CN108949622B - A Streptomyces strain producing chitinase and its application - Google Patents
A Streptomyces strain producing chitinase and its application Download PDFInfo
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Abstract
一株产几丁质酶的链霉菌及其应用，本发明涉及一株产几丁质酶的链霉菌及其应用。本发明的目的是要解决现有控制大豆灰霉病、黑斑病的方法存在着农残隐患的问题，本发明的一株产几丁质酶的链霉菌为链霉菌(Streptomyces sp.)G6，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址是北京市朝阳区北辰西路1号院3号，保藏日期是2018年02月02日，保藏编号为CGMCC No.15334。本发明的产几丁质酶的链霉菌的应用是指在大豆病害防治上的应用。菌株G6不仅具有几丁质酶活，且对大豆灰霉病、黑斑病有很好的拮抗作用，无农药残留，不产生抗药性。本发明应用于环境微生物技术领域。
A chitinase-producing Streptomyces strain and its application, the invention relates to a chitinase-producing Streptomyces strain and its application. The object of the present invention is to solve the problem that existing methods for controlling soybean gray mold and black spot have hidden dangers of pesticide residues, and a Streptomyces sp. chitinase-producing strain of the present invention is Streptomyces sp. G6 , preserved in the General Microbiology Center of the China Microorganism Culture Collection Management Committee, the preservation address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, the preservation date is February 2, 2018, and the preservation number is CGMCC No.15334. The application of the chitinase-producing Streptomyces in the present invention refers to the application in the prevention and control of soybean diseases. Strain G6 not only has chitinase activity, but also has a good antagonistic effect on soybean gray mold and black spot, and has no pesticide residues and no drug resistance. The invention is applied to the technical field of environmental microorganisms.
Description
Technical Field
The invention relates to a streptomyces strain for producing chitinase and application thereof.
Background
Soybean is a main grain crop in China, and is also a main economic crop and feed crop. With the increasing population and the improvement and promotion of living standard, the yield and quality of soybean are the focus of attention. The gray mold and black spot of the soybean can infect the whole growth period of the soybean, thereby causing the soybean diseases, and the soybean diseases are distributed in Jilin, Liaoning, Heilongjiang, Jiangsu, Sichuan and other provinces. It is known from the existing reports that the gray mold and the black spot of the soybean not only infect the soybean, but also have pathogenicity on plants such as ginseng, citrus, sweet potato, cabbage, date fruit, Chinese rose and the like.
At present, the main method for controlling the gray mold and the black spot of the soybean still takes chemical pesticides as main materials, but the chemical pesticides have hidden danger of pesticide residues.
Disclosure of Invention
The invention aims to solve the problem of pesticide residue hidden danger in the existing method for controlling gray mold and black spot disease of soybean, and provides a streptomyces strain for producing chitinase and application thereof.
The Streptomyces for producing chitinase is Streptomyces (Streptomyces sp.) G6, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, has the preservation address of No. 3 Xilu-Shih-1 of Beijing Korean area, the preservation date of 2018, 02 and 34, and the preservation number of CGMCC No. 15334.
The application of the streptomyces for producing chitinase of the invention refers to the application in prevention and treatment of soybean diseases.
The invention adopts wild rice rhizosphere soil to separate and purify chitinase-producing strains:
primary screening: weighing 5g of rhizosphere soil sample from wild rice in east and rural areas of Jiangxi province in 45ml of sterile water by adopting a plate dilution coating method, standing at 120rpm for 20min, and taking supernatant for 10-time concentration gradient dilution. Respectively take 10-1、10-2、10-3Dilutions were run at 80 μ L each, and each gradient was plated onto 3 plates of chitin medium, using sterile water instead of dilutions as a blank. The culture medium is placed in an incubator at 25 ℃ for 2-3 days in an inverted mode, and the growth of bacterial colonies and the generation of transparent rings are observed. (chitin Medium: K)2HPO41g，MgSO4·7H2O0.5g，NaCl 0.5g，NH4Cl 1g, agar 15g, distilled water to 1000ml, wherein 20ml 1% colloidal chitin is added into each 80ml chitin culture medium. )
Re-screening: on a chitin culture medium, 60 colonies with larger transparent circles but different shapes such as morphology, color and the like are respectively selected, plate streaking is carried out until the colonies are single, Streptomyces sp.G6 producing chitinase is selected, and the physiological and biochemical characteristics of the G6 strain are identified according to part of culture media and methods recommended in a common bacteria system identification manual and a Bergey system identification manual, so that the G6 strain is found to be positive in catalase, negative in methyl red reaction, negative in V-P reaction, positive in starch hydrolysis, liquefied in gelatin, free of hydrogen sulfide, negative in indole and 10% in NaCl tolerance.
The genome of the strain was extracted using a genome extraction Kit (FastDNA Spin Kit for Soil) and 16SrDNA was amplified. After PCR amplification, the product was checked by 1.5% agarose gel electrophoresis, and the result showed an obvious characteristic band at 1500 bp. The amplified product is then sent to Biotechnology engineering (Shanghai) Co., Ltd for sequencing, and the sequence is uploaded to Genbank. Homology alignment with sequences in the gene bank was performed by Blast analysis and phylogenetic evolutionary trees were constructed using Neihbor-joining in MEGA5.1 software (fig. 2). The G6 strain has the highest homology with Streptomyces, reaching 99 percent, and is finally named as Streptomyces sp G6.
The invention provides a streptomycete G6 strain which is obtained by screening in wild rice rhizosphere soil and has chitinase activity and an inhibiting effect on soybean gray mold and black spot, wherein the preservation number is CGMCC NO.15334, the strain is obtained by primarily selecting in wild rice rhizosphere soil in different places of the west, east and rural areas, and then repeatedly screening the strain with good antagonistic effect on soybean gray mold and black spot, a large number of experiments prove that the strain G6 not only has chitinase activity, but also has good antagonistic effect on soybean gray mold and black spot, the inhibiting effect of G6 fermentation liquor on gray mold reaches 91.7%, and the inhibiting effect on black spot reaches 85.5%.
The invention utilizes the means of biological control, namely, utilizes organisms or metabolites thereof to effectively control plant diseases, is more environment-friendly compared with other traditional chemical control, has no pesticide residue and no drug resistance, and provides theoretical basis for the development and effective utilization of microorganism resources for control. Thereby laying a long-term development for green organic agriculture.
Drawings
FIG. 1 is a map of the morphology of the G6 strain on PDA plates;
FIG. 2 is a molecular identification clustering map of strain G6;
FIG. 3 is a graph showing the antagonistic effect of G6 strain against gray mold;
FIG. 4 is a graph showing the antagonistic effect of G6 strain on melasma;
FIG. 5 is a graph showing the effect of fermentation broth G6 and Botrytis cinerea specific on Botrytis cinerea;
FIG. 6 is a graph showing the inhibitory effect of G6 fermentation broth and difenoconazole on black spot;
FIG. 7 is a graph showing the comparison of the effect of fermentation broth G6 and Botrytis cinerea specific on the inhibition of Botrytis cinerea; wherein a is G6 fermentation liquor, b is Botrytis cinerea specific element;
FIG. 8 is a graph comparing the inhibitory effect of G6 broth and difenoconazole on black spot; wherein a is G6 fermentation liquor, and c is difenoconazole.
Detailed Description
The first embodiment is as follows: the streptomyces strain for producing chitinase is streptomyces (streptomyces sp.) G6, and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 Xilu No.1 of Beijing Korean district, the preservation date is 2018, 02 and days, and the preservation number is CGMCC No. 15334.
In the embodiment, the chitinase-producing strains are separated and purified in wild rice rhizosphere soil:
primary screening: weighing 5g of rhizosphere soil sample from wild rice in east and rural areas of Jiangxi province in 45ml of sterile water by adopting a plate dilution coating method, standing at 120rpm for 20min, and taking supernatant for 10-time concentration gradient dilution. Respectively take 10-1、10-2、10-3Dilutions were run at 80 μ L each, and each gradient was plated onto 3 plates of chitin medium, using sterile water instead of dilutions as a blank. The culture medium is placed in an incubator at 25 ℃ for 2-3 days in an inverted mode, and the growth of bacterial colonies and the generation of transparent rings are observed. (chitin Medium: K)2HPO41g，MgSO4·7H2O0.5g，NaCl 0.5g，NH4Cl 1g, agar 15g, distilled water to 1000ml, wherein 20ml 1% colloidal chitin is added into each 80ml chitin culture medium. )
Re-screening: on a chitin culture medium, 60 colonies with larger transparent circles but different shapes such as morphology, color and the like are respectively selected, plate streaking is carried out until the colonies are single, Streptomyces sp.G6 producing chitinase is selected, and the physiological and biochemical characteristics of the G6 strain are identified according to part of culture media and methods recommended in a common bacteria system identification manual and a Bergey system identification manual, so that the G6 strain is found to be positive in catalase, negative in methyl red reaction, negative in V-P reaction, positive in starch hydrolysis, liquefied in gelatin, free of hydrogen sulfide, negative in indole and 10% in NaCl tolerance.
The genome of the strain was extracted using a genome extraction Kit (FastDNA Spin Kit for Soil) and 16SrDNA was amplified. After PCR amplification, the product was checked by 1.5% agarose gel electrophoresis, and the result showed an obvious characteristic band at 1500 bp. The amplified product is then sent to Biotechnology engineering (Shanghai) Co., Ltd for sequencing, and the sequence is uploaded to Genbank. Homology alignment with sequences in the gene bank was performed by Blast analysis and phylogenetic evolutionary trees were constructed using Neihbor-joining in MEGA5.1 software (fig. 2). The G6 strain has the highest homology with Streptomyces, reaching 99 percent, and is finally named as Streptomyces sp G6.
The embodiment utilizes a biological control means, namely, utilizes organisms or metabolites thereof to effectively control plant diseases, is more environment-friendly compared with other traditional chemical control, does not generate drug resistance, and provides a theoretical basis for the development and effective utilization of microorganism resources for control. Thereby laying a long-term development for green organic agriculture.
The second embodiment is as follows: the application of the streptomyces for producing chitinase of the embodiment is the application in prevention and treatment of soybean diseases.
The embodiment provides a streptomycete G6 strain which has chitinase activity and has an inhibiting effect on soybean gray mold and black spot disease and is obtained by screening in wild rice rhizosphere soil, wherein the preservation number is CGMCC NO.15334, the streptomycete G6 strain is obtained by primarily selecting in wild rice rhizosphere soil in different places of the west, the east and the rural areas to obtain a strain with strong ability of decomposing chitin, and then repeatedly screening a strain with good antagonistic effect on soybean gray mold and black spot disease, a large number of experiments prove that the strain G6 not only has chitinase activity, but also has good antagonistic effect on soybean gray mold and black spot disease, the inhibiting effect of G6 fermentation liquor on gray mold reaches 91.7%, and the inhibiting effect on black spot disease reaches 85.5%.
The third concrete implementation mode: the second embodiment is different from the first embodiment in that: the streptomyces producing chitinase is applied to inhibiting melasma. The rest is the same as the second embodiment.
The fourth concrete implementation mode: the second or third embodiment is different from the first or second embodiment in that: the streptomyces producing chitinase is applied to the inhibition of gray mold. The other embodiments are the same as the second or third embodiment.
The fourth concrete implementation mode: the true bookThe method for screening Streptomyces sp.G6 for producing chitinase by the application mode comprises the following steps: primary screening: weighing 5g of rhizosphere soil sample from wild rice in east and rural areas of Jiangxi province in 45ml of sterile water by adopting a plate dilution coating method, standing at 120rpm for 20min, and taking supernatant for 10-time concentration gradient dilution. Respectively take 10-1、10-2、10-3Dilutions were run at 80 μ L each, and each gradient was plated onto 3 plates of chitin medium, using sterile water instead of dilutions as a blank. The culture medium is placed in an incubator at 25 ℃ for 2-3 days in an inverted mode, and the growth of bacterial colonies and the generation of transparent rings are observed. (chitin Medium: K)2HPO41g，MgSO4·7H2O0.5g，NaCl 0.5g，NH4Cl 1g, agar 15g, distilled water to 1000ml, wherein 20ml 1% colloidal chitin is added into each 80ml chitin culture medium. )
Re-screening: on a chitin culture medium, 60 colonies with large transparent circles but different shapes such as morphology, color and the like are respectively selected, plate streaking is carried out until the colonies are single, and the form diagram of the screened Streptomyces sp.G6 and G6 strains producing chitinase on a PDA plate is shown in figure 1. According to the partial culture medium and the method recommended in the handbook for identifying common bacteria systems and the handbook for identifying Bergey systems, the physiological and biochemical characteristics of the G6 strain are identified, and the G6 strain is positive in catalase, negative in methyl red reaction, negative in V-P reaction, positive in starch hydrolysis, liquefied in gelatin, free of hydrogen sulfide, negative in indole and 10% in NaCl tolerance.
The fifth concrete implementation mode: the identification of Streptomyces sp.G6 producing chitinase in the embodiment:
1. physiological and biochemical identification
According to the partial culture medium and the method recommended in the handbook for identifying common bacteria systems and the handbook for identifying Bergey systems, the physiological and biochemical characteristics of the G6 strain are identified, and the G6 strain is positive in catalase, negative in methyl red reaction, negative in V-P reaction, positive in starch hydrolysis, liquefied in gelatin, free of hydrogen sulfide, negative in indole and 10% in NaCl tolerance.
TABLE 1 physiological and biochemical Properties of Strain G6
Note: "+" indicates positive, and "-" indicates negative.
2. Molecular identification
The genome of the strain was extracted using a genome extraction Kit (FastDNA Spin Kit for Soil) and 16SrDNA was amplified. The PCR universal primer is: 27F (5 '-agaggtttgattctcggctcag-3'), 1492R (5 '-ggttactctgttacgactt-3'), reaction 25 μ l, Premix version 2.012.5 μ l, 27F (10nM)1 μ l, 1492R (10nM)1 μ l, DNA template 1 μ l, sterile water deficient to 25 μ l. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 50s, extension at 72 ℃ for 90s, and 35 cycles; final extension at 72 ℃ for 10 min.
After PCR amplification, the product was checked by 1.5% agarose gel electrophoresis, and the result showed an obvious characteristic band at 1500 bp. The amplified product is then sent to Biotechnology engineering (Shanghai) Co., Ltd for sequencing, and the sequence is uploaded to Genbank. Homology alignment with sequences in the gene bank was performed by Blast analysis and phylogenetic evolutionary trees were constructed using Neihbor-joining in MEGA5.1 software (fig. 2). The G6 strain has the highest homology with Streptomyces, reaching 99 percent, and is finally named as Streptomyces sp G6.
The functionality of Streptomyces sp.G6 producing chitinase in antagonism of soybean gray mold and black spot is verified as follows:
experiment 1, antagonism of the plate confrontation method on gray mold and black spot of soybean
And (3) determining the bacteriostatic activity of the antagonistic strain on the gray mold and the black spot by adopting a plate confrontation method. Firstly, respectively activating the gray mold and the black spot of the soybean on a PDA flat plate, and selecting a pathogenic bacteria flat plate with vigorous growth vigor. Preparing a bacterial cake with diameter of 7mm with a puncher, placing upside down on a PDA plate, inoculating the bacterial cake of antagonistic bacterial strain at a position about 2cm away from pathogenic bacteria, wherein the plate without antagonistic bacterial strain is used as a control, each group is repeated for 3 times, placing in a constant temperature incubator at 26 deg.C, culturing for 2-6 days, and observing the size of the inhibition zone (figure 3, figure 4). And recording the minimum radius of the colony of the pathogenic bacteria and the radius of the control colony at the time of 2,4 and 6 days, and calculating the bacteriostasis rate.
Bacteriostatic ratio (%) - (control colony radius-pathogen colony radius)/control colony radius × 100
From the bacteriostatic rate data of different days, the inhibition effect of the G6 strain on gray mold still can reach 82.3% on day 6, and the inhibition effect on black spot can reach 79.9%.
Test 2: g6 fermentation liquor respectively has inhibition effect on gray mold and black spot with gray mold specific element and difenoconazole
Preparation of G6 fermentation broth: 100ml of fermentation medium (fermentation medium: 10.0g of glucose, 7.5g of yeast extract, distilled water to a constant volume of 1000ml) is filled into a 250ml triangular flask, and the mixture is autoclaved at 121 ℃. Inoculating G6 bacterial cake into cooled liquid culture medium in a clean bench, fermenting and culturing for 3 days in a constant temperature shaking table at 160rpm to obtain fermentation liquid, centrifuging the fermentation liquid at 3500rpm for 5 minutes to collect thallus, washing with sterile water for 2 times, resuspending in sterile water, measuring concentration with a blood counting plate, and diluting to 107cfu/ml for use.
Preparing a pesticide diluent: the commercial gray mold special effect element (North America Nongda) and the difenoconazole (50%) (North America Nongda) are diluted by 500-fold and 1000-fold according to the instructions, and in order to verify the inhibiting effect of G6 on pathogenic bacteria, the gray mold special effect element and the difenoconazole are respectively diluted by 250-fold and 500-fold.
Antagonistic test: respectively taking 100 mu l G6 of fermentation filter, 250 times of gray mold specific element diluent, 500 times of gray mold diluent, 250 times of 50% difenoconazole diluent and 500 times of difenoconazole diluent to coat on a PDA (personal digital assistant) plate, respectively inoculating 7mm gray mold and black spot pathogenic bacteria cakes in the center of the plate, taking sterile water as a reference (figure 5 and figure 6), repeating each group for 3 times, culturing at constant temperature of 26 ℃, measuring the diameters of pathogenic bacteria on days 2, 3, 4 and 5, and calculating the bacteriostasis rate.
The bacteriostatic ratio (%) - (control colony diameter-pathogen colony diameter)/control colony diameter × 100%
The results show that: since 500-fold diluted gray mold special effect element and difenoconazole have almost no inhibition effect on gray mold and black spot, the inhibition effects of G6 fermentation liquor and 250-fold gray mold special effect element and difenoconazole are only compared in the figure (figure 7, figure 8), wherein the inhibition effects of G6 fermentation liquor on gray mold and black spot are gradually enhanced along with the increase of days, the inhibition effect of G6 fermentation liquor on gray mold reaches 91.7% and the inhibition effect on black spot reaches 85.5% on day 5, while the inhibition effects of gray mold special effect element and difenoconazole are gradually reduced, and the inhibition effects of gray mold special effect element and difenoconazole on day 2 are the best, respectively 95.9% and 47.3%.
Sequence listing
<110> institute of geography and agroecology of northeast China academy of sciences
<120> streptomycete strain for producing chitinase and application thereof
<160> 1
<210> 1
<211> 1428
<212> DNA
<213> Streptomyces sp
<400> 1
cgccggcggg cgtgctacac atgcagtcga cgatgaagcc cttcggggtg gattagtggc 60
gaacgggtga gtaacacgtg ggcaatctgc cctgcactct gggacaagcc ctggaaacgg 120
ggtctaatac cggatatgac catcttgggc atccttgatg gtgtaaagct ccggcggtgc 180
aggatgagcc cgcggcctat cagcttgttg gtgaggtaac ggctcaccaa ggcgacgacg 240
ggtagccggc ctgagagggc gaccggccac actgggactg agacacggcc cagactccta 300
cgggaggcag cagtggggaa tattgcacaa tgggcgaaag cctgatgcag cgacgccgcg 360
tgagggatga cggccttcgg gttgtaaacc tctttcagca gggaagaagc gagagtgacg 420
gtacctgcag aagaagcgcc ggctaactac gtgccagcag ccgcggtaat acgtagggcg 480
caagcgttgt ccggaattat tgggcgtaaa gagctcgtag gcggcttgtc gcgtcggttg 540
tgaaagcccg gggcttaacc ccgggtctgc agtcgatacg ggcaggctag agttcggtag 600
gggagatcgg aattcctggt gtagcggtga aatgcgcaga tatcaggagg aacaccggtg 660
gcgaaggcgg atctctgggc cgatactgac gctgaggagc gaaagcgtgg ggagcgaaca 720
ggattagata ccctggtagt ccacgccgta aacggtgggc actaggtgtg ggcaacattc 780
cacgttgtcc gtgccgcagc taacgcatta agtgccccgc ctggggagta cggccgcaag 840
gctaaaactc aaaggaattg acgggggccc gcacaagcgg cggagcatgt ggcttaattc 900
gacgcaacgc gaagaacctt accaaggctt gacatacacc ggaaagcatt agagatagtg 960
ccccccttgt ggtcggtgta caggtggtgc atggctgtcg tcagctcgtg tcgtgagatg 1020
ttgggttaag tcccgcaacg agcgcaaccc ttgtcccgtg ttgccagcag gcccttgtgg 1080
tgctggggac tcacgggaga ccgccggggt caactcggag gaaggtgggg acgacgtcaa 1140
gtcatcatgc cccttatgtc ttgggctgca cacgtgctac aatggccggt acaatgagct 1200
gcgataccgt gaggtggagc gaatctcaaa aagccggtct cagttcggat tggggtctgc 1260
aactcgaccc catgaagtcg gagtcgctag taatcgcaga tcagcattgc tgcggtgaat 1320
acgttcccgg gccttgtaca caccgcccgt cacgtcacga aagtcggtaa cacccgaagc 1380
cggtggccca accccttgtg ggagggagct tcgaagtgac catcctcg 1428
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence of PCR primer 27F.
<400> 2
<210> 3
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> PCR primer 1492R nucleotide sequence.
<400> 3
GGTTACCTTG TTACGACTT 19
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| CN106434493A (en) * | 2016-12-01 | 2017-02-22 | 北京市农林科学院 | Strain of biocontrol Streptomyces and application thereof |
| CN107083349A (en) * | 2017-06-28 | 2017-08-22 | 北京市农林科学院 | The white black streptomycete of one plant of disease prevention growth-promoting and its preparation and application of metabolite |
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| CN106434493A (en) * | 2016-12-01 | 2017-02-22 | 北京市农林科学院 | Strain of biocontrol Streptomyces and application thereof |
| CN107083349A (en) * | 2017-06-28 | 2017-08-22 | 北京市农林科学院 | The white black streptomycete of one plant of disease prevention growth-promoting and its preparation and application of metabolite |
Non-Patent Citations (2)
| Title |
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| Construction of a Streptomyces lydicus A01 transformant with a chit42 gene from Trichoderma harzianum P1 and evaluation of its biocontrol activity against Botrytis cinerea;Qiong Wu等;《Journal of Microbiology》;20130427;第51卷(第2期);全文 * |
| 链霉菌769与百日草黑斑病病菌诱导百日草几丁质酶基因的表达差异;张振鲁等;《中国植保导刊》;20130930;第33卷(第9期);第5页摘要，第8页左栏最后1段 * |
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