CN116716225A - Radiation-resistant streptomycete and application thereof - Google Patents
Radiation-resistant streptomycete and application thereof Download PDFInfo
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- CN116716225A CN116716225A CN202310785378.6A CN202310785378A CN116716225A CN 116716225 A CN116716225 A CN 116716225A CN 202310785378 A CN202310785378 A CN 202310785378A CN 116716225 A CN116716225 A CN 116716225A
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Abstract
The invention provides radiation-resistant streptomycete and application thereof. The invention firstly provides radiation-resistant streptomyces (Streptomyces radiopugnans) with the preservation number of CGMCC No.26717. The radiation-resistant streptomycete CGMCC No.26717 is a non-pathogenic streptomycete, can inhibit or prevent potato scab, can obviously promote the root system of potato seedlings to be more developed, thicken stems, enhances the resistance to soil-borne disease potato scab, obviously improves the yield and quality of potato tubers, reduces environmental pollution and improves the economic benefit of potatoes.
Description
Technical Field
The invention relates to radiation-resistant streptomyces (Streptomyces radiopugnans) and application thereof, in particular to radiation-resistant streptomyces and related application thereof in inhibiting growth of pathogenic bacteria of potato scab.
Background
The potato has strong environmental adaptability, high economic benefit, rich nutrition of tubers and great yield-increasing potential, and is a fourth main grain crop which is used as a global food and is used for rice, wheat and corn and is the most important. In potato production, disease severely affects potato quality and yield, scab is one of the most serious potato diseases in recent years worldwide. The reported pathogenic bacteria causing potato scab are mainly Streptomyces of actinomycetes, and the prominent characteristic of the tubers is that the surface of the tubers is concave, convex or net scab shape, which seriously affects the quality of the potatoes, thereby causing serious economic loss. In the traditional control mode, the soil fumigation method has a certain control effect, but the environmental pollution is serious; pesticide control has no benefit on crops and human health.
In recent years, biological control of potato scab is the most potential control route for comprehensive research. At present, most of biological control technologies relate to control approaches for controlling disease infection by using chemical pesticides, and a small number of biological control technologies relate to control of plant pathogen diseases by using resistant microorganisms.
CN110205258A discloses a streptomycete (Streptomyces pratensis) PBS9 for preventing and treating potato scab, the prevention effect of soaking seed potato seeds in PBS9 on potato scab reaches 57.88%, and the prevention effect of ditch application treatment can reach 62.01%. CN110200016a discloses a streptomyces circulans (Streptomyces anulatus) PBSH9 strain that can control potato scab and promote potato growth. Experiments prove that the seed soaking treatment of the streptomyces circulans strain PBSH9 has a control effect on potato scab of 73.97 percent, can increase the yield of potatoes by 15.93 percent, has a control effect of 64.77 percent by ditch application of the strain PBSH9, can increase the yield by 7.58 percent, and has the effects of promoting the germination of the potato seed and the growth of seedlings. PBSH9 also has an inhibition effect on the growth of verticillium potato, and the inhibition rate reaches 79.07%. CN109303067a discloses a streptomycete composition for preventing and treating potato scab, which is formed by combining streptomycete (Streptomyces anulatus) CGMCC No.15826 and streptomycete (Streptomyces pratensis) CGMCC No. 15829. The seed soaking treatment of the composition on potato seeds has a control effect on potato scab up to 98.47%, and the ditch treatment has a control effect up to 97.15%. However, these prior art Streptomyces species for controlling potato scab are not very ideal for potato virus-free seedlings (rapid propagation seedlings) and have low specificity for potato scab pathogens.
Disclosure of Invention
An object of the present invention is to provide a microorganism which inhibits the growth of pathogenic bacteria of potato scab.
The inventor obtains a strain capable of inhibiting the growth of pathogenic bacteria of potato scab by collecting rhizosphere soil of potato soil sample sites with scab and separating by a coating streaking technology of separated strains and utilizing various culture mediums such as Gao's first number, phosphate solubilizing, nitrogen fixing, 1/2R2A, oat, LB, NA and the like through separation and purification, thereby achieving very effective biological control effect, and the strain is radiation-resistant streptomyces (Streptomyces radiopugnans) through species identification, and is also named radiation-resistant streptomyces KL2 in the invention, and the strain is preserved in China general microbiological culture collection center (CGMCC), and the preservation date is: 2023, 02, 28; preservation unit: china general microbiological culture Collection center (CGMCC); deposit unit address: the institute of microbiology, national institute of sciences, 1, 3, north chen west way, north, south, face, chinese, postal code: 100101; preservation number: CGMCC No.26717; classification naming: streptomyces radiodurans (Streptomyces radiopugnans).
Experiments of the invention show that the radiation-resistant streptomyces (Streptomyces radiopugnans) KL2 is applied to potato rapid propagation seedlings (test tube seedlings) and potting results, shows lower scab incidence and disease degree, shows better biological prevention and treatment potential of potato scab, and the KL2 does not show any inhibition in the plane opposition to other microorganisms (such as common growth-promoting fungi trichoderma harzianum, growth-promoting bacteria pseudomonas and azotobacter and the like), and shows that the radiation-resistant streptomyces (Streptomyces radiopugnans) KL2 is at least highly specific and specific to common scab pathogens (Streptomyces scabiei), has higher biological safety and can be used simultaneously with common plant growth-promoting bacteria or biological prevention and treatment strains (such as trichoderma harzianum). The whole genome of the KL2 strain was determined by the megaina Miseq platform of the metage biosequencing company, and analysis of the results gave the 11 Genomic islands (Genomic island), 3 prophages (Prophage) and 18 CRISPRs found in the strain KL 2. The strain is predicted to be capable of generating a plurality of active substances such as antibiotics and the like, and directly killing or inhibiting growth; reducing pathogenic agent expression and related product production; inducing plants to increase systemic resistance to pathogenic microorganisms; regulating and controlling soil microbial community structure and enhancing disease resistance function expression; nutrition and niche competition. Can obviously inhibit the growth of pathogenic bacteria of potato scab; can obviously prevent and inhibit the occurrence of potato tuber scab symptoms, thereby promoting the development and establishment of root systems of potato rapid propagation seedlings and potted seedlings, leading the root systems to be more developed, thickening stems, thickening leaves and increasing chlorophyll, enhancing the resistance to soil-borne diseases, enhancing the resistance to drought, plant diseases and insect pests and salt stress, promoting the growth and obviously improving the yield and quality of potato tubers.
Thus, in one aspect, the invention provides a strain of radiation resistant streptomyces (Streptomyces radiopugnans) with the preservation number of CGMCC No.26717.
On the other hand, the invention also provides a radiation-resistant streptomycete preparation, which contains the acinetobacter with the preservation number of CGMCC No.26717 and is a solid or liquid fungus preparation.
In another aspect, the invention also provides a fermentation product of Streptomyces radiodurans, which is produced by fermentation of the Streptomyces radiodurans of the invention or the Streptomyces radiodurans preparation of claim 2.
In another aspect, the invention also provides a preparation for inhibiting the growth of pathogenic bacteria of potato scab, which comprises the radiation-resistant streptomyces disclosed by the invention, the radiation-resistant streptomyces preparation disclosed by the invention or the radiation-resistant streptomyces fermentation product disclosed by the invention. Preferably, the concentration of the radiation resistant streptomyces is 10 in the preparation 7 ～10 9 CFU/g or 10 7 ～10 9 CFU/ml。
On the other hand, the invention also provides the application of the radiation-resistant streptomyces, the radiation-resistant streptomyces preparation or the radiation-resistant streptomyces fermentation product in preparation of the preparation for inhibiting the growth of pathogenic bacteria of potato scab.
On the other hand, the invention also provides the application of the radiation-resistant streptomyces, the radiation-resistant streptomyces preparation or the radiation-resistant streptomyces fermentation product or the preparation in preparation of the preparation for specifically inhibiting the growth of pathogenic bacteria of potato scab.
According to a specific embodiment of the invention, the formulation according to the invention also has at least one of the following effects:
generating active substances such as antibiotics;
reducing pathogenic agent expression and related product production;
inducing plants to increase systemic resistance to pathogenic microorganisms;
regulating and controlling soil microbial community structure, enhancing disease resistance function expression, nutrition and ecological niche competition
Promoting the development and establishment of potato root systems;
thickening potato stems, thickening leaves and increasing chlorophyll;
enhancing the resistance of potatoes to soil-borne diseases;
enhancing resistance of potatoes to drought, disease and insect pests, and salt stress;
promoting potato growth;
improving the yield and quality of potato tubers.
According to a specific embodiment of the invention, the radiation-resistant streptomyces preparation or the radiation-resistant streptomyces (Streptomyces radiopugnans) KL2 of the invention has specificity to potato scab pathogenic bacteria and can inhibit the growth of potato scab pathogenic bacteria, thereby promoting the growth of disease-resistant tuber planting cut pieces of potatoes; the prepared antagonistic bacterial suspension can obviously promote the development and establishment of the root system of the potato seedling, the root system is developed, the stems are thickened, the leaves are thickened, the chlorophyll is increased, the resistance to soil-borne diseases is enhanced, the resistance to drought and salt stress is enhanced, and the yield and quality of potato tubers are obviously improved.
Drawings
FIG. 1 shows the results of screening experiments for the antagonistic bacteria against scab pathogenic bacteria in the present invention. And (3) upper pictures: the width of the antagonistic bacteria inhibition zone is 7mm, 5mm and 7mm respectively according to the primary screening result; the following pictures: verifying and re-screening effects, wherein the widths of the antagonistic bacteria inhibition zones are 33mm, 32mm and 35mm respectively.
Fig. 2 shows the experimental results of KL2 strain antagonism control group.
FIG. 3 shows morphological analysis of fast-propagating seedlings inoculated with pathogenic bacteria, inoculated with KL2 strain and non-inoculated with CK control.
FIG. 4 is a graph showing the final product of the antagonistic control results of the potato (stock) obtained after about 3 months of the verification experiment of adding antagonistic bacteria liquid after transplanting potato rapid propagation seedlings (test tube seedlings) into potato scab pathogen soil. Left picture: the invention is characterized in that; right picture, control group.
Fig. 5 and 6 are potato samples collected in a field continuous cropping system of the present invention.
Microbial preservation for patent procedures:
streptomyces radiodurans KL2 of the invention:
preservation date: 2023, 02, 28;
preservation unit: china general microbiological culture Collection center (CGMCC);
deposit unit address: the institute of microbiology, national institute of sciences, 1, 3, north chen west way, north, south, face, chinese, postal code: 100101;
preservation number: CGMCC No.26717;
classification naming: streptomyces radiodurans (Streptomyces radiopugnans).
Detailed Description
The technical solution of the present invention will be described in detail below for a clearer understanding of technical features, objects and advantageous effects of the present invention, but should not be construed as limiting the scope of the present invention. The experimental procedures, which do not address specific conditions in each example, are performed under conditions conventional in the art. Preservation, resuscitation and seed solution preparation of the X006 strain were carried out by conventional methods of Acinetobacter.
The culture medium and the formulation thereof used in the following examples have the following proportions of the components:
culture medium No.1, gao: soluble starch 20g, potassium nitrate 1g, dipotassium hydrogen phosphate 0.5g and MgSO 4 ·7H 2 O0.5g，NaCl 0.5g，FeSO 4 ·7H 2 0.01g of O, 20.0g of agar, 1000mL of distilled water and pH 7.2-7.4.
1/2R2A Medium: glucose 0.5g, starch 0.5g, acid hydrolyzed casein peptone 0.5g, bacteriological peptone 0.5g, yeast extract 0.5g, sodium pyruvate 0.3g, dipotassium phosphate 0.3g, magnesium sulfate heptahydrate 0.3g, TES 2mL. Agar solid medium was additionally supplemented with 15g of agar.
PDA medium: 160g of potato, 20g of sucrose and 20g of agar. 160g of peeled potatoes are weighed, cut into small pieces, added with a proper amount of distilled water to be boiled until the potato pieces are gently stirred up by a glass rod, filtered by four layers of gauze to collect filtrate, and added with glucose and agar to be supplemented with water to 1000mL.
Rye medium: 50g of rye, 20g of sucrose and 20g of agar. Weighing 50g of rye, cleaning, soaking in 1000mL of deionized water overnight, sterilizing at 121deg.C for 40min under high pressure, filtering with 4 layers of gauze, collecting filtrate, adding sucrose and agar, supplementing water to 1000mL, sterilizing at 121deg.C under high pressure, and naturally maintaining pH.
Oat medium: oat 60g, sucrose 20g and agar 8g. Weighing 60g of oat, cleaning, soaking in distilled water for 24 hours, sterilizing at 121 ℃ for 40 minutes under high pressure, filtering with 4 layers of gauze, collecting filtrate, adding sucrose and agar, supplementing water to 1000mL, sterilizing at 121 ℃ under high pressure, and naturally adjusting pH.
LB medium: 10g of tryptone, 5g of yeast extract, 10g of NaCl, 15g of agar, 1000mL of deionized water and pH 7.4.
Example 1 screening and identification of Streptomyces radiodurans KL2
Collecting rhizosphere soil with potato scab in field, taking 10g of soil sample, adding 90ml of sterile water, shaking table at 180r/min for 30min, taking 1ml of soil suspension, and adding 9ml of sterile water to obtain 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 Six gradient dilutions, multiple, etc.), 100 μl of each concentration was taken, the same amount of dilution was applied to the surface of a plate of solid medium No. one Gao, and after 5d incubation in a constant temperature incubator at 28 ℃, single Streptomyces colonies were picked from the plate streak culture, and purification was performed by plate streak culture until the colonies became single pure colonies. Then, the numbers of the individual colonies were recorded, a part of Streptomyces colonies was nipped with forceps to a 2ml test tube, 25% glycerol was added, and the test tube was placed in a refrigerator at-80℃for preservation for use.
The bacterial strain genome DNA obtained by separation and purification is further used as a template, the DNA is extracted, a bacterial universal primer 27F/1492R is adopted to amplify the 16 srdnas, the amplified products are detected by agarose gel electrophoresis of 1 percent (containing SYBR Green I fluorescent dye), sequencing is completed by the division of biological engineering (Shanghai), the sequences obtained by sequencing are subjected to Blast comparison (or EzTaxon) on NCBI, and sequences with higher similarity in a database (the primary determination that the sequence similarity of the 16S rRNA gene is more than 97 percent) are prepared for identification.
In the invention, a strain is obtained by screening, the invention is named KL2, and the similarity between the 16S rRNA gene sequence of the KL2 and the model strain radiation-resistant streptomyces (Streptomyces radiopugnans) (with the accession number NR_ 044013) reaches 99.713 percent through identification. The results are shown in Table 1.
TABLE 1
| Strain numbering | The nearest species | Similarity (%) |
| KL2 | Streptomyces radiopugnans | 99.713 |
Preliminary identification the strain KL2 screened by the invention is radiation-resistant streptomycete (Streptomyces radiopugnans), and the strain is preserved in China general microbiological culture Collection center (CGMCC), and the preservation date is as follows: 2023, 02, 28; preservation unit: china general microbiological culture Collection center (CGMCC); deposit unit address: the institute of microbiology, national institute of sciences, 1, 3, north chen west way, north, south, face, chinese, postal code: 100101; preservation number: CGMCC No.26717; classification naming: streptomyces radiodurans (Streptomyces radiopugnans).
While KL2 does not show any inhibition in the plane opposition to other microorganisms (such as the common growth-promoting fungi Trichoderma harzianum, the growth-promoting bacteria Pseudomonas and azotobacter, etc.), it shows that at least the most common scab pathogen (Streptomyces scabiei) is highly specific and specific, has higher biological safety, and can be used simultaneously with the common plant growth-promoting bacteria or biological control strains (such as Trichoderma harzianum). The whole genome of the KL2 strain was determined by the megaina Miseq platform of the metage biosequencing company, and analysis of the results gave the 11 Genomic islands (Genomic island), 3 prophages (Prophage) and 18 CRISPRs found in the strain KL 2. The strain is predicted to be capable of generating a plurality of active substances such as antibiotics and the like, and directly killing or inhibiting growth; reducing pathogenic agent expression and related product production; inducing plants to increase systemic resistance to pathogenic microorganisms; regulating and controlling soil microbial community structure and enhancing disease resistance function expression; nutrition and niche competition.
The virulence factor predictive classification statistics of strain KL2 are shown in Table 2. The comparison of the secondary metabolite synthesis gene cluster in strain KL2 with the reference strain is shown in Table 3.
TABLE 2 prediction classification statistics of strain KL2 virulence factors
Table 3, comparison of the secondary metabolite Synthesis Gene Cluster in Strain KL2 and reference Strain
Example 2: KL2 strain activation, culture and collection
100 mu L of the mixed preservation (the radiation-resistant streptomyces KL2 of the example 1) of 500 mu L of glycerol which is absorbed by 25% and bacterial liquid in a ratio of 1:1 is coated on a culture medium of Gao's first and cultured at 28 ℃, single colony with typical colony characteristics and rapid growth is selected to be placed in a corresponding liquid culture medium (potassium dichromate is added) and is cultured at 25 ℃; centrifuging at 8000g centrifugal force during late logarithmic phase, and culturing at K 2 HPO 4 -KH 2 PO 4 After repeated washing 3 times with buffer (pH 7.2), the cells were resuspended to cell concentration with the same buffer10 7 ～10 9 CFU/mL。
Example 3: KL2 bacteria antagonism potato pathogenic microorganism
100. Mu.L of Streptomyces scab spore suspension was pipetted onto solid medium, and a sterile filter paper sheet (d=6 mm) was placed on the plate for further use. Inoculating the separated KL2 strain to a liquid LB culture medium or a Gaoshan first culture medium (the two culture mediums are respectively used for primary screening and secondary screening), culturing for 24 hours at 28 ℃ at 200r/min, sucking 6 mu L of culture liquid drop at the center of a filter paper sheet, taking the sterile liquid LB/Gaoshan first culture medium as a blank control, culturing for 5-7 days at 28 ℃, and measuring the diameter of a bacteriostasis ring to calculate the bacteriostasis rate.
The radiation-resistant streptomyces KL2 has remarkable inhibition effect on the growth of pathogenic microorganisms which are particularly serious in potato production, namely potato scab pathogenic bacteria; the potato seedling growing agent has a growth promoting function for the growth of potato rapid propagation seedlings; compared with common streptomyces, the strain has strong radiation resistance (the strain is classified as the radiation-resistant streptomyces Streptomyces radiopugnans just because of the strong ultraviolet radiation resistance).
As shown in the results of figures 1, 2 and 4, the radiation resistant streptomyces KL2 has remarkable effect on inhibiting scab, has no remarkable inhibition effect on fungi such as phytophthora potato, aspergillus flavus, rhizopus oryzae and the like, and potato black nevus and fusarium wilt through detection verification, and shows that the fungus has high specificity on common scab pathogenic bacteria.
TABLE 4 Table 4
| Bacterial strain | Antibacterial rate |
| KL2 | 48.50％ |
| CK (blank) | 20.20％ |
The results of the plate-facing experiments in Table 4 show that: the radiation-resistant streptomyces KL2 has a bacteriostasis rate of 40% -50% on potato common scab Streptomyces scabies, has a certain potential for preventing and treating potato soil-borne diseases, promotes sustainable development of national agriculture, especially inner Mongolia agriculture, and improves the biological industry, and has important theoretical research significance and practical application value.
Example 4: effect of KL2 strain on potato rapid propagation seedling and potted scab potato
Effect experiment of KL2 strain on potato rapid propagation seedling (containing scab pathogen suspension): streptomyces radiopugnans KL2 is used for preparing bacterial suspension, and the effective viable count of the bacterial suspension is 10 7 ～10 9 CFU/g or 10 7 ～10 9 CFU/mL, when the OD value of the bacterial liquid reaches 0.8-1.0 by using an ultraviolet spectrophotometer under the condition of 600nm, 1mL:50mL (bacterial liquid: MS culture medium) is added into the rapid propagation seedling, and the growth condition of the seedling is observed in 25 days period.
As shown in fig. 3 and table 5, the invention has the capability of promoting the growth of the potato rapid propagation seedlings and enhancing the resistance of the rapid propagation seedlings.
TABLE 5
| Treatment of | Plant height | Root length | Stolon stem | Number of blades |
| KL 2-grafted strain | 10.83±0.19a | 10.2±0.87a | 4.28±0.58a | 8.44±0.46a |
| Non-inoculation CK | 9.41±0.30b | 8.47±0.39b | 3.92±0.41a | 6.88±0.11b |
| Inoculating pathogenic bacteria | 7.50±0.28c | 7.77±0.48b | 2.32±0.27b | 6.32±0.18c |
Effect experiment of KL2 strain on potted scab potato: according to soil: vermiculite=1:1/2 (the mixed soil is not sterilized, the soil is a soil sample collected in scab disease), a pot is firstly filled with 30cm×30cm, and the pot is just poured into a rectangular flowerpot with the length of 57cm, the width of 31cm and the height of 13.5 cm. Healthy potato stock seeds are planted firstly, 50mL of water is added into each pit, then soil is covered, and finally 1500mL of water is added into each flowerpot, so that the soil is wet. Streptomyces radiopugnans KL2 bacterial suspension (effective viable count 10) 7 ～10 9 CFU/g or 10 7 ～10 9 The CFU/mL) was watered once every other week (100 mL each time).
Experimental results show that the radiation-resistant streptomyces KL2 can remarkably promote the establishment of potato root systems, enhance the resistance to drought and diseases, promote the growth and improve the yield and appearance quality of potato tubers (figure 4).
According to the experiment of the potted scab potato, common biological control strain trichoderma harzianum is added into soil, and experimental results show that the inhibition effect of radiation-resistant streptomyces KL2 on potato scab pathogenic bacteria is basically the same as experimental results of promoting root system establishment, enhancing drought and disease resistance, promoting growth and improving potato tuber yield and appearance quality.
Fig. 5 and 6 show potato samples collected by continuous cropping in the field, and it can also be seen that the radiation-resistant streptomycete KL2 has an inhibiting effect on potato scab pathogenic bacteria, and can significantly promote the establishment of potato root systems, enhance drought and disease resistance, promote growth, and improve potato tuber yield and appearance quality.
Claims (10)
1. A radiation resistant Streptomyces (Streptomyces radiopugnans) has a preservation number of CGMCC No.26717.
2. A radiation-resistant streptomycete preparation contains Acinetobacter with a preservation number of CGMCC No.26717, which is a solid or liquid fungus preparation.
3. A fermentation product of Streptomyces radiodurans produced by fermentation of the Streptomyces radiodurans of claim 1 or the Streptomyces radiodurans formulation of claim 2.
4. A formulation for inhibiting the growth of pathogenic bacteria of potato scab comprising the streptomyces radiodurans of claim 1, the streptomyces radiodurans formulation of claim 2, or the streptomyces radiodurans fermentation product of claim 3.
5. The formulation of claim 4, wherein the concentration of Streptomyces radiodurans is 10 7 ～10 9 CFU/g or 10 7 ～10 9 CFU/ml。
6. Use of the radiation-resistant streptomyces of claim 1, the radiation-resistant streptomyces formulation of claim 2 or the radiation-resistant streptomyces fermentation product of claim 3 for the preparation of a formulation for inhibiting the growth of potato scab pathogens.
7. Use of a streptomyces radiodurans according to claim 1, a streptomyces radiodurans preparation according to claim 2 or a streptomyces radiodurans fermentation product according to claim 3, or a preparation according to claim 4 or 5 for the preparation of a preparation for specifically inhibiting the growth of pathogenic bacteria of potato scab.
8. The use according to claim 6 or 7, wherein the formulation further has at least one of the following efficacy:
generating active substances such as antibiotics;
reducing pathogenic agent expression and related product production;
inducing plants to increase systemic resistance to pathogenic microorganisms;
regulating and controlling soil microbial community structure, enhancing disease resistance function expression, nutrition and ecological niche competition
Promoting the development and establishment of potato root systems;
thickening potato stems, thickening leaves and increasing chlorophyll;
enhancing the resistance of potatoes to soil-borne diseases;
enhancing resistance of potatoes to drought, disease and insect pests, and salt stress;
promoting potato growth;
improving the yield and quality of potato tubers.
9. The use according to claim 8, wherein the radiation-resistant streptomyces of claim 1, the radiation-resistant streptomyces formulation of claim 2 or the radiation-resistant streptomyces fermentation product of claim 3, or the formulation of claim 4 or 5 is acting on a potato detoxified seedling.
10. The use according to claim 8, wherein the radiation-resistant streptomyces species is specific for potato scab pathogens.
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