tested by adopting a cup and dish method. The results are shown in Table 3, and the bacterial strain HN60 has antibacterial activity on the four indicator bacteria, and the antibacterial effect is shown in Table 3.
TABLE 3 bacteriostatic circle diameter of strain HN60 fermentation liquid against four pathogenic bacteria
Figure DEST_PATH_IMAGE003
Example 3: prevention and treatment effect of strain HN60 fermentation liquor on aspergillus flavus
Taking Aspergillus flavus infection as an example, the peanut and the corn are infected by germs, and the fermentation liquor in the example 2 is used as pesticide after being sterilized. The surface of the peanuts was disinfected with 0.1% mercuric chloride, and the sterilized distilled water was washed 3 times to remove mercuric chloride. Preparation of Aspergillus flavus spore suspension with spore concentration of 108CFU/mL. Blank group, control group and experimental group are set in the experiment. The blank group is only subjected to surface disinfection, the control group is only added with 200 uL of aspergillus flavus spore suspension, and the experimental group is respectively added with the strain HN60 fermentation liquor except for the aspergillus flavus suspension. The surfaces of the peanuts and the corns are fully contacted with the spore suspension and the fermentation liquor, and the experimental result at the temperature of 28 ℃ is shown in a table 4.
TABLE 4 inhibition of Aspergillus flavus by fermentation broth
Figure DEST_PATH_IMAGE004
Example 4: strain HN60 fermentation liquor antibacterial substance stability research
The stability of the antibacterial substance in the fermentation liquor is measured from three aspects of temperature, acid, alkali and heredity.
Temperature stability: respectively placing the supernatant of the fermentation liquid in water bath at 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C, 90 deg.C and 100 deg.C for 1 hr, placing the control group at room temperature for 1 hr, measuring antibacterial activity of the fermentation liquid after the treatment, and setting 3 parallel groups. And (4) determining whether the antibacterial activity of the treated fermentation liquor is changed or not by comparing with a control group.
pH stability: adjusting pH of the supernatant of the fermentation liquor to 2, 4, 6, 8, 10, 12, respectively, placing in a water bath kettle at 25 deg.C for 1h, maintaining original fermentation liquor pH in a water bath kettle at 25 deg.C for 1h, adjusting pH to original fermentation liquor pH after treatment, measuring antibacterial activity of the fermentation liquor, and setting 3 parallel groups. And (4) determining whether the antibacterial activity of the treated fermentation liquor is changed or not by comparing with a control group.
Genetic stability: the strain HN60 was inoculated on a solid YMG medium and subcultured 1 time every 5 days for 7 consecutive generations. Inoculating each generation of strain in liquid YMG culture medium, and performing shake culture at 28 deg.C and 180r/min for 7 d. And (4) after the culture is finished, measuring the antibacterial activity of the fermented supernatant, and setting 3 parallel groups.
The experimental results of the temperature, the pH and the genetic stability of the antibacterial substance in the fermentation liquid are respectively shown in tables 5, 6 and 7, and the experimental results show that the antibacterial substance has good temperature stability, good stability in the range of pH 2-10 and good genetic stability.
TABLE 5 temperature stability of antibacterial substances in fermentation broths
Figure DEST_PATH_IMAGE005
TABLE 6 pH stability of antibacterial substances in fermentation broths
Figure DEST_PATH_IMAGE006
TABLE 7 genetic stability of antibacterial substances in fermentation broths
Figure DEST_PATH_IMAGE007
<110> university of Qufu Master
Application of <120> marine actinomycete HN60 in preparation of antifungal active substances
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1421
<212> DNA
<213> Streptomyces griseofulensis HN60
<400> 1
gtgcttacac atgcaggtcg aacgatgaag ccacttgcgg tggtggatta gtggcgaacg 60
ggtgagtaac acgtgggcaa tctgcccttc actctgggac aagccctgga aacggggtct 120
aataccggat aacactctgt cccgcatggg acggggttga aagctccggc ggtgaaggat 180
gagcccgcgg cctatcagct tgttggtggg gtaatggcct accaaggcga cgacgggtag 240
ccggcctgag agggcgaccg gccacactgg gactgagaca cggcccagac tcctacggga 300
ggcagcagtg gggaatattg cacaatgggc gaaagcctga tgcagcgacg ccgcgtgagg 360
gatgacggcc ttcgggttgt aaacctcttt cagcagggaa gaagcgaaag tgacggtacc 420
tgcagaagaa gcgccggcta actacgtgcc agcagccgcg gtaatacgta gggcgcaagc 480
gttgtccgga attattgggc gtaaagagct cgtaggcggc ttgtcacgtc ggatgtgaaa 540
gcccggggct taaccccggg tctgcattcg atacgggcta gctagagtgt ggtaggggag 600
atcggaattc ctggtgtagc ggtgaaatgc gcagatatca ggaggaacac cggtggcgaa 660
ggcggatctc tgggccatta ctgacgctga ggagcgaaag cgtggggagc gaacaggatt 720
agataccctg gtagtccacg ccgtaaacgt tgggaactag gtgttggcga cattccacgt 780
cgtcggtgcc gcagctaacg cattaagttc cccgcctggg gagtacggcc gcaaggctaa 840
aactcaaagg aattgacggg ggcccgcaca agcagcggag catgtggctt aattcgacgc 900
aacgcgaaga accttaccaa ggcttgacat ataccggaaa gcatcagaga tggtgccccc 960
cttgtggtcg gtatacaggt ggtgcatggc tgtcgtcagc tcgtgtcgtg agatgttggg 1020
ttaagtcccg caacgagcgc aacccttgtt ctgtgttgcc agcatgccct tcggggtgat 1080
ggggactcac aggagactgc cggggtcaac tcggaggaag gtggggacga cgtcaagtca 1140
tcatgcccct tatgtcttgg gctgcacacg tgctacaatg gccggtacaa tgagctgcga 1200
tgccgcgagg cggagcgaat ctcaaaaagc cggtctcagt tcggattggg gtctgcaact 1260
cgaccccatg aagtcggagt tgctagtaat cgcagatcag cattgctgcg gtgaatacgt 1320
tcccgggcct tgtacacacc gcccgtcacg tcacgaaagt cggtaacacc cgaagccggt 1380
ggcccaaccc cttgtggagg gagctgtcga aggtggaccc g 1421

Claims (1)

1. Application of marine actinomycetes HN60 in preparation of antifungal active substances is characterized in that marine actinomycetes HN60 is streptomyces griseofulensis (Streptomyces griseus) (A. clarkii, A. clarkii and A. clarkii, B. clarkii, A. clarkii and A. clarkiiStreptomyces griseoplanus) HN60, deposited in China general microbiological culture Collection center on 13.11.2019 with the preservation number of CGMCC No. 18948;
the antibacterial active substance is derived from fermentation liquor obtained by liquid fermentation of a bacterial strain HN60, and the fermentation conditions are as follows: performing shake culture in culture medium at 30 deg.C and 180r/min for 6 d; wherein the culture medium comprises the following components: 4g of yeast extract powder, 4g of glucose, 10g of malt extract powder, 1L of seawater and pH 7.2-7.4;
the fermentation liquor of the strain HN60 is used for inhibiting the biological activity of aspergillus flavus, rice blast mold or soybean anthrax.
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Citations (2)

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Publication number Priority date Publication date Assignee Title
DE2904628A1 (en) * 1978-02-08 1979-09-13 Meiji Seika Kaisha DERIVATIVES OF SUBSTANCE SF-1739, PROCESS FOR THE PREPARATION OF THE SAME AND AGENTS WITH ANTIBACTERIAL EFFECT AND ANTITUMOR EFFECT, CONTAINING THESE DERIVATIVES
CN106676040A (en) * 2016-12-16 2017-05-17 吉林农业大学 Streptomyces griseoplanus, application thereof and microbial agent

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2904628A1 (en) * 1978-02-08 1979-09-13 Meiji Seika Kaisha DERIVATIVES OF SUBSTANCE SF-1739, PROCESS FOR THE PREPARATION OF THE SAME AND AGENTS WITH ANTIBACTERIAL EFFECT AND ANTITUMOR EFFECT, CONTAINING THESE DERIVATIVES
CN106676040A (en) * 2016-12-16 2017-05-17 吉林农业大学 Streptomyces griseoplanus, application thereof and microbial agent

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
optimization and characterization of biosurfactant from streptomyces griseoplanus NRRL-ISP5009(ms1);M.A.Elkhawaga;《Journal of applied microbiology》;20171231;第691-707页 *
Psychrotolerant antifungal Streptomyces isolated from Tawang, India and the shift in chitinase gene family;Rajal Debnath等;《Extremophile》;20131231;第1046页右栏第4段，表1，第1053页右栏第3段以及图3 *

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