CN109182216B

CN109182216B - Marine streptomyces SCFJ-05 with inhibition effect on succulent plant stem rot

Info

Publication number: CN109182216B
Application number: CN201811210334.6A
Authority: CN
Inventors: 余德亿; 姚锦爱; 黄鹏
Original assignee: Institute of Plant Protection of FAAS
Current assignee: Institute of Plant Protection of FAAS
Priority date: 2018-10-17
Filing date: 2018-10-17
Publication date: 2021-11-09
Anticipated expiration: 2038-10-17
Also published as: CN109182216A

Abstract

The invention provides a marine streptomyces SCFJ-05 with an inhibiting effect on succulent plant stem rot, and belongs to the technical field of microbial biocontrol. The marine streptomyces SCFJ-05 is streptomyces viridis (S. viridis: (A)Streptomyces prasinopilosus) The culture medium is registered and preserved in China general microbiological culture Collection center (CGMCC) at 6 and 4 months in 2018, and the preservation number is CGMCC No. 15860. The marine streptomyces SCFJ-05 has stronger inhibition effect on succulent plant phomopsis longissima and has the potential of being developed into biological pesticide.

Description

Marine streptomyces SCFJ-05 with inhibition effect on succulent plant stem rot

Technical Field

The invention relates to a marine streptomyces SCFJ-05 with an inhibiting effect on succulent plant stem rot, and belongs to the technical field of microbial biocontrol.

Background

Succulent plants are higher plants with large vegetative organs, usually with three vegetative organs, root, stem and leaf, and three reproductive organs, flower, fruit and seed. In horticulture, the plants are also called succulent plants or succulent flowers, but succulent plants are most commonly used. Succulent plants as potted plants are widely popular around the world, and most of the common species at present belong to the Crassulaceae and Cactaceae families.

The pathogenic bacteria of succulent plant stalk rot is usually Fusarium oxysporum (F. oxysporum) ((R))Fusarium oxysporum) The fungus is a main fungus which is harmful to succulent plants, belongs to Deuteromycotina, is a kind of fungi distributed worldwide, is commonly present on soil and animal and plant organisms, even exists in severe cold arctic and arid and hot desert, and has parasitic or saprophytic life. The pathogenic bacteria not only can live through winter and summer in soil, but also can live through summerDifferent specialization types are expressed on different plants; it specifically causes vascular bundle disease of plants, destroys plant conducting tissues, and produces toxins to harm crops in the processes of plant growth, development and metabolism, causing plant wilting death and affecting yield and quality. The stem rot is one of the important diseases difficult to control in the current succulent plant production, the succulent plant can be suddenly fallen in a large amount and withered to die once suffering from the disease, brown disease spots can begin to appear on the stem of the plant, and then the inside of the plant is rotted until the whole plant is suddenly fallen to die. In the prior art, succulent plant stem rot is generally controlled by chemical bactericides such as carbendazim, thiophanate methyl, zineb and the like. Although the chemical bactericides have certain prevention effect on stem rot of succulent plants, the chemical bactericides are easy to generate drug resistance after long-term use, have toxicity and are easy to cause damage to the environment and human bodies in the use process. The agricultural antibiotic as an important part of biological pesticide has the advantages of no environmental pollution, safety to people and livestock, easy decomposition by soil microorganisms and the like, so that the application of the novel agricultural antibiotic in the prevention and treatment of plant diseases has wide application prospect. At present, a plurality of important agricultural antibiotics which are widely applied are separated from actinomycetes, the actinomycetes mainly come from a terrestrial environment, and due to continuous repeated separation and screening, the difficulty in screening the actinomycetes with a novel disease-resistant mechanism is very high. Therefore, people gradually turn their eyes to oceans with abundant microbial resources, and it is expected to screen out biocontrol microorganisms with a novel disease resistance mechanism from the ocean environment for the prevention and treatment of plant diseases. The sea is used as a huge biological resource bank, the diversity and uniqueness of marine organisms different from terrestrial organisms are determined due to the special living environment, such as high salinity, high pressure, low temperature and special illumination, about 99 percent of marine microorganisms are not known at present, more and more marine microorganisms are developed and utilized along with the development of biotechnology, and the marine microorganisms and secondary metabolites thereof provide new resources for the prevention and treatment of plant diseases. Therefore, the method for extracting active substances from the marine streptomyces and the metabolic products thereof to be applied to the prevention and treatment of the succulent plant stem rot is a novel and feasible new path.

Disclosure of Invention

The invention aims to provide a marine streptomyces SCFJ-05 with an inhibiting effect on succulent plant stem rot, and the strain has an obvious inhibiting effect on pathogenic bacteria of succulent plant stem rot and has the potential of being developed into a biological pesticide.

The purpose of the invention is realized by the following technical scheme:

the marine streptomyces SCFJ-05 provided by the invention is streptomyces viridis (A. viridis: (A. viridis)Streptomyces prasinopilosus) The microbial culture has been registered and preserved in China general microbiological culture Collection center (CGMCC) at 6.4.2018, the accession number is CGMCC No.15860, at the institute of microbiology, No.1, national institute of sciences, North Cheng Xilu, Chaoyang, Beijing.

The invention provides application of the marine streptomyces SCFJ-05, namely application of the marine streptomyces SCFJ-05 in inhibition of succulent plant stalk rot diseases.

The invention also provides application of the marine streptomyces SCFJ-05 in preparation of biological pesticides for inhibiting succulent plant diseases.

The marine streptomyces SCFJ-05 is obtained by separating sea mud collected from mangrove forest tidal flat wetland in skies county of Yuanzhou city, Fujian province, and the culture and morphological characteristics, physiological and biochemical characteristics, 16S rDNA sequence and strain classification results of the strain are as follows:

1. culturing and morphological characteristics of marine streptomycete SCFJ-05: the growth of Streptomyces marinus SCFJ-05 on the 7 media tested is shown in Table 1.

TABLE 1 cultivation and morphological characterization of the strains

2. Physiological and biochemical characteristics of Streptomyces marinus SCFJ-05: of the 6 carbon sources tested, the marine streptomyces SCFJ-05 can utilize glucose, fructose, and arabinose as carbon sources. Can hydrolyze starch, gelatinize milk, reduce nitrate, prevent gelatin liquefaction and generation of H₂S and melanin, see table 2.

TABLE 2 physiological and biochemical Properties of the strains

3. 16S rDNA sequence of Streptomyces marinus SCFJ-05: the 16S rDNA sequence of the marine streptomycete SCFJ-05 is shown in a sequence table SEQ ID NO. 1. The obtained sequence is subjected to Blast comparison analysis on database resources on the website of National Center for Biotechnology Information (NCBI) of the United states, and the detected sequence has higher homology with a plurality of species of streptomyces in Genbank, wherein the homology with the species of streptomyces in GenbankS. prasinopilosusHas the highest homology (GeneBank accession numbers: KR703669, KU647247 and KU 647227), and has a sequence similarity of more than 99%.

4. And (3) strain classification results: according to the culture and morphological characteristics, physiological and biochemical characteristics and 16S rDNA sequence of the marine streptomycete SCFJ-05, the marine streptomycete SCFJ-05 is identified as streptomycete viridissimulus (S. viridis:)Streptomyces prasinopilosus )。

Compared with the prior art, the invention has the advantages and beneficial effects that: (1) the marine streptomyces SCFJ-05 provided by the invention is separated from the sea mud collected by mangrove forest tidal flat wetland in campsis county of campsis, Zhangzhou city, Fujian province, and reports are fresh about separation of antagonistic streptomyces from the sea mud and application of the antagonistic streptomyces to prevention and control of succulent plant diseases, and related researches on the strains are also few at present; (2) the crude extract of the fermentation liquor of the marine streptomycete SCFJ-05 has bacteriostatic activity on various plant pathogenic fungi diseases, and has the characteristics of low toxicity, low residue, no pollution and difficult generation of drug resistance compared with the currently used chemical bactericide; (3) the marine streptomycete SCFJ-05 has good inhibition effect on succulent plant phomopsis solani, fusarium solani, phytophthora infestans, orchid phomopsis, succulent plant colletotrichum gloeosporioides and the like. (4) The marine streptomycete SCFJ-05 is easy to culture, and the crude extract of the fermentation liquor is easy to extract and prepare. Therefore, the marine streptomyces SCFJ-05 can be easily developed into a biological control agent, and has good market application prospect for preventing and treating plant fungal diseases.

Drawings

FIG. 1 inhibition of Phomopsis marinus SCFJ-05 against Phoma succulentPreparation (spore concentration from left to right is 10 respectively⁹、10⁸、10⁷、10⁶、10⁵spores/mL).

FIG. 2 shows the bacteriostatic activity of Streptomyces marinus SCFJ-05 (pathogenic bacteria include succulent plant phomopsis, Fusarium solani, phytophthora infestans, orchid phomopsis, and succulent plant anthrax).

FIG. 3 culture characteristics of Streptomyces marinus SCFJ-05 on PDA medium.

FIG. 4 is a phylogenetic tree constructed.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.

The marine streptomyces with strong bacteriostatic activity is named as marine streptomyces SCFJ-05, and is classified and named as follows: streptomyces viridochromogenes (A), (B)Streptomycesprasinopilosus) The preservation number is: CGMCC No.15860, preservation date: 6/4/2018, depository: china general microbiological culture Collection center, preservation Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.

Example 1: isolated culture of marine actinomycetes

1. Biological sample and culture medium: the sample sea mud is collected in mangrove tidal flat wetland in campsis county of campsis county, Zhangzhou, Fujian province, the collected sample is taken back to a laboratory for the isolated culture of actinomycetes, 6 isolated culture mediums are selected in total, the components are shown in Table 3, 15 mu g/mL nalidixic acid, 50 mu g/mL nystatin and 50 mu g/mL cycloheximide are added into the 6 culture mediums.

TABLE 3 composition of the isolation Medium

2. Sample treatment: the collected sea mud sample was isolated and cultured by the following treatment method. The treatment method comprises the following steps: taking 1g of sea mud sample, putting the sea mud sample into 4 mL of sterile water, thermally shocking for 10 min at 55 ℃, violently shaking, diluting the sample by 1:4, taking 20 mu L of diluent, uniformly coating the diluent on 6 separation culture medium flat plates, and culturing for 7 d-12 d at 28 ℃ until actinomycetes grow out.

3. And (3) purifying and culturing actinomycetes: picking single colony hypha growing on the separation culture medium plate for transfer purification, removing repeated strains according to the culture and morphological characteristics of the strains, and finally separating to obtain 26 marine actinomycete strains in total. Selecting purified strain, inoculating mycelia to Gao synthesized No.1 (formula is 20 g soluble starch, KNO)₃ 1 g、K₂HPO₄0.5 g、MgSO₄·7H₂O 0.5 g、FeSO₄·7H₂0.01 g of O, 0.5 g of NaCl, 20 g of agar, 1000 mL of distilled water and pH of 7.2-7.4), culturing in a constant temperature incubator at 28 ℃ for 5 d-7 d, and storing in a refrigerator at 4 ℃ for later use.

Example 2: screening of antagonistic strain of marine actinomycetes

1. Preparing an indicator bacterium plate: the succulent plant stem rot pathogenic bacteria are selected from succulent plant Rhododendron simsii (A. pulchr.) DC (A. pulchrum. DC.) (A. pulchr.) DCEcheveria'Perle Von N ü rnberg') from the rhizomes thereof, belonging to the genus Fusarium oxysporum (F. oxysporum;)Fusarium oxysporum). Transferring preserved succulent plant phomopsis into PDA culture medium (preparation method comprises weighing fresh peeled potato 200 g, cutting up, adding water 1000 mL, boiling for half an hour, filtering with gauze, adding filtrate to 1000 mL, adding glucose 20 g and agar 20 g, heating to dissolve completely, packaging into conical flask or glass test tube, autoclaving at 121 deg.C for 28 min), placing on a microorganism incubator at 28 deg.C, culturing until bacterial colony produces spore, adding sterile water 10 mL into the culture dish to collect spore, filtering with sterile gauze, adjusting spore suspension concentration to 10 with sterile water⁵-10⁹And (4) sucking 20 mu L spore suspension liquid with different concentrations by a pipette, and uniformly coating the spore suspension liquid on a PDA (personal digital assistant) plate for later use (the experiment is repeated for 3 times).

2. Screening of strains with inhibitory activity: the 26 marine actinomycetes isolated in example 1 were inoculated on a PDA medium plate, cultured at 28 ℃ for 7 days, and then a cake of 5 mm in diameter was taken out with a punch at the growth site, placed in the center of the plate inoculated with the indicator, and cultured in a microbial incubator at 28 ℃. And after 48 h, observing the existence and the size of a transparent bacteriostatic zone around the bacterial cake, and judging the bacteriostatic activity and the antagonistic capability of the 26 marine actinomycetes.

Through the method, 1 strain with strong inhibitory activity to succulent plant phomopsis longissima is screened from 26 marine actinomycetes obtained by separation, and is named as marine streptomyces SCFJ-05 (figure 1).

Example 3: culture, morphological characteristics, physiological and biochemical characteristics of marine streptomycete SCFJ-05

1. Culture and morphological characteristics of the strain SCFJ-05: adopting 7 kinds of international culture media, which are respectively inorganic salt starch agar (ISP-4, the formula is 10 g of soluble starch and K₂HPO₄ 1 g、MgSO₄·7H₂O 1 g、NaCl 1 g、(NH₄)₂SO₄ 2 g、CaCO₃ 2 g、FeSO₄·7H₂O 0.001g、MnCl₂·7H₂O 0.001 g、ZnSO4·7H₂0.001g of O, 20 g of agar, 1000 mL of distilled water, pH 7.0-7.4), glucose asparagine agar (ISP-5, formula: 1g of L-aspartic acid, 10 g of glucose and K₂HPO₄1g, 1 mL of trace element solution, 20 g of agar, 1000 mL of distilled water, pH 7.2, and the content of the trace element solution: FeSO₄·7H₂O 0.1 g、MnCl₂·4H₂O 0.1 g、ZnSO₄·7H₂O0.1 g), oat flour agar (ISP-3, formula: oat flour 20 g, agar 20 g, trace element solution 1 mL, pH 7.2, trace element solution: FeSO₄·7H₂O 0.1 g、MnCl₂·4H₂O 0.1 g、ZnSO₄·7H₂0.1 g of O), yeast refined maltose agar (ISP-2, the formula is as follows: 4 g of yeast extract, 10 g of wort, 4 g of glucose, 20 g of agar, 1000 mL of distilled water and pH 7.0), and nutrient agar (NA, the formula is as follows: 10 g of peptone, 3 g of beef extract, 5 g of sodium chloride, 20 g of agar, 1000 mL of distilled water and pH 7.3), and glycerol asparagines agar (formula: l-asparagines 1g, K₂HPO₄1g, 1 mL of saline solution, and 10 g of glycerolg. Agar 20 g, distilled water 1000 mL, pH 7.0-7.4) and starch agar (formula: soluble starch 10 g, MgCO₃ 1 g、K₂HPO₄ 0.3 g、NaCl 0.5 g、NaNO₃1g of agar, 20 g of agar and 1000 mL of distilled water, and the pH value is 7.0), inoculating the strain SCFJ-05 on the culture medium, then placing the strain in a constant-temperature incubator at 28 ℃ for inverted culture, and observing the growth conditions of aerial hyphae and hyphae in the medium and the production conditions of soluble pigments of the SCFJ-05 strain after 7 days.

The result shows that the strain SCFJ-05 can grow on 7 culture media, and aerial mycelium and matrix mycelium are produced; no soluble pigment was produced in any of the 4 media, except starch agar, glucose asparagine agar and nutrient agar (Table 1). The strain SCFJ-05 grew well on PDA medium (fig. 3), with loose and open spores, regular short helices, sometimes just the initial ones, and with fine hairs as long as 2 um on the surface of the spores.

2. Physiological and biochemical characteristics of the strain SCFJ-05: refer to "fast Actinomycetes identification and Classification of systems" (Ruan-Tu-Ying, Huang-Ying. fast Actinomycetes identification and Classification of systems [ M)]Beijing: scientific publishing agency, 2011) and Streptomyces identification Manual (Streptomyces classification group of institute of microbiology, national academy of sciences, Streptomyces identification Manual [ M)]Beijing: scientific Press, 1975) on the physiological and biochemical characteristics of the strain SCFJ-05, such as carbon source utilization, melanin production, H₂And measuring indexes such as S generation, gelatin liquefaction, starch hydrolysis, milk coagulation peptonization, nitrate reduction and the like.

The results show that of the 6 carbon sources tested, S.marinus SCFJ-05 can utilize glucose, fructose and arabinose as carbon sources (Table 2). Can hydrolyze starch, gelatinize milk, reduce nitrate, prevent gelatin liquefaction and generation of H₂S and melanin (table 2).

In conclusion, the marine streptomycete SCFJ-05 is preliminarily identified as the streptomycete viridissimus by the detection and analysis of the culture, morphological characteristics and physiological and biochemical characteristics (Streptomyces prasinopilosus )。

Example 4: 16S rDNA sequence analysis of marine streptomycete SCFJ-05

The strain SCFJ-05 is cultured in a Gao's No. one liquid culture medium for 4 d-5 d of bacterial liquid, and the bacterial liquid is centrifuged in a sterile centrifuge tube to collect bacterial precipitation. Weighing 0.1 g-0.2 g of thallus in a sterile mortar, adding a small amount of PVP, grinding into powder by using liquid nitrogen, and extracting the total DNA of the thallus by using an actinomycete genome extraction kit. With the universal primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', 1541R: 5'-AAGGAGGTGATCCAGCCGCA-3' the actinomycete 16S rDNA was amplified. The PCR product is loaded into agarose gel containing GelRed dye for electrophoresis, and then a target band is recovered and sent to the company of biological engineering (Shanghai) GmbH for sequencing. The sequence was analyzed by NCBI Blast, the 16S rDNA sequence of the strain with higher homology to the sequence in GenBank was selected, multiple alignments were performed by Clustal X software, the sequence similarity between the strain SCFJ-05 and the reference strain was analyzed, and phylogenetic trees were constructed by Neighbor join method in Mega4.1 software (FIG. 4).

The result shows that the sequence of the 16S rDNA of the marine streptomycete SCFJ-05 is shown in a sequence table SEQ ID NO. 1. The obtained sequence is subjected to Blast comparison analysis on the website of National Center for Biotechnology Information (NCBI) of the United states, and the detected sequence has higher homology with a plurality of species of streptomyces in Genbank, wherein the species of streptomyces has higher homology with the species of the GenbankS. prasinopilosusHas the highest homology (GeneBank accession numbers: KR703669, KU647247 and KU 647227), and has a sequence similarity of more than 99%.

According to the primary identification result of the strain SCFJ-05 with culture, morphological characteristics and physiological and biochemical characteristics, the marine streptomycete SCFJ-05 is finally identified as streptomycete viridissimulus (S. viridis:)Streptomycesprasinopilosus)。

Example 5: preparation of marine streptomycete SCFJ-05 fermentation liquor crude extract

1. Activating and culturing the strain: the isolated and preserved marine streptomycete SCFJ-05 is transferred to a Gao's synthetic No.1 culture medium plate and cultured for 7 d in an incubator at 28 ℃.

2. Fermentation culture of the strain: the marine streptomyces on the Gao's synthetic No.1 culture medium SCFJ-05 which is activated is inoculated into the Gao's synthetic No.1 liquid fermentation culture medium (100 mL culture medium/250 mL conical flask, the formula is the same as that of the Gao's synthetic No.1 solid culture medium, and agar is not contained), and the shaking fermentation culture is carried out for 4 d at the temperature of 28 ℃ by a shaker at 200 rpm/min.

3. Preparation of a crude extract of fermentation liquor: taking the shake culture fermentation broth, and performing suction filtration through sterile filter paper to respectively obtain a supernatant and mycelia. Taking supernatant, extracting with ethyl acetate for 3 times, and mixing extractive solutions to obtain ethyl acetate extractive solution of supernatant. Soaking mycelium in 80% acetone water solution overnight, extracting with ultrasonic wave for 3 times, mixing extractive solutions, concentrating under reduced pressure until no acetone is present, and extracting the obtained water phase with equal volume of ethyl acetate for 3 times to obtain ethyl acetate extract of mycelium. And combining the supernatant and the ethyl acetate extract of the mycelium, and concentrating under reduced pressure to dryness to obtain an ethyl acetate extract of the strain. Weighing 1g of extract, adding 1 mL of dimethyl sulfoxide to dissolve the extract, adding sterile water to a constant volume of 10 mL, and obtaining the crude extract of the marine streptomyces SCFJ-05 fermentation liquor.

Example 6: determination of marine streptomycete SCFJ-05 bacterial inhibition spectrum

1. Test strains and culture media: the pathogenic bacteria of the test plants include succulent plant phomopsisFusarium oxysporum) Fusarium solani (A) and (B)Fusarium solani) Phytophthora infestans (A), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C)Phytophthora cactorum) Orchid stem rot bacteria (Fusarium oxysporum) And succulent plant anthrax (B) ((B))Colletotrichum gloeosporioides). The culture medium used for the cultivation of phytopathogens was the PDA medium, and its preparation method is described in example 2.

2. Activation of test strains: and (3) respectively heating and dissolving the PDA culture medium, and pouring the PDA culture medium into a sterile culture dish with the diameter of 9 cm to prepare a PDA culture medium flat plate. Picking out each plant pathogenic bacteria block stored in a slant culture medium of a refrigerator at 4 ℃ by using a sterile inoculating needle, inoculating the plant pathogenic bacteria block on a PDA culture medium flat plate, and culturing and activating the plant pathogenic bacteria block in a constant temperature incubator at 28 ℃.

3. And (3) determining the antibacterial activity: the method is characterized in that 5 plant pathogenic bacteria such as succulent plant phomopsis solani, fusarium solani, phytophthora infestans, orchid phomopsis longissima and succulent plant colletotrichum are taken as target bacteria, and the antibacterial spectrum of marine streptomycete SCFJ-05 is determined and screened by adopting a punching method. A target bacteria cake with the diameter of 5 mm is taken by a puncher and is placed at a position 2.5 cm away from the center of a culture dish, then the hole is punched at the center of a PDA (personal digital assistant) flat plate mixed with the target bacteria, 200 mu L of sterile fermentation liquor crude extract is added into the hole and is respectively placed in an HGZ-150 type illumination incubator with the temperature of (28 +/-1) DEG C and the photoperiod of 12L: 12D for culture, the antibacterial bandwidth is measured after 6D, and the test is repeated for 3 times (figure 2).

By the method, the strain SCFJ-05 has strong inhibition effects on succulent plant phomopsis solani, phytophthora infestans and orchid phomopsis longissima, also has strong inhibition effects on fusarium solani and succulent plant colletotrichum (Table 4), and can show that the inhibition spectrum of the marine streptomycete SCFJ-05 is wider.

TABLE 4 inhibition of plant pathogenic bacteria by crude extract of fermentation broth of marine streptomycete SCFJ-05

Note: the data in the same column are marked with different lower case letters, which are shown in the test of Duncan's new repolarization methodPThe level < 0.05 was significantly different.

SEQUENCE LISTING

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Claims

1. A strain of marine streptomyces SCFJ-05 is characterized in that: the bacterium is streptomyces viridis (A), (B) and (C)Streptomyces prasinopilosus) SCFJ-05, which has been registered and preserved in China general microbiological culture Collection center (CGMCC) at 6.4.2018, with the preservation number of CGMCC No. 15860.

2. The use of Streptomyces marinus SCFJ-05 according to claim 1 for inhibiting succulent plant diseases, characterized in that: the succulent plant disease is a stalk rot caused by fusarium oxysporum.

3. The use of Streptomyces marinus SCFJ-05 according to claim 1 in the preparation of biopesticides for inhibiting succulent plant diseases.