CN108949622A - One plant of streptomycete for producing chitinase and its application - Google Patents

Abstract

A strain of streptomyces producing chitinase and its application, the invention relates to a strain of streptomyces producing chitinase and its application. The purpose 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. A strain of chitinase-producing Streptomyces of the present invention is Streptomyces (Streptomyces sp.) G6 , preserved in the General Microorganism Center of China Microbiological 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 of 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 good antagonistic effect on soybean gray mold and black spot, has no pesticide residue, and does not produce drug resistance. The invention is applied to the technical field of environmental microorganisms.

Description

A strain of Streptomyces producing chitinase and its application

technical field

The invention relates to a strain of streptomyces producing chitinase and its application.

Background technique

Soybean is the main food crop in my country, and it is also the main economic crop and feed crop. With the continuous growth of the population and the improvement and improvement of living standards, the yield and quality of soybeans are the focus of attention. Botrytis cinerea and black spot of soybeans can infect soybeans throughout the growth period, causing soybean diseases, and are distributed in Jilin, Liaoning, Heilongjiang, Jiangsu, Sichuan and other provinces. According to existing reports, soybean gray mold and black spot not only infect soybeans, but also have pathogenicity on plants such as ginseng, citrus, sweet potato, cabbage, jujube fruit, and rose.

At present, the main method of controlling soybean gray mold and black spot is still based on chemical pesticides, but in view of the hidden dangers of pesticide residues in chemical pesticides.

Contents of the invention

The purpose of the present invention is to solve the problem of hidden dangers of pesticide residues in the existing methods for controlling soybean gray mold and black spot, and provide a strain of streptomyces producing chitinase and its application.

A strain of chitinase-producing Streptomyces of the present invention is Streptomyces sp. G6, which is preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee, and the preservation address is No. 1, Beichen West Road, Chaoyang District, Beijing. No., the deposit date is February 02, 2018, and the deposit number is CGMCC No.15334.

The application of the chitinase-producing streptomyces of the present invention refers to the application in the prevention and control of soybean diseases.

The present invention adopts wild rice rhizosphere soil to carry out the separation and purification of chitinase-producing strains:

Preliminary screening: Using the plate dilution coating method, weigh 5g of the rhizosphere soil sample from Dongxiang wild rice in Jiangxi, put it in 45ml of sterile water, 120rpm, let it stand for 20min, and take the supernatant for 10-fold concentration gradient dilution. Take 80 μL of the diluents with concentrations of 10 ^-1 , 10 ^-2 , and 10 ^-3 respectively, spread each gradient on 3 chitin medium plates, and use sterile water instead of diluents as a blank control. Place it upside down in an incubator at 25°C for 2-3 days, and observe the growth of colonies and the generation of transparent circles. (Chitin medium: K ₂ HPO ₄ 1g, MgSO ₄ 7H ₂ O 0.5g, NaCl 0.5g, NH ₄ Cl 1g, agar 15g, add distilled water to 1000ml, add 20ml 1% colloidal chitin.)

Re-screening: On the chitin medium, more than 60 colonies with large transparent circles but different shapes, colors, etc. were selected separately, and streaked on the plate until the colony was single, and the streptomyces producing chitinase were screened out Streptomycessp.G6, referring to some culture media and methods recommended in the "Common Bacteria System Identification Manual" and "Berger System Identification Manual", identified the physiological and biochemical characteristics of the G6 strain and found that the catalase of the G6 strain was positive, and the methyl red reaction was Negative, V-P reaction negative, starch hydrolysis positive, gelatin liquefied, no hydrogen sulfide produced, indole negative, tolerance to NaCl 10%.

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 there was an obvious characteristic band at 1500bp. Then the amplified product was sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing, and the obtained sequence was uploaded to Genbank. The homology comparison with the sequence in the gene bank was carried out by Blast analysis, and the phylogenetic tree was constructed by Neihbor-Joinhing in MEGA5.1 software (Figure 2). The strain G6 had the highest homology with Streptomyces, reaching 99%, and was finally named Streptomyces sp. G6.

The present invention provides a strain of Streptomyces G6 which has chitinase activity and has inhibitory effect on soybean gray mold and black spot obtained by screening in wild rice rhizosphere soil. The preservation number is CGMCC NO.15334, which is in Jiangxi In the rhizosphere soil of wild rice in different places in Dongxiang, the strains with strong ability to decompose chitin were obtained through primary selection, and then the strains with good antagonistic effects against soybean gray mold and black spot were repeatedly screened. A large number of experiments proved that , the strain G6 not only has chitinase activity, but also has a good antagonistic effect on soybean gray mold and black spot. The inhibitory effect of G6 fermentation broth on gray mold reaches 91.7%, and the inhibitory effect on black spot reaches 85.5%.

The present invention utilizes the means of biological control, that is, uses organisms or their metabolites to effectively prevent and control plant diseases. Compared with other traditional chemical control methods, it is more environmentally friendly, has no pesticide residues, and does not produce drug resistance. Effective utilization provides a theoretical basis. So as to lay a long-term development for green organic agriculture.

Description of drawings

Fig. 1 is the morphological figure of G6 bacterial strain on PDA plate;

Fig. 2 is the molecular identification clustering map of the G6 strain;

Fig. 3 is the antagonistic effect figure of G6 bacterial strain and Botrytis cinerea;

Fig. 4 is the antagonism effect figure of G6 bacterial strain and black spot;

Fig. 5 is G6 fermented liquid and Botrytis cinerea special effect factor to the inhibition effect figure of Botrytis cinerea;

Fig. 6 is the graph of G6 fermented liquid and difenoconazole to black spot inhibition effect;

Fig. 7 is the comparison chart of G6 fermented liquid and botrytis cinerea specific effect factor to Botrytis cinerea; Wherein a is G6 fermented liquid, b is botrytis cinerea specific effect factor;

Figure 8 is a comparison chart of the inhibitory effect of G6 fermentation broth and difenoconazole on black spot; wherein a is G6 fermentation broth, and c is difenoconazole.

Detailed ways

Specific embodiment one: a strain of chitinase-producing streptomyces in this embodiment is streptomyces (Streptomycessp.) G6, which is preserved in the General Microbiology Center of China Committee for the Collection of Microbial Strains, and the preservation address is Beichen West, Chaoyang District, Beijing No. 3, Courtyard No. 1, the preservation date is February 02, 2018, and the preservation number is CGMCCNo.15334.

In this embodiment, the wild rice rhizosphere soil is used to separate and purify chitinase-producing strains:

Preliminary screening: Using the plate dilution coating method, weigh 5g of the rhizosphere soil sample from Dongxiang wild rice in Jiangxi, put it in 45ml of sterile water, 120rpm, let it stand for 20min, and take the supernatant for 10-fold concentration gradient dilution. Take 80 μL of the diluents with concentrations of 10 ^-1 , 10 ^-2 , and 10 ^-3 respectively, spread each gradient on 3 chitin medium plates, and use sterile water instead of diluents as a blank control. Place it upside down in an incubator at 25°C for 2-3 days, and observe the growth of colonies and the generation of transparent circles. (Chitin medium: K ₂ HPO ₄ 1g, MgSO ₄ 7H ₂ O 0.5g, NaCl 0.5g, NH ₄ Cl 1g, agar 15g, add distilled water to 1000ml, add 20ml 1% colloidal chitin.)

Re-screening: On the chitin medium, more than 60 colonies with large transparent circles but different shapes, colors, etc. were selected separately, and streaked on the plate until the colony was single, and the streptomyces producing chitinase were screened out Streptomycessp.G6, referring to some culture media and methods recommended in the "Common Bacteria System Identification Manual" and "Berger System Identification Manual", identified the physiological and biochemical characteristics of the G6 strain and found that the catalase of the G6 strain was positive, and the methyl red reaction was Negative, V-P reaction negative, starch hydrolysis positive, gelatin liquefied, no hydrogen sulfide produced, indole negative, tolerance to NaCl 10%.

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 there was an obvious characteristic band at 1500bp. Then the amplified product was sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing, and the obtained sequence was uploaded to Genbank. The homology comparison with the sequence in the gene bank was carried out by Blast analysis, and the phylogenetic tree was constructed by Neihbor-Joinhing in MEGA5.1 software (Figure 2). The strain G6 had the highest homology with Streptomyces, reaching 99%, and was finally named Streptomyces sp. G6.

This embodiment uses the means of biological control, that is, the use of organisms or their metabolites to effectively prevent and control plant diseases. Compared with other traditional chemical control methods, it is more environmentally friendly and does not produce drug resistance. theoretical basis. So as to lay a long-term development for green organic agriculture.

Embodiment 2: The application of a strain of Streptomyces producing chitinase in this embodiment refers to the application in the prevention and control of soybean diseases.

This embodiment provides a strain of Streptomyces G6 that has chitinase activity and has inhibitory effects on soybean gray mold and black spot in wild rice rhizosphere soil. The preservation number is CGMCC NO.15334, which is in In the rhizosphere soil of wild rice in different locations in Dongxiang, Jiangxi, strains with strong ability to decompose chitin were obtained through primary selection, and then the strains with good antagonistic effects against soybean gray mold and black spot were screened repeatedly. After a large number of experiments It has been proved that the strain G6 not only has chitinase activity, but also has a good antagonistic effect on soybean gray mold and black spot. Reached 85.5%.

Embodiment 3: This embodiment is different from Embodiment 2 in that: Streptomyces producing chitinase is used to suppress black spot. Others are the same as in the second embodiment.

Embodiment 4: This embodiment is different from Embodiment 2 or 3 in that: Streptomyces producing chitinase is used to inhibit Botrytis cinerea. Others are the same as the second or third specific embodiment.

Specific embodiment four: the screening method of Streptomyces sp.G6 that produces chitinase in this embodiment is as follows: primary screening: adopt plate dilution coating method, weigh 5 g of rhizosphere soil samples derived from Jiangxi Dongxiang wild rice in 45ml sterile water, 120rpm, let stand after 20min, take the supernatant and carry out 10-fold concentration gradient dilution. Take 80 μL of the diluents with concentrations of 10 ^-1 , 10 ^-2 , and 10 ^-3 respectively, spread each gradient on 3 chitin medium plates, and use sterile water instead of diluents as a blank control. Place it upside down in an incubator at 25°C for 2-3 days, and observe the growth of colonies and the generation of transparent circles. (Chitin medium: K ₂ HPO ₄ 1g, MgSO ₄ 7H ₂ O 0.5g, NaCl 0.5g, NH ₄ Cl 1g, agar 15g, add distilled water to 1000ml, add 20ml 1% colloidal chitin.)

Re-screening: On the chitin medium, more than 60 colonies with large transparent circles but different shapes, colors, etc. were selected separately, and streaked on the plate until the colony was single, and the streptomyces producing chitinase were screened out Streptomycessp.G6, the morphology of the G6 strain on the PDA plate is shown in Figure 1. Referring to some culture media and methods recommended in the "Common Bacterial System Identification Manual" and "Berger System Identification Manual", the physiological and biochemical characteristics of the G6 strain were identified. It was found that the catalase of the G6 strain was positive, the methyl red reaction was negative, and the V-P reaction was negative. Negative, positive for starch hydrolysis, liquefaction of gelatin, no production of hydrogen sulfide, negative for indole, 10% tolerance to NaCl.

Specific embodiment five: the identification of Streptomyces sp.G6 that produces chitinase in this embodiment:

1. Physiological and biochemical identification

Referring to some culture media and methods recommended in the "Common Bacterial System Identification Manual" and "Berger System Identification Manual", the physiological and biochemical characteristics of the G6 strain were identified. It was found that the catalase of the G6 strain was positive, the methyl red reaction was negative, and the V-P reaction was negative. Negative, positive for starch hydrolysis, liquefaction of gelatin, no production of hydrogen sulfide, negative for indole, 10% tolerance to NaCl.

Table 1 Physiological and biochemical characteristics of strain G6

Note: "+" means positive, "-" means 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 general primers for PCR are: 27F (5ˊ-AGAGTTTGATCCTGGCTCAG-3ˊ), 1492R (5ˊ-GGTTACCTTGTTACGACTT-3ˊ), reaction system 25μl, Premix version 2.0 12.5μl, 27F (10nM) 1μl, 1492R (10nM) 1μl, DNA template 1μl, no Bacterial water is insufficient to 25μl. The reaction conditions were: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 56°C for 50 s, extension at 72°C for 90 s, 35 cycles; final extension at 72°C for 10 min.

After PCR amplification, the product was checked by 1.5% agarose gel electrophoresis, and there was an obvious characteristic band at 1500bp. Then the amplified product was sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing, and the obtained sequence was uploaded to Genbank. The homology comparison with the sequence in the gene bank was carried out by Blast analysis, and the phylogenetic tree was constructed by Neihbor-Joinhing in MEGA5.1 software (Figure 2). The strain G6 had the highest homology with Streptomyces, reaching 99%, and was finally named Streptomyces sp. G6.

Functional verification of the resistance of the chitinase-producing Streptomyces sp.G6 to soybean gray mold and black spot in this embodiment:

Experiment 1. Antagonism of Botrytis cinerea and black spot by plate confrontation method

The antibacterial activity of antagonistic strains against botrytis cinerea and black spot was determined by plate confrontation method. First, soybean gray mold and black spot were respectively activated on the PDA plate, and the pathogen plate with vigorous growth was selected. Use a puncher to make a 7mm-diameter bacterial cake, place it upside down on a PDA plate, inoculate the bacterial cake of the antagonistic bacteria strain about 2 cm away from the pathogenic bacteria, and use the plate without the antagonistic bacteria as a control, with 3 repetitions in each group, Place them in a constant temperature incubator at 26° C. for 2-6 days, and observe the size of the inhibition zone ( FIG. 3 , FIG. 4 ). And on the 2nd, 4th, and 6th days, record the minimum radius of the colony of the pathogenic bacteria and the radius of the control colony, and calculate the bacteriostatic rate.

Bacterial inhibition rate (%)=(control colony radius-pathogen colony radius)/control colony radius×100

According to the antibacterial rate data of different days, the inhibitory effect of G6 strain on Botrytis cinerea can still reach 82.3%, and the inhibitory effect on black spot can reach 79.9% on the 6th day.

Experiment 2: Inhibitory effects of G6 fermentation broth, botrytis cinerea specific effect factor and difenoconazole on botrytis cinerea and black spot

Preparation of G6 fermentation broth: 100ml of fermentation medium (fermentation medium: 10.0g of glucose, 7.5g of yeast extract, distilled water to 1000ml) was placed in a 250ml Erlenmeyer flask, and autoclaved at 121°C. Put the G6 bacteria cake into the cooled liquid medium in the ultra-clean workbench, ferment and cultivate it in a constant temperature shaking shaker at 160rpm for 3 days, and obtain the fermentation broth, centrifuge the fermentation broth at 3500rpm for 5 minutes to collect the bacteria, and wash with sterile water After 2 times, resuspend in sterile water, measure the concentration with a hemocytometer and dilute it to 10 ⁷ cfu/ml for later use.

Preparation of pesticide dilution: Difenoconazole (50%) (50%) (North American Agricultural University) and difenoconazole (50%) (North American Agricultural University) were diluted 500-1000 times according to the instructions. In order to verify the inhibitory effect of G6 on pathogenic bacteria, we Botrytis cinerea-specific effects and difenoconazole were diluted 250 times and 500 times respectively.

Antagonism test: take 100 μl of G6 fermentation filter, 250 times of botrytis cinerea specific effect dilution, 500 times of gray mold dilution, 250 times of 50% difenoconazole dilution, 500 times of difenoconazole dilution Spread on PDA plates, inoculate 7mm gray mold and black spot pathogenic bacteria cakes in the center of the plate respectively, use sterile water as the control (Figure 5, Figure 6), each group has 3 replicates, and culture at a constant temperature of 26°C. , 3, 4, 5 days to measure the diameter of pathogenic bacteria, calculate the rate of inhibition.

Bacterial inhibition rate (%)=(control colony diameter-pathogen colony diameter)/control colony diameter×100%

The results show that: since the 500-fold diluted botrytis cinerea and difenoconazole have almost no inhibitory effect on gray mold and black spot, only the G6 fermentation broth and 250-fold botrytis cinerea are compared in the figure and the inhibitory effect of difenoconazole (Fig. 7, Fig. 8), wherein as the number of days increases, the inhibitory effect of G6 fermented liquid on Botrytis cinerea and black spot is gradually enhanced, and on the 5th day, G6 fermented liquid has an effect on gray mold The inhibitory effect on mildew reached 91.7%, and the inhibitory effect on black spot reached 85.5%, while the effects of botrytis cinerea and difenoconazole were gradually decreasing, and both botrytis cinerea and difenoconazole Inhibition was best at day 2, 95.9% and 47.3%, respectively.

sequence listing

<110>Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences

<120>A Strain of Streptomyces Producing Chitinase and Its Application

<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 acggggccc 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 tcacggggaga 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

AGAGTTTGATCCTGGCTCAG 20

<210> 3

<211> 19

<212>DNA

<213> Artificial sequence

<220>

<223>PCR primer 1492R nucleotide sequence.

<400> 3

GGTTACCTTGTTACGACTT 19

Claims (4)

1. the streptomycete of one plant of production chitinase, it is characterised in that it is streptomycete (Streptomyces sp.) G6, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address are BeiChen West Road, Chaoyang District, BeiJing Cities 1 Institute 3, preservation date are on February 02nd, 2018, and deposit number is CGMCC No.15334.

2. the application of the streptomycete of one plant of production chitinase as described in claim 1, it is characterised in that produce the chain of chitinase Application of the mould in soybean diseases prevention and treatment.

3. the application of the streptomycete of one plant of production chitinase according to claim 2, it is characterised in that produce chitinase Streptomycete is applied in the inhibition to black spot.

4. the application of the streptomycete of one plant of production chitinase according to claim 2, it is characterised in that produce chitinase Streptomycete is applied in the inhibition to gray mold.