CN110144302B - High-yield siderophin-producing extreme pseudomonas orientalis, microbial inoculum and application - Google Patents
High-yield siderophin-producing extreme pseudomonas orientalis, microbial inoculum and application Download PDFInfo
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Abstract
The invention relates to the field of biology, in particular to pseudomonas extreme orientalis with high yield of siderophin, a microbial inoculum and application. The strain is SWNY4-5, and the strain is registered and preserved in China general microbiological culture Collection center (CGMCC); the preservation date is 2018, 10 months and 10 days; the serial number is CGMCC No.16563. Compared with the prior art, the invention adopting the technical scheme has the following beneficial effects: the microbial inoculum can effectively prevent and treat the physiological iron deficiency yellows of plants; the plant pathological yellows caused by root-knot nematodes are effectively prevented and treated; effectively inhibiting plant diseases utilizing iron element nutrition; effectively preventing and treating other plant diseases caused by root-knot nematodes.
Description
Technical Field
The invention relates to the field of biology, in particular to pseudomonas extreme orientalis with high yield of siderophin, a microbial inoculum and application.
Background
Physiological iron deficiency and pathological yellowing disease are serious plant diseases, after plants are yellowed, leaves are green and yellow, photosynthesis is blocked, tree vigor is weak, yield and fruit quality are reduced slightly, and plants die seriously. The physiological iron deficiency yellows is caused by the following reasons: firstly, because the iron content in the soil is too low; secondly, iron in the soil exists in a ferric iron form, and plants cannot absorb and utilize the iron; thirdly, the transportation of the iron element in the plant is problematic, and the iron element cannot reach the plant leaves. The pathological yellowing disease is caused by the pathological changes of plant roots, such as root knot nematode or other fungal root diseases causing the absorption of nutrients and trace metal elements by plants.
Siderophins are low molecular weight substances that bind ferric ions (ferricion) and supply microbial cells. The siderophore is mainly present in microorganisms (bacteria, fungi), and additionally, gramineae, such as barley, wheat, etc., can synthesize siderophore. The microorganisms need iron elements to synthesize cytochromes and enzymes, but the earth is an oxygen-rich environment, and iron in nature exists in the form of oxides and is extremely insoluble (extreme insoluble). Siderophore is defined briefly as: a low molecular weight compound for taking up iron element, which chelates iron, synthesized and secreted by microorganisms or plants. Pseudomonas is a type of plant growth-promoting rhizobacteria.
Disclosure of Invention
The purpose of the invention is as follows: in order to provide the high-yield siderophore extreme pseudomonas orientalis with better effect, the microbial inoculum and the application, the specific purpose is seen in a plurality of substantial technical effects of a specific implementation part.
In order to achieve the purpose, the invention adopts the following technical scheme:
a high-yield siderophore extreme oriental pseudomonas is characterized in that a strain is SWNY4-5, and the strain is registered and preserved in CGMCC (China general microbiological culture Collection center); the preservation date is 2018, 10 months and 10 days; the serial number is CGMCC No.16563.
The culture medium for the high-yield siderophin extreme oriental pseudomonas is characterized in that the culture medium for slant culture preservation is an NA culture medium or a KMB culture medium, wherein the NA culture medium comprises 3g of beef extract, 10g of peptone, 5g of NaCl, 1000mL of water and agarSterilizing at pH7.0-7.2 of 15-20g at 121 deg.C for 20min; KMB Medium, i.e. Kings Medium B: peptone 20.0g, K 2 HPO 4 1.5g，MgSO 4 ·7H 2 1.5g of O, 10mL of glycerol, 1000mL of water, 15-20g of agar, 7.2 +/-0.2 of pH7, and sterilizing for 20min at 121 ℃. The culture medium for producing the SWNY4-5 microbial inoculum by fermentation is an improved MSA culture medium: sucrose 20.0g/L, aspartic acid 2.0g/L, K 2 HPO 4 0.5g/L，MgSO 4 ·7H 2 O0.5 g/L, pH7.2-7.5, sterilizing at 121 deg.C for 20min.
A high-yield siderophore extreme oriental pseudomonas is characterized in that the microscopic morphological characteristics of SWNY4-5 are as follows: gram reaction is negative, and the thallus is long-rod-shaped and does not produce spores; the physiological and biochemical characteristics are as follows:
the application of SWNY4-5 strain with preservation number of CGMCC No.16563 in preparing substance for killing plant pathogenic bacteria Fusarium oxysporum and its spore.
The application of SWNY4-5 strain with preservation number of CGMCC No.16563 in preparing substance for killing root-knot nematode larva and reducing hatching rate of nematode eggs.
The application of SWNY4-5 strain with preservation number of CGMCC No.16563 in preparing medicine for inhibiting plant diseases utilizing iron element nutrition.
A novel sterilization method is characterized in that SWNY4-5 strain with the preservation number of CGMCC No.16563 generates extracellular protease and cellulase, and the physiological activity of pathogenic microorganisms is destroyed by the extracellular protease and the cellulase.
A novel microbial inoculum is characterized in that the microbial inoculum is a fermentation microbial inoculum, and the main components of the microbial inoculum comprise SWNY4-5 strain living bodies with the preservation number of CGMCC No.16563 and fermentation metabolites of the SWNY4-5 strain with the preservation number of CGMCC No.16563; the microbial agent may be a liquid fermentation broth or a solid powder.
The production method of the microbial agent is characterized in that the SWNY4-5 strain with the preservation number of CGMCC No.16563 is subjected to slant culture → shake flask culture → seed tank culture → production tank zymocyte liquid → solid bacterial powder;
the culture medium for slant strains is an NA culture medium; the culture medium for shaking the bottle strain is a conventional NB culture medium, and the NB culture medium is an NA culture medium without agar; the culture medium for seeding tank strain and fermentation production is an improved MSA culture medium; the culture temperature is 30-37 ℃, the slant culture time is 24h, the culture time of shake flask strains and seed tank strains is 18-24h, and the fermentation time of the produced microbial inoculum is 24-36h.
Compared with the prior art, the invention adopting the technical scheme has the following beneficial effects: the microbial inoculum can effectively prevent and treat the physiological iron deficiency yellowing disease of plants; the plant pathological yellows caused by root-knot nematodes are effectively prevented and treated; effectively inhibiting plant diseases utilizing iron element nutrition; effectively preventing and treating other plant diseases caused by root-knot nematodes.
Drawings
To further illustrate the present invention, further description is provided below with reference to the accompanying drawings:
FIG. 1 colony morphology of strain SWNY4-5 on NA medium;
FIGS. 2-3 colony morphology of strain SWNY4-5 on KMB medium;
FIG. 4 microscopic morphology (100X) of strain SWNY 4-5;
FIG. 5-6 Strain SWNY4-5 the siderophore chelation color change circles generated on CAS plates;
FIG. 7 is a clear circle generated by the strain SWNY4-5 on the protease detection plate;
FIG. 8 is a transparent circle generated by the strain SWNY4-5 on a cellulase detection plate;
FIG. 9 results of plate inhibition assay of strain SWNY4-5 on Fusarium oxysporum;
FIG. 10-11 inhibitory Effect of the Strain SWNY4-5 on Fusarium oxysporum hypha development FIG. 10 is CK;
FIG. 12-14 inhibitory effect of strain SWNY4-5 on Fusarium oxysporum spore germination FIG. 12 is CK;
FIG. 15 shows the physiological and biochemical characteristics of strain SWNY4-5 of Table 1;
FIG. 16 is a table 2SWNY4-5 microbial inoculum for root-knot nematode poisoning;
FIG. 17 shows the inhibition of the root knot nematode egg hatchability by the microbial inoculum of Table 3SWNY 4-5;
FIG. 18 is a preservation certificate and a survival certificate.
The extreme oriental pseudomonas strain is SWNY4-5, and the preservation number of the strain is CGMCC NO.16563; the preservation unit is called China general microbiological culture Collection center (CGMCC), for short. Address: xilu No.1 Hospital No. 3, beijing, chaoyang, north; survival: is that; the preservation date is as follows: 10 months and 10 days in 2018; and (3) classification and naming: pseudomonas orientalis SWNY4-5 Pseudomonas extremomentalia.
Detailed Description
The invention is further elucidated with reference to the drawings and the detailed description.
The patent provides a plurality of parallel schemes, and different expressions belong to improved schemes based on the schemes or parallel schemes. Each solution has its own unique features.
A high-yield siderophore extreme Pseudomonas orientalis (Pseudomonas exotriementalis) SWNY4-5 is registered and preserved in China general microbiological culture Collection center (CGMCC); the preservation date is 2018, 10 months and 10 days; the serial number is CGMCC No.16563.
A high-yield siderophore extreme Pseudomonas orientalis (Pseudomonas extrementallis) SWNY4-5 is separated from the rhizosphere soil of a kiwi fruit tree, and a bacterial colony is characterized in that the bacterial colony is cultured on a NA culture medium (beef extract 3g, peptone 10g, naCl 5g, water 1000mL, agar 15-20g, pH7.0-7.2, and sterilization is carried out for 20min at 121 ℃) plate, and the bacterial colony is milky white, nearly circular, protruding, neat in edge, smooth and moist in surface, and does not produce pigments. In KMB Medium (Kings Medium B: peptone 20.0g 2 HPO 4 1.5g，MgSO 4 ·7H 2 O1.5g, glycerol 10mL, water 1000mL, agar 15-20g, pH7.2 + -0.2, sterilization at 121 ℃ for 20 min) plate, the colony morphology is similar to that on the NA culture medium, but bright yellow-green pigment is produced. The colony morphology of SWNY4-5 is characterized in FIG. 1,2,3.
The microscopic morphological characteristics of high-yield siderophore extreme Pseudomonas orientalis (Pseudomonas extremomentalis) SWNY4-5 are as follows: gram reaction is negative, the thallus is long rod-shaped, does not produce spores, and has the size of length multiplied by width =1.5-2.0 multiplied by 0.3-0.5 μm. The photomicrograph of the strain is shown in FIG. 4.
The physiological and biochemical characteristics of high-yield siderophore extreme Pseudomonas orientalis (Pseudomonas extremomentalis) SWNY4-5 are shown in Table 1.
Experiments prove that the high-yield siderophore extreme Pseudomonas orientalis (Pseudomonas exotrymentalis) SWNY4-5 can generate siderophore with high activity, an obvious light-soil yellow color change ring can be formed on a CAS detection plate, and the siderophore color change ring is shown in figures 5 and 6. The result of quantitative determination shows that the concentration of Siderophore is expressed by Siderophore Unit (SU) and the strain SWNY4-5 is in modified MSA culture solution (sucrose 20.0g/L, aspartic acid 2.0g/L, K) by CAS detection solution method and ultraviolet spectrophotometer at wavelength 680nm 2 HPO 4 0.5g/L，MgSO 4 ·7H 2 O0.5 g/L, pH7.2-7.5, sterilized at 121 ℃ for 20 min) for 24-48 h, wherein the concentration of the siderophin in the fermentation broth is SU =81.1%, as/Ar =0.1891, and the siderophin producing capacity is grade +++++. Through full-band scanning, siderophore in the improved MSA culture solution of the strain SWNY4-5 has a maximum absorption peak at 400 nm.
Experiments prove that high-yield siderophin extreme Pseudomonas orientalis (Pseudomonas extremomentalis) SWNY4-5 can produce extracellular protease and cellulase. Obvious transparent rings can be formed on the protease and cellulase detection plate. The results are shown in FIGS. 7 and 8.
Experiments prove that the fermentation microbial agent for high-yield siderophil extreme Pseudomonas orientalis (Pseudomonas extorementalis) SWNY4-5 has an antagonistic effect on plant pathogenic bacteria fusarium oxysporum, and can obviously inhibit the hypha development and spore germination of the fusarium oxysporum. The results are shown in FIGS. 9, 10, 11, 12, 13 and 14.
Experiments prove that the fermentation microbial agent for high-yield siderophin extreme Pseudomonas orientalis (Pseudomonas extorquential) SWNY4-5 has an inhibition effect on the actinidia root-knot nematode, can kill 2-instar larvae of the root-knot nematode and reduce the hatching rate of eggs. The results are shown in tables 2 and 3.
In order to realize the purpose, the pseudomonas aeruginosa with high yield of siderophore and the microbial inoculum adopt the following technical scheme:
in agricultural production application, a main product form of the high-yield siderophore extreme oriental pseudomonas is a fermentation microbial agent of a bacterial strain SWNY4-5, and the main components of the microbial agent comprise bacterial strain living bodies of the SWNY4-5 and fermentation metabolites of the SWNY 4-5; the microbial agent can be liquid fermentation bacteria liquid or solid bacteria powder, and the solid bacteria powder is obtained by adding carriers and auxiliary materials commonly used for preparing microbial agents in industrial production into the fermentation bacteria liquid.
A production mode of a microbial agent of high-yield siderophil extreme oriental pseudomonas SWNY4-5 is a conventional microbial fermentation mode, and comprises the following steps: slant strain → shake flask strain → seeding tank strain → production tank fermented bacterial liquid → solid bacterial powder.
The culture medium for the slant strain of the microbial agent of the high-yield siderophore extreme oriental pseudomonas SWNY4-5 is a conventional NA culture medium; the culture medium for shaking flask strains is a conventional NB culture medium (NA culture medium does not contain agar); the culture medium for seeding tank strain and fermentation production is improved MSA culture medium, the culture temperature is 30-37 ℃, the stirring speed is 180-250rpm, and the ventilation volume is 0.5-1.0vvm. The slant culture time is 24h, the shake flask culture time and the seeding tank culture time is 18-24h, and the fermentation time of the produced microbial inoculum is 24-36h.
Creatively, the above effects exist independently, and the combination of the above results can be completed by a set of structure.
It should be noted that the solutions provided in this patent include their own basic solutions, which are independent of each other and not restricted to each other, but they may be combined with each other without conflict, so as to achieve a plurality of effects.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are intended to illustrate the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, and the invention is to be limited to the embodiments described above.
Claims (8)
1. The high-yield siderophin extreme oriental pseudomonas is characterized in that a strain is SWNY4-5, and the strain is registered and preserved in CGMCC (China general microbiological culture Collection center); the preservation date is 2018, 10 months and 10 days; the serial number is CGMCC No.16563.
2. The pseudomonas extremely oriental highly productive of siderophiles according to claim 1, characterized by the microscopic morphological characteristics of SWNY 4-5: gram reaction is negative, and the thallus is long-rod-shaped and does not produce spores; the physiological and biochemical characteristics are as follows:
3. the use of the pseudomonas extremely oriental highly productive siderophore as claimed in claim 1, in the preparation of a substance for killing the plant pathogen fusarium oxysporum and its spores.
4. Use of the pseudomonads extremely oriental hybrids of high-yielding siderophilus according to claim 1 for preparing a substance for killing larvae of root-knot nematodes and reducing hatching rate of eggs of the larvae.
5. Use of the pseudomonas extremely oriental highly productive siderophore as claimed in claim 1 in the preparation of a medicament for inhibiting plant diseases utilizing iron element nutrition.
6. A sterilization method, characterized in that the high-yield siderophore extreme Pseudomonas orientalis as claimed in claim 1 produces extracellular protease and cellulase, and the physiological activity of pathogenic microorganisms is destroyed by the extracellular protease and cellulase.
7. A microbial agent, characterized in that it is a fermentation microbial agent, the main components of which comprise the living body of a high-yield siderophin-producing pseudomonas extreme orientalis strain as claimed in claim 1 and the fermentation metabolite of the high-yield siderophin-producing pseudomonas extreme orientalis strain as claimed in claim 1; the microbial agent is a liquid state zymogen liquid or a solid state bacterial powder.
8. The method for producing a microbial agent, characterized in that the high-yield siderophin-producing pseudomonas extremely oriental strain of claim 1 is subjected to slant strain → shake flask strain → seed tank strain → production tank zymogen → solid bacterial powder;
the slant culture medium is NA culture medium; the culture medium for shaking the bottle strain is a conventional NB culture medium, the NB culture medium is an NA culture medium, and agar is not added; the culture medium for seeding tank strain and fermentation production is an improved MSA culture medium, the culture temperature is 30-37 ℃, the stirring speed is 180-250rpm, the ventilation volume is 0.5-1.0vvm, the slant strain culture time is 24h, the shaking flask strain and seeding tank strain culture time is 18-24h, and the production microbial inoculum fermentation time is 24-36h.
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| WO2014089951A1 (en) * | 2012-12-14 | 2014-06-19 | 江苏耕耘化学有限公司 | Trichoderma asperellum strain gy20 and paste preparation for preventing and treating fusarium oxysporum schl. f. sp. fragariae |
| CN104017759A (en) * | 2014-06-10 | 2014-09-03 | 天津双联科鑫生物科技有限公司 | Pseudomonas strain capable of producing siderophore with high yield and application thereof |
| CN104254611A (en) * | 2012-02-28 | 2014-12-31 | 马罗内生物创新公司 | Control of phytopathogenic microorganisms with pseudomonas sp. and substances and compositions derived therefrom |
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|---|---|---|---|---|
| CN104254611A (en) * | 2012-02-28 | 2014-12-31 | 马罗内生物创新公司 | Control of phytopathogenic microorganisms with pseudomonas sp. and substances and compositions derived therefrom |
| WO2014089951A1 (en) * | 2012-12-14 | 2014-06-19 | 江苏耕耘化学有限公司 | Trichoderma asperellum strain gy20 and paste preparation for preventing and treating fusarium oxysporum schl. f. sp. fragariae |
| CN104017759A (en) * | 2014-06-10 | 2014-09-03 | 天津双联科鑫生物科技有限公司 | Pseudomonas strain capable of producing siderophore with high yield and application thereof |
Non-Patent Citations (2)
| Title |
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| 嗜铁素在促进植物生长及病害防治等方面的应用;余贤美等;《中国农学通报》;20070805(第08期);摘要 * |
| 荧光假单胞菌植物病害防治及研究进展;李等;《分子植物育种》;20180614(第11期);摘要 * |
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