CN110819561B - Actinomycete TL-007 and application thereof - Google Patents
Actinomycete TL-007 and application thereof Download PDFInfo
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Abstract
一株放线菌TL‑007及其应用，涉及一株放线菌TL‑007及其应用。本发明的目的是要提供一株具有广谱拮抗作用的放线菌TL‑007及其应用。本发明一株放线菌TL‑007为黑胡桃链霉菌(Streptomyces nogalater)TL‑007，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2019年9月29日，保藏编号CGMCC No.18633。本发明的一株放线菌TL‑007的应用是指在作物病害防治上的应用。TL‑007对锈腐病的抑菌率为85.93％，为高效拮抗人参锈腐病的菌株且具有广谱拮抗作用。本发明应用于环境微生物技术领域。
An actinomycete TL-007 and its application, relating to an actinomycete TL-007 and its application. The purpose of the present invention is to provide an actinomycete TL-007 with broad-spectrum antagonism and its application. An actinomycete TL-007 of the present invention is Streptomyces nogalater TL-007, which is preserved in the General Microorganism Center of the China Microbial Culture Collection and Administration Commission, and the preservation address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. No., the deposit date is September 29, 2019, and the deposit number is CGMCC No.18633. The application of the actinomycetes TL-007 of the present invention refers to the application in the prevention and control of crop diseases. The bacteriostatic rate of TL‑007 against rust rot is 85.93%, which is a highly effective strain against ginseng rust rot and has a broad-spectrum antagonistic effect. The invention is applied to the technical field of environmental microorganisms.
Description
Technical Field
The invention relates to an actinomycete TL-007 and application thereof.
Background
In recent years, the chemical method for preventing and treating crop diseases has the characteristics of short time and quick response, but has the serious problem of chemical pesticide residue in the prevention and treatment process, and the problems of environmental pollution and drug resistance of germs caused by excessive use of chemical pesticides. For example, ginseng is one of the famous and precious Chinese traditional medicinal materials for preventing and treating ginseng rust rot, but various diseases often occur in the cultivation process to influence the yield and quality of ginseng. The ginseng rust rot is a soil-borne disease of ginseng roots, and chlamydospores of the ginseng rust rot can survive in soil for a long time in a dormant mode under a host-free condition to cause soil bacteria, so that the planting of the next batch of ginseng is directly influenced, and the ginseng is prevented from continuous cropping. The chemical method is adopted for preventing and treating the ginseng root from accumulating a large amount of toxic substances, which seriously affects the quality of the ginseng; the traditional agricultural control adopts measures of applying organic fertilizer, reasonably rotating crop, changing autumn planting into spring planting, changing old ginseng land into farmland and the like, but most of the measures can only play a role in prevention. Therefore, biological control is one of the main approaches for controlling crop diseases, and a biocontrol bacterium with broad-spectrum antagonism is urgently needed.
Disclosure of Invention
The invention aims to provide an actinomycete TL-007 with broad-spectrum antagonistic action and application thereof.
The actinomycete TL-007 is streptomycete nigricans (Streptomyces niger) TL-007 which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 Siro No.1 of Beijing Korean district, the preservation date is 2019, 9 and 29 days, and the preservation number is CGMCC No. 18633.
The application of the actinomycete TL-007 is the application in crop disease control.
The invention adopts a plate dilution method to separate and obtain 54 ginseng rhizosphere actinomycetes from mountain city in Jilin province, and carries out screening of antagonistic rust rot.
And (3) separation and purification of actinomycetes: taking 1.0g of a soil sample, air-drying, grinding and dispersing uniformly, and mixing the materials in a proportion of 1: dissolving in 10% sterile water, filtering to obtain filtrate 10-1Starting solution, successively obtaining 10-2And 10-3And (5) soil gradient dilution. Respectively diluting and coating on Gao's 1 and PDA isolated culture medium, culturing at 28 deg.C for 7d until single colony grows out, selecting single colony, and purifying on culture medium to obtain single strain.
Screening antagonistic actinomycetes: the method comprises inoculating rust rot bacteria on PDA culture medium for activation by plate confrontation. On a sterile operating platform, a 1cm puncher is used for taking a rusty rot fungi cake to be placed in the center of a PDA flat plate, antagonistic bacteria hyphae are taken to draw a straight line at a position about 2cm away from pathogenic bacteria, and the flat plate without antagonistic bacteria hyphae is used as a contrast. Each set of 3 replicates was incubated in a 28 ℃ incubator and colony growth was observed and the minimum radius of the bacteria and the radius of the control colony were recorded at 5 d. The calculation formula ═ (control colony radius-pathogen colony radius)/control colony radius × 100%. The bacteriostatic rate of TL-007 to rust rot is 85.93%.
ITS sequence and phylogenetic analysis of actinomycete TL-007: the obtained 16S rDNA gene sequence of the strain is compared with known sequences in a GenBank database by BLAST, and the MEGA 5.1 is adopted to construct a phylogenetic tree TL-007 strain which belongs to the same branch of Streptomyces nogalaters, and the similarity of the strain reaches 100 percent. Finally named as Streptomyces nigricans (Streptomyces nogalaters) TL-007 through morphological characteristics and molecular biological identification results.
The invention discloses a ginseng rhizosphere actinomycete TL-007 strain with the preservation number of CGMCC NO. 18633. Among a large amount of ginseng rhizosphere microorganisms, a plurality of strains capable of antagonizing ginseng rust rot are obtained through primary screening, and then the strains with the best antagonistic effect are obtained through repeated screening, wherein the bacteriostatic rate of TL-007 to the rust rot is 85.93%. Through identification, the strain TL-007 is black walnut Streptomyces (Streptomyces nogalaters), and the high-efficiency antagonistic strain can effectively inhibit rust rot of ginseng and is free of pathogenicity. Lays a good foundation for the development and the utilization of bacterial manure for preventing and treating ginseng rust rot. The bacterial strain is subjected to an antibacterial spectrum experiment, and the experiment shows that the antibacterial rates of TL-007 on soybean black spot, soybean gray mold, Chinese cabbage black rot and ginseng fusarium are respectively as follows: 80.37 percent, 76.37 percent, 81.48 percent and 82.66 percent, which shows that the compound has antagonism effect on diseases of crops such as soybean, Chinese cabbage, ginseng and the like and has broad-spectrum antagonism effect. The strain TL-007 is a strain for efficiently antagonizing ginseng rust rot and has a broad-spectrum antagonistic effect, and the biological control means, namely, the organisms or metabolites thereof are utilized to effectively control plant diseases, so that compared with other traditional chemical control means, the strain TL-007 is more environment-friendly, has no pesticide residue and does not generate drug resistance.
Drawings
FIG. 1 is the colony morphology of TL-007 strain on PDA medium;
FIG. 2 is a microscopic picture of TL-007 strain;
FIG. 3 is a graph of antagonism of the TL-007 strain to ginseng rust rot;
FIG. 4 is the bacterial inhibition spectrum of TL-007 strain;
FIG. 5 is a molecular identification map of TL-007 strain;
FIG. 6 is a preliminary back-grafting validation of TL-007 strain on ginseng sections.
Detailed Description
The first embodiment is as follows: the actinomycete TL-007 of the embodiment is streptomycete nigricans (Streptomyces niger) TL-007 which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 Siro No.1 of Beijing Kogyo area north Chen, the preservation date is 2019, 29 days 9 and 18633, and the preservation number is CGMCC No. 18633.
In the embodiment, 54 ginseng rhizosphere actinomycetes from mountain city, Jilin province and 54 ginseng rhizosphere actinomycetes are obtained by adopting a flat plate dilution method, and are screened for resisting rust rot.
And (3) separation and purification of actinomycetes: taking 1.0g of a soil sample, air-drying, grinding and dispersing uniformly, and mixing the materials in a proportion of 1: dissolving in 10% sterile water, filtering to obtain filtrate 10-1Starting solution, successively obtaining 10-2And 10-3And (5) soil gradient dilution. Respectively diluting and coating on Gao's 1 and PDA isolated culture medium, culturing at 28 deg.C for 7d until single colony grows out, selecting single colony, and purifying on culture medium to obtain single strain.
Screening antagonistic actinomycetes: the method comprises inoculating rust rot bacteria on PDA culture medium for activation by plate confrontation. On a sterile operating platform, a 1cm puncher is used for taking a rusty rot fungi cake to be placed in the center of a PDA flat plate, antagonistic bacteria hyphae are taken to draw a straight line at a position about 2cm away from pathogenic bacteria, and the flat plate without antagonistic bacteria hyphae is used as a contrast. Each set of 3 replicates was incubated in a 28 ℃ incubator and colony growth was observed and the minimum radius of the bacteria and the radius of the control colony were recorded at 5 d. The calculation formula ═ (control colony radius-pathogen colony radius)/control colony radius × 100%. The bacteriostatic rate of TL-007 to rust rot is 85.93%.
ITS sequence and phylogenetic analysis of actinomycete TL-007: the obtained 16S rDNA gene sequence of the strain is compared with known sequences in a GenBank database by BLAST, and the MEGA 5.1 is adopted to construct a phylogenetic tree TL-007 strain which belongs to the same branch of Streptomyces nogalaters, and the similarity of the strain reaches 100 percent. Finally named as Streptomyces nigricans (Streptomyces nogalaters) TL-007 through morphological characteristics and molecular biological identification results.
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 actinomycete TL-007 in the embodiment is the application in crop disease control.
The strain TL-007 of the embodiment is the Streptomyces nigricans (Streptomyces niger), and the efficient antagonistic strain can effectively inhibit the rust rot of ginseng and is free of pathogenicity. Lays a good foundation for the development and the utilization of bacterial manure for preventing and treating ginseng rust rot. The bacterial strain is subjected to an antibacterial spectrum experiment, and the experiment shows that the antibacterial rates of TL-007 on soybean black spot, soybean gray mold, Chinese cabbage black rot and ginseng fusarium are respectively as follows: 80.37 percent, 76.37 percent, 81.48 percent and 82.66 percent, which shows that the compound has antagonism effect on diseases of crops such as soybean, Chinese cabbage, ginseng and the like and has broad-spectrum antagonism effect.
The third concrete implementation mode: the second embodiment is different from the first embodiment in that: the actinomycete TL-007 is applied to the inhibition of soybean black spot pathogenic bacteria. 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 actinomycete TL-007 is applied to the inhibition of the botrytis cinerea. The other embodiments are the same as the second or third embodiment.
The fifth concrete implementation mode: this embodiment is different from one of the second to fourth embodiments in that: the actinomycete TL-007 is applied to the inhibition of Chinese cabbage black rot pathogen. The other is the same as one of the second to fourth embodiments.
The sixth specific implementation mode: the present embodiment is different from one of the second to fifth embodiments in that: the actinomycete TL-007 is applied to the inhibition of ginseng fusarium pathogenic bacteria. The rest is the same as one of the second to fifth embodiments.
The seventh embodiment: the present embodiment is different from one of the second to sixth embodiments in that: the actinomycete TL-007 is applied to inhibiting ginseng rust rot pathogenic bacteria. The rest is the same as one of the second to sixth embodiments.
The specific implementation mode is eight: in the embodiment, 54 ginseng rhizosphere actinomycetes from mountain city, Jilin province and 54 ginseng rhizosphere actinomycetes are obtained by adopting a flat plate dilution method, and are screened for resisting rust rot.
And (3) separation and purification of actinomycetes: taking 1.0g of a soil sample, air-drying, grinding and dispersing uniformly, and mixing the materials in a proportion of 1: dissolving in 10% sterile water, filtering to obtain filtrate 10-1Starting solution, successively obtaining 10-2And 10-3And (5) soil gradient dilution. Respectively performing dilution coating on Gao's 1 and PDA separation culture media, culturing at 28 ℃ for 7d until a single colony grows out, picking the single colony on the culture media, and purifying until single strains (figure 1) and (figure 2) are finally obtained.
Screening antagonistic actinomycetes: the method comprises inoculating rust rot bacteria on PDA culture medium for activation by plate confrontation. On a sterile operating platform, a 1cm puncher is used for taking a rusty rot fungi cake to be placed in the center of a PDA flat plate, antagonistic bacteria hyphae are taken to draw a straight line at a position about 2cm away from pathogenic bacteria, and the flat plate without antagonistic bacteria hyphae is used as a contrast. Each set of 3 replicates was incubated in a 28 ℃ incubator and colony growth was observed and the minimum radius of the bacteria and the radius of the control colony were recorded at 5d (FIG. 3). The calculation formula ═ (control colony radius-pathogen colony radius)/control colony radius × 100%. The bacteriostatic rate of TL-007 to rust rot is 85.93%.
The fifth concrete implementation mode: identification of Streptomyces sp.g6 with broad-spectrum antagonistic effect of the present embodiment:
1. physiological and biochemical identification
The physiological and biochemical characteristics of the TL-007 strain are measured by referring to the culture medium and the method recommended in the handbook of identifying common bacteria system and the handbook of identifying Bergey system, and the tolerance of the strain for liquefying gelatin, producing catalase, starch hydrolase, producing indoleacetic acid and NaCl is measured to be 7 percent (Table 1).
TABLE 1 Actinomycetes TL-007 physiological and biochemical test
Note: the physiological and biochemical test results of actinomycete TL-007: "+" is positive; "-" is negative
2. ITS sequence of actinomycete TL-007 and phylogenetic analysis
Obtaining a 16S rDNA gene sequence of the strain, wherein the DNA sequence is shown as SEQ ID NO: 1, then performing BLAST comparison analysis with known sequences in a GenBank database, and adopting MEGA 5.1 to construct a phylogenetic tree TL-007 strain, which belongs to the same branch as a Streptomyces nogalaters, wherein the similarity reaches 100%. As a result of morphological characteristics and molecular biological identification, the TL-007 strain was a Streptomyces nigricans strain (Streptomyces nogalaters) (FIG. 5).
The Streptomyces sp.G6 of the embodiment has the following functional verification on crop disease control:
experiment 1, antagonism of soybean black spot, soybean gray mold, cabbage black rot, ginseng fusarium by plate confrontation method
Calculating the bacteriostatic rate of TL-007 to soybean black spot, soybean gray mold, cabbage black rot and ginseng fusarium (figure 4) by adopting a plate confronting method and referring to a method for screening antagonistic actinomycetes (respectively): 80.37%, 76.37%, 81.48%, 82.66%. The product has antagonistic effect on diseases of soybean, Chinese cabbage, ginseng and other crops, and has broad-spectrum antagonistic effect.
Test 2: primary back grafting verification of TL-007 strain on ginseng slice
Culturing the obtained TL-007 bacteria in a Gao's I liquid at 28 ℃ for 3d, centrifuging at 10000rpm to obtain bacteria, adding water, blowing, centrifuging again for 1 time, and cleaning the bacteria. The obtained cells were prepared into 10 with sterile water8cfu/mL. Placing Ginseng radix stem block in sterilized culture dish, processing in 4 groups without any CK group, inoculating rusty corrosion bacteria on Ginseng radix stem block, adding Ginseng radix biocontrol bacteria suspension, and processing with Ginseng radix biocontrol bacteria suspension alone. The vessel was placed in an incubator at 28 ℃ for 5 days, and then the ginseng stem pieces were observed for decay (FIG. 6). The results show that: the growth of the rust rot fungi can be obviously inhibited by adding the antagonistic bacteria TL-007, and the fungi mass of the antagonistic bacteria TL-007 which is added independently has no rot, which indicates that the antagonistic bacteria TL-007 has no damage to the ginseng stems and the antagonistic bacteria TL-007 has the biological control effect.
Sequence listing
<110> institute of geography and agroecology of northeast China academy of sciences
<120> actinomycete TL-007 and application thereof
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<213> Streptomyces nigricans strain (Streptomyces nogalaters)
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agggcgtgcg ggcgtgctta cacatgcaag tcgacgatga acctccttcg ggaggggatt 60
agtggcgaac gggtgagtaa cacgtgggca atctgccctg cactctggga caagccctgg 120
aaacggggtc taataccgga tacgagcctc caccgcatgg tgggggttgg aaagctccgg 180
cggtgcagga tgagcccgcg gcctatcagc ttgttggtga ggtaacggct caccaaggcg 240
acgacgggta gccggcctga gagggcgacc ggccacactg ggactgagac acggcccaga 300
ctcctacggg aggcagcagt ggggaatatt gcacaatggg cgaaagcctg atgcagcgac 360
gccgcgtgag ggatgacggc cttcgggttg taaacctctt tcagcaggga agaagcgaga 420
gtgacggtac ctgcagaaga agcgccggct aactacgtgc cagcagccgc ggtaatacgt 480
agggcgcaag cgttgtccgg aattattggg cgtaaagagc tcgtaggcgg cttgtcacgt 540
cggttgtgaa agcccggggc ttaaccccgg gtctgcagtc gatacgggca ggctagagtt 600
cggtagggga gatcggaatt cctggtgtag cggtgaaatg cgcagatatc aggaggaaca 660
ccggtggcga aggcggatct ctgggccgat actgacgctg aggagcgaaa gcgtggggag 720
cgaacaggat tagataccct ggtagtccac gccgtaaacg gtgggcacta ggtgtgggca 780
acattccacg ttgtccgtgc cgcagctaac gcattaagtg ccccgcctgg ggagtacggc 840
cgcaaggcta aaactcaaag gaattgacgg gggcccgcac aagcggcgga gcatgtggct 900
taattcgacg caacgcgaag accttaccaa ggcttgacat acaccggaaa gcattagaga 960
tagtgccccc cttgtggtcg gtgtacaggt 990
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| CN108977382A (en) * | 2018-08-07 | 2018-12-11 | 广西壮族自治区农业科学院 | Streptomyces for producing volatile antibacterial substances and application thereof |
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| CN108977382A (en) * | 2018-08-07 | 2018-12-11 | 广西壮族自治区农业科学院 | Streptomyces for producing volatile antibacterial substances and application thereof |
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