CN115806913A - Streptomyces nojiri (Streptomyces nojiriensis) strain 9-13 and application thereof - Google Patents

Streptomyces nojiri (Streptomyces nojiriensis) strain 9-13 and application thereof Download PDF

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CN115806913A
CN115806913A CN202211504881.1A CN202211504881A CN115806913A CN 115806913 A CN115806913 A CN 115806913A CN 202211504881 A CN202211504881 A CN 202211504881A CN 115806913 A CN115806913 A CN 115806913A
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杜宜新
石妞妞
陈福如
邱德柱
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Abstract

The invention relates to a Streptomyces nojiri (Streptomyces nojiriensis) strain 9-13 and application thereof, belonging to the technical field of crop disease control. Streptomyces nojirimi strain 9-13 was deposited at 28 months 6 at 2021 in the China general microbiological culture Collection center (CGMCC) with the collection number of CGMCC No.22798. Streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13 has antagonistic action on 3 verruca fungus causing agaricus bisporus verruculosis, the fermentation liquor has inhibition effect on the hypha growth of the 3 verruca fungus, and the fermentation liquor has excellent prevention and treatment effect on the agaricus bisporus verruculosis and is safe to agaricus bisporus.

Description

Streptomyces nojiri (Streptomyces nojiriensis) strain 9-13 and application thereof
Technical Field
The invention relates to the technical field of crop disease control, in particular to a Streptomyces nojiri (Streptomyces nojiriensis) strain 9-13 and application thereof.
Background
Agaricus bisporus, also known as white mushroom, is an important edible and medicinal fungus due to its good taste, nutritive value and medicinal value. China is the largest agaricus bisporus producing country in the world, and the yield of the agaricus bisporus in China in 2013 reaches 54 percent of the total yield all over the world.
The mycosporidium (eczema) disease of the agaricus bisporus caused by the fungus of the genus Mycogone of the family Moniliaceae occurs in the world and becomes one of the most main diseases of the agaricus bisporus. The prevention and treatment of the wart spore mold present two characteristics: lack of commercial cultivars of disease-resistant agaricus bisporus; the verruca vulgaris and host agaricus bisporus belong to the same fungus, and the bactericide has an inhibition effect on both verruca vulgaris and host agaricus bisporus, so that the safety of the agaricus bisporus needs to be considered while the efficacy of the bactericide is evaluated.
Agricultural antibiotics are used as biopesticides with good development prospect, and become an indispensable variety in pesticide structures in China. China is a large agricultural antibiotic producing country and a large agricultural antibiotic applying country, and is also an early country for agricultural antibiotic research development. Research has been initiated as early as the 50's of the 20 th century, with slow pace, and much progress has been made by the 70's. The jinggangmycin, kasugamycin, agro-120, polyoxin, blasticidin, prince ridge mycin, jiyang mycin, monensin and plant growth are successively and successfully developedGibberellins are long-regulating agents. 50-100 mg/kg of a dual-purpose antibiotic for sterilization selected from Nippon combinatorial chemistry industries, ltd -1 Effective on mites, has ovicidal effect, and has a residual period of more than 20 days; and has better control effect on rice blast, peach brown rot, sclerotinia rot of cucumber and vegetable, powdery mildew of apple and melon, strawberry silk shell disease, gray mold of flowers and plants, mosaic disease of cucumber and tobacco and the like. Zhongshengmycin (kejunkang), which is developed successfully by the institute of biological control of the Chinese academy of agricultural sciences, is a novel agricultural antibiotic and is an N-glycoside alkaline water-soluble substance. The producing strain is Streptomyces lavendulae Hainan variety (Streptomyces 1avendiulans var. Hainanensis n. Var.). The processed preparation of the strain is a protective bactericide with wider bactericidal spectrum, and is effective in preventing and treating fungal diseases of crops such as rice, apples, vegetables, oranges, gingers and the like.
The agricultural antibiotic is safe and non-toxic to human and livestock, does not pollute the environment, does not destroy ecological balance, and has remarkable social benefit and ecological benefit.
Disclosure of Invention
The invention aims at solving the problems that the generation of the agaricus bisporus wart mildew hinders the safe production of the agaricus bisporus industry, the safety of the agaricus bisporus needs to be considered while the bactericide is used, and the like, and provides a Streptomyces nojiri (Streptomyces nojiriensis) strain 9-13 which has antagonism on 3 pathogenic bacteria causing the agaricus bisporus wart mildew.
The invention also aims to provide application of fermentation liquor of Streptomyces nojiri (Streptomyces nojiriensis) strains 9-13 in preventing and treating agaricus bisporus wart.
The purpose of the invention is realized by the following technical scheme: streptomyces nojiri (Streptomyces nojiriensis) strain 9-13, wherein the Streptomyces nojirimensis strain 9-13 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.22798 and the preservation date: year 2021, 6 months and 28 days.
The 16s rRNA sequence and rpoB gene sequence of Streptomyces nojiri (Streptomyces nojiriensis) strains 9-13 are shown in SEQ ID NO 1 and SEQ ID NO 2, respectively.
Streptomyces nojiri (Streptomyces nojiriensis) strains 9-13 have yellow basal hyphae and white aerial hyphae on a Gao's No. one culture medium, and no soluble pigment is produced. Spore silk is straight, flexible, hook-shaped, loose and compact spiral, and spore is oval and cylindrical.
The physiological and biochemical characteristics of the Streptomyces nojiri (Streptomyces nojiriensis) strains 9-13 are as follows: melanin can be produced, nitrate cannot be reduced; glucose, mannose, mannitol, lactose, galactose, etc. can be used; can utilize alanine; threonine, lysine and proline cannot be utilized.
Streptomyces nojirimensis (Streptomyces nojiriensis) strains 9-13 have antagonistic effects on the harmful Mycoplasma (Mycogone perniciosa), mycospora erythraea (M.rosea) and M.xinjiangensis which cause the agaricus bisporus wartwort mold.
Application of Streptomyces nojiri (Streptomyces nojiriensis) strain 9-13 in preventing and treating Agaricus bisporus wart spore mold is provided.
A preparation method of a fermentation liquid of a Streptomyces nojiri (Streptomyces nojiri) strain 9-13 comprises the steps of inoculating the Streptomyces nojiri (Streptomyces nojiri) strain 9-13 into an ISP2 liquid culture medium, fermenting at 26-28 ℃ for 7-8d, and filtering to obtain the fermentation liquid.
Application of fermentation liquor of Streptomyces nojiri (Streptomyces nojiriensis) strain 9-13 in preventing and treating Agaricus bisporus wart spore mold is provided.
Fermentation broth of Streptomyces nojiri (Streptomyces nojiriensis) strain 9-13 is used for inhibiting hypha growth of Agaricus bisporus wart spore mold pathogenic bacteria;
the pathogenic bacteria of the agaricus bisporus and the verruculospora are harmful verruculospora (Mycogone perniciosa), verruculospora rubripes (M.rosea) and M.xinjiangensis.
Compared with the prior art, the invention has the advantages that: streptomyces nojirimensis (Streptomyces nojiriensis) strains 9-13 have antagonistic action on 3 pathogenic bacteria causing agaricus bisporus wart mildew, fermentation liquor of the strains can obviously inhibit the hypha growth of the 3 wart mildews, and the prepared fermentation liquor has good prevention and treatment effect on the agaricus bisporus wart mildew and is safe to agaricus bisporus.
In addition, the Streptomyces nojiri (Streptomyces nojiriensis) strain 9-13 is obtained from soil, is harmonious and compatible with the soil environment, and has good application prospect.
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FIG. 1 shows the characteristics of the culture of Streptomyces nojiri (Streptomyces nojiriensis) strains 9 to 13 on the medium of Homoji No. one.
FIG. 2 shows the cultivation of Streptomyces nojiri (Streptomyces nojiriensis) strain 9-13 in opposition to 3 species of Mycoplasma japonicas of Agaricus bisporus. Note: a is Verticillium perniciosa (Mycogone perniciosa), B is Verticillium erythraeum (M.rosea), C is M.xinjiangensis, and CK is control group.
FIG. 3 shows the inhibitory effect of Streptomyces nojiri (Streptomyces nojiriensis) strain 9-13 fermentation broth on 3 verruculomyces of Agaricus bisporus in vitro. Note: a is Verticillium perniciosa (Mycogone perniciosa), B is Verticillium erythraeum (M.rosea), C is M.xinjiangensis, and CK is control group.
Detailed Description
The invention is described in detail below with reference to the drawings and examples of the specification:
the first embodiment is as follows: isolation and identification of Streptomyces nojiri (Streptomyces nojiriensis) strains 9-13.
1. Collection of soil samples
Collecting 3 parts of soil sample from Jiangxi Jinggang mountain, removing the soil on the surface, collecting the soil sample in the depth of 5-20cm, marking, and taking back to the laboratory for natural air drying.
2. Isolation of Actinomycetes
The separation was performed by plate dilution. Grinding the air-dried soil sample with a mortar, weighing 1g of the sample, suspending the sample in 9mL of sterile water, shaking at 40 ℃ and 180rpm for 30min, standing for 5min, sequentially diluting by 10 times, and respectively preparing into 10 -2 、10 -3 、10 -4 Respectively sucking 0.1mL of each suspension with different concentrations, adding into a modified HVA culture medium (adding potassium dichromate with a final concentration of 100-200 ppm) plate, uniformly coating, inverting at 28 deg.C, culturing for observation, selecting different single colonies after 5-7 days, streaking, purifying, and collecting the purified bacteriaThe plants were stored in a-80 ℃ freezer using the glycerol method.
3. Identification of Streptomyces nojiri (Streptomyces nojiriensis) Strain 9-13
(1) Morphological feature observation
Streptomyces nojiri (Streptomyces nojiriensis) strains 9-13 grew well on most media, all producing no soluble pigments (Table 1). Streptomyces nojiri (Streptomyces nojiriensis) strain 9-13 under an optical microscope has straight, flexible, hooked, loose and compact spiral spores and oval and cylindrical spores.
TABLE 1 cultivation characteristics of Streptomyces nojiri (Streptomyces nojiriensis) 9-13
Figure BDA0003967804010000051
(2) Physiological and biochemical characteristics
The results of measuring the characteristics of Streptomyces nojiri (Streptomyces nojiriensis) 9-13 nitrate reduction and carbon and nitrogen source utilization by the method described in the Streptomyces identification manual are shown in Table 2.
TABLE 2 physio-biochemical characteristics of Streptomyces nojiri strains 9-13
Figure BDA0003967804010000052
Figure BDA0003967804010000061
(3) Sequence analysis
After extracting genome DNA of the strain 9-13 by the bacterial genome extraction kit, respectively carrying out 16S rRNA and rpoB gene amplification to obtain sequences with full lengths of 1412bp and 859bp, submitting the obtained sequences to a GenBank database, and carrying out BLAST comparison analysis to obtain the strain 9-13 which is Streptomyces nojiri (Streptomyces nojiriensis).
The second embodiment: the antagonistic effect of Streptomyces nojiri (Streptomyces nojiriensis) strains 9-13 on 3 verruculospora molds was determined.
By adopting a plate opposing culture method, antagonistic determination is carried out on harmful verrucaria, verrucaria rosea and M.xinjiangensis by using Streptomyces nojirimensis strain 9-13. Firstly, streaking and inoculating Streptomyces nojiri (Streptomyces nojiriensis) strains 9-13 and 3d on two sides of the edge of a PDA culture medium, inoculating a pathogenic bacteria cake with the diameter of 5mm into the center of a flat plate, culturing 5d at 25 ℃, and measuring the bacteriostatic bandwidth and the pathogenic bacteria diameter (table 3) of the Streptomyces nojiri (Streptomyces nojiriensis) strains 9-13 to the pathogenic bacteria to be tested, wherein the pathogenic bacteria to be tested are not inoculated with antagonistic bacteria. The results showed that Streptomyces nojiri (Streptomyces nojiriensis) strains 9-13 all had very strong antagonistic effects against the harmful species Verticillium, neurospora roseoflavus and M.xinjiangensis (FIG. 2).
TABLE 3 antagonistic Effect of Streptomyces nojirimi strains 9-13 on 3 Verticillium molds
Figure BDA0003967804010000062
Example three: streptomyces nojiri (Streptomyces nojiriensis) strain 9-13 fermentation liquor has inhibitory effect on 3 verrucous moulds.
Inoculating spores of Streptomyces nojiri (Streptomyces nojiriensis) strain 9-13 activated by a Gao's I culture medium into ISP2 liquid culture medium, fermenting at 26-28 deg.C for 7-8 days, filtering to obtain fermentation liquor, and sterilizing with 0.22 μm microporous filter membrane.
Taking 5mL of fermentation liquor of Streptomyces nojiri (Streptomyces nojiriensis) strain 9-13, adding into 50mL of PDA culture medium, mixing uniformly, pouring into a culture dish, inoculating a pathogenic bacteria cake with the diameter of 5mm into the center of a PDA culture medium plate, culturing at 25 ℃ for 5d, and measuring the diameter of a bacterial colony. The inhibition rate of the fermentation broth on the hyphal growth of 3 verruculospora molds was calculated by using a blank PDA medium as a control (table 4). The results show that the fermentation liquor of Streptomyces nojiri (Streptomyces nojiriensis) strains 9-13 has strong inhibition effect on the hyphal growth of harmful verrucaria, verrucaria rubra and M.xinjiangensis (figure 3).
TABLE 4 inhibitory Effect of Streptomyces nojirimi Strain 9-13 fermentation broth on the hyphal growth of 3 verruculospora molds
Figure BDA0003967804010000071
Example four: application of Streptomyces nojiri (Streptomyces nojiriensis) strain 9-13 fermentation liquor in preventing and treating agaricus bisporus wart mildew.
Culturing Mycosporium pernicicum, mycosporium roseum and M.xinjiangensis at 25 deg.C for 5 days, punching bacterial cake with 5mm diameter punch at colony edge, transferring the bacterial cake into PDB liquid culture medium, culturing at 150r/min and 25 deg.C under dark oscillation for 7 days, detecting chlamydospore concentration with blood counting plate to obtain a product with concentration of 1 × 10 5 The suspension was mixed for each spore/mL.
The test site is selected from Cambodia village in Fuzhou, fujian province, and the Agaricus bisporus variety is W192. The experiment set up 3 treatments, respectively: a.50% of prochloraz manganese salt WP1.0 g/m, B fermentation liquor of Streptomyces nojiri (Streptomyces nojiriensis) strain 9-13, and C clear water as control. Each treatment is repeated 4 times, the total number is 12 cells, the cells are arranged in random block groups, and each cell is 4m 2 . Quantitatively spraying the liquid medicine with the tested concentration on quantitative soil particles to be covered with soil (about 25kg of soil is covered on each square meter of mushroom bed, 1.5L of the liquid medicine is sprayed), uniformly stirring, keeping the water content of the soil to be about 70%, covering the soil with a plastic film for 24h, then covering the soil, and then inoculating 100mL of the spore mixed suspension in each cell. And (4) after covering soil for 7 days, respectively carrying out second pesticide application, and uniformly spraying pesticide liquid with the same dosage on soil particles on the bed surface until the soil particles are wet and do not flow into the bacteria material. Investigation is carried out from the first tide of mushrooms, 3 tides of mushrooms are investigated, a full-cell investigation mode is adopted, the number of diseased mushroom grains or colonies (the number of the diseased mushroom grains or the colonies is more than or equal to 1 cm) on the compost is investigated and recorded during each harvest, the number of the diseased mushroom grains in each cell is accumulated, and the control effect is calculated.
Control effect (%) = [ (control area mushroom number-treatment area mushroom number)/control area mushroom number ] × 100
TABLE 5 field efficacy test of fermentation broth of Streptomyces nojirimi strain 9-13 for preventing and treating Agaricus bisporus wart
Figure BDA0003967804010000081
Experimental results show that the control effects of fermentation liquor of A.50% of prochloraz manganese salt WP1.0 g/square meter and B. Streptomyces nojiri (Streptomyces nojiriensis) strains 9-13 on the agaricus bisporus wart spore mold are 77.53% and 75.62% respectively, and the fermentation liquor is safe to agaricus bisporus without chemical injury. The control effect of the fermentation liquor of Streptomyces nojiri (Streptomyces nojiriensis) strains 9-13 on the agaricus bisporus wart spore mold is not obviously different from the control effect of 1.0 g/square meter of 50 percent of prochloraz manganese WP on the obvious level of 5 percent and the very obvious level of 1 percent. Therefore, the control effect of the fermentation liquor of the Streptomyces nojiri (Streptomyces nojiriensis) strains 9-13 on the agaricus bisporus wart is equivalent to the control effect of 50 percent of prochloraz manganese salt WP1.0 g/square meter, and the method has good application prospect.
The 16s rRNA sequence and rpoB gene sequence of Streptomyces nojiri (Streptomyces nojiriensis) strains 9-13 are shown in SEQ ID NO 1 and SEQ ID NO 2, respectively.
SEQ ID NO:1
TGCGGCGTGCTTACCATGCAGTCGAACGATGAACCTCCTTCGGGAGGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTTCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATATGACACGGGGTCGCATGATCTCCGTGTGGAAAGCTCCGGCGGTGAAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGAGGTAGTGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGAGAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCGAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGATGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCATTCGATACGGGCAGGCTAGAGTTCGGTAGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGTTGGGAACTAGGTGTGGGCGACATTCCACGTCGTCCGTGCCGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGCCCGCACAAGCAGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATACACCGGAAAACCGTGGAGACACGGTCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTCTGTGTTGCCAGCATGCCTTTCGGGGTGATGGGGACTCACAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGATACCGCGAGGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGTCTGCAACTCGACCCCATGAAGTCGGAGTTGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAACCCCTTGTGGGAGGGAATCGTCGAAGG
SEQ ID NO:2
GTACGGCTAAAAAAGCGCGCCTGTCGGCGCTGGGCCCCGGCGGTCTGTCCCGTGAGCGGGCCGGCTTCGAGGTCCGTGACGTTCACCCGTCGCACTACGGCCGCATGTGCCCGATCGAGACCCCTGAAGGCCCGAACATCGGTCTGATCGGCTCGCTCGCCTCCTACGGTCGCGTCAACGCGTTCGGTTTCGTCGAGACCCCGTACCGCAAGGTCATCGACGGTGTCGTCACCGACGAGGTCGACTACCTGACGGCCGACGAGGAAGACCGCTTCGTCATCGCCCAGGCCAACGCCGGCCTGTCCGAGGACATGCGCTTCACCGAGAACCGCGTGCTGGTGCGCCGTCGTGGCGGCGAGATCGACTACATCGCCGGCGACGACGTCGACTACATGGACGTCTCCCCGCGCCAGATGGTGTCCGTCGCGACCGCGATGATCCCCTTCCTGGAGCACGACGACGCCAACCGTGCCCTCATGGGCGCGAACATGATGCGCCAGGCCGTTCCGCTCATCAAGGCGGAGGCCCCGCTCGTCGGCACCGGCATGGAGTACCGCTGTGCGGTCGACGCCGGTGACTCGATCCGTGCGGAGAAGGACGGTGTCGTCCAGGAGGTCTCGGCCGACTACATCACCGTCGCCAACGACGACGGCACGTACACCACGTACCGCGTCGCCAAGTTCTCCCGCTCGAACCAGGGCACCTCGGTCAACCAGAAGGTCATCGTCAACGAGGGTGACCGGATCATCGAGTCCCAGGTCCTCGCCGACGGTCCCGCGACCGAAGAGGGCGAAATGG
CCCTCGGCAAGAACCTGCTCGTCGCGTTCATGCCGTGGGAGGGCCCAACT
TATACAAAGGA。

Claims (7)

1. A Streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13 is characterized in that:
the Streptomyces nojiri (Streptomyces nojiriensis) strain 9-13 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.22798 and the preservation date: 6 and 28 months in 2021.
2. A Streptomyces nojiri (Streptomyces nojiriensis) strain 9-13 according to claim 1, characterized in that:
streptomyces nojirimensis (Streptomyces nojiriensis) strains 9-13 have antagonistic effects on the harmful Mycoplasma (Mycogone perniciosa), mycospora erythraea (M.rosea) and M.xinjiangensis which cause the agaricus bisporus wartwort mold.
3. Use of a Streptomyces nojirimensis strain 9-13 as defined in claim 1 for the control of Agaricus bisporus warty mildew.
4. A process for the preparation of a fermentation broth of Streptomyces nojiri strain 9-13 as claimed in claim 1, characterized in that: streptomyces nojiri (Streptomyces nojiriensis) strain 9-13 is inoculated in ISP2 liquid culture medium, the fermentation temperature is 26-28 ℃, the fermentation culture time is 7-8 days, and the fermentation broth is obtained by filtration.
5. A fermentation broth of Streptomyces nojiri (Streptomyces nojiriensis) strain 9-13 prepared by the process of claim 4.
6. Use of a fermentation broth of Streptomyces nojiri strain 9-13 as claimed in claim 5, wherein: application of fermentation liquor of Streptomyces nojiri (Streptomyces nojiriensis) strain 9-13 in preventing and treating Agaricus bisporus wart spore mold is provided.
7. Use of a fermentation broth of Streptomyces nojiri strain 9-13 according to claim 6, characterized in that:
fermentation broth of Streptomyces nojiri (Streptomyces nojiriensis) strain 9-13 is used for inhibiting hypha growth of Agaricus bisporus wart spore mold pathogenic bacteria;
the pathogenic bacteria of the agaricus bisporus and the verruculospora are harmful verruculospora (Mycogone perniciosa), verruculospora rubripes (M.rosea) and M.xinjiangensis.
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