CN111944710A - Shewanella and application thereof in improving stress resistance of plants - Google Patents
Shewanella and application thereof in improving stress resistance of plants Download PDFInfo
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Abstract
The invention discloses Shewanella and application thereof in improving stress resistance of plants. The Shewanella is Shewanella sp YJD-CS2, which has a preservation number of CCTCC NO: and M2019330. Experiments demonstrated that Shewanella sp YJD-CS2CCTCC NO: m2019330 can improve the saline-alkali stress resistance of the plant, and is particularly characterized in that the dry weight, the fresh weight, the total root length and the root surface area of the plant are obviously increased. The invention has important application value.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to Shewanella and application thereof in improving stress resistance of plants.
Background
The salinization of soil has become a serious problem affecting agricultural production, and crops are often stressed by abiotic stress factors such as high alkali and high salt in the growth and development process of the crops, and the stress factors cause a large amount of yield reduction of the crops in the world. In recent years, adverse factors such as abnormal climate, scarcity of water resources, reduction of available cultivated land and increase of saline-alkali soil seriously threaten national food safety of China. At present, people carry out a great deal of research on the mechanism of the plant responding to the adverse stress such as saline-alkali stress and the like from the aspects of physiology, biochemistry, metabolism, ecology, heredity, evolution and the like, accumulate abundant data, and particularly along with the development of molecular biology, people can know the adverse resistance mechanism of the plant to the saline-alkali stress on the molecular level of gene composition, expression regulation, signal conduction and the like, thereby opening up a new way for improving the adverse resistance of the plant by utilizing a genetic engineering means. Because of the complexity of the plant stress resistance character, the traditional breeding method is very difficult to improve the stress resistance of the plant, and although the genetic engineering means opens up a new way for the plant stress resistance breeding, the separation of the high-efficiency stress resistance gene becomes a main factor for limiting the plant stress resistance genetic engineering.
Disclosure of Invention
The invention aims to improve the stress resistance of plants.
The invention firstly protects Shewanella sp YJD-CS2, the strain is preserved in China center for type culture Collection (CCTCC for short, address: Wuhan university, Wuhan, China) in 2019 at the number of 07 th month, the preservation number is CCTCC NO: and M2019330. Shewanella (Shewanella sp.) YJD-CS2CCTCC NO: m2019330 is abbreviated as Shewanella (Shewanella sp) YJD-CS2 or Shewanella YJD-CS 2.
The invention also protects a microbial inoculum which contains the Shewanella YJD-CS 2.
The microbial inoculum can be used for improving the saline-alkali stress resistance of plants.
The preparation method of the microbial inoculum comprises the following steps: shewanella YJD-CS2 was inoculated into bacterial culture medium and performedCulturing to obtain OD600nmThe bacterial liquid with the value of 0.5-1.0 is the microbial inoculum.
The bacterial culture medium can be LB liquid culture medium.
In the preparation method of the microbial inoculum, the culture conditions can be as follows: 25-35 deg.C (such as 25-30 deg.C, 30-35 deg.C, 25 deg.C, 30 deg.C or 35 deg.C), 100-.
The microbial inoculum may include a carrier in addition to the active ingredient. The carrier may be a solid carrier or a liquid carrier. The solid carrier may be a mineral material, a plant material or a polymeric compound. The mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth. The plant material may be at least one of corn flour, bean flour and starch. The high molecular compound may be polyvinyl alcohol. The liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water. The organic solvent may be decane and/or dodecane. In the microbial inoculum, the active ingredient may be present in the form of cultured living cells, a fermentation broth of living cells, a filtrate of a cell culture, or a mixture of cells and a filtrate. The composition can be prepared into various dosage forms, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules.
According to the requirement, the microbial inoculum can also be added with a surfactant (such as Tween 20, Tween 80 and the like), a binder, a stabilizer (such as an antioxidant), a pH regulator and the like.
The Shewanella (Shewanella sp.) YJD-CS2CCTCC NO: the application of M2019330 or any of the microbial inoculum in regulating and controlling the stress resistance of plants also belongs to the protection scope of the invention.
In the application, the regulation and control of the plant stress resistance can be the improvement of the saline-alkali resistance of the plant (namely the improvement of the saline-alkali stress resistance of the plant).
The Shewanella (Shewanella sp.) YJD-CS2CCTCC NO: the application of M2019330 or any one of the above bactericides in preparation of products for improving stress resistance of plants.
In the above application, the product may be a microbial fertilizer.
In any of the above applications, the stress resistance may be three, any two or any one of salt resistance, alkali resistance and salt and alkali resistance.
In the use of any of the above, the plant may be any of the following c1) to c 11): c1) a dicotyledonous plant; c2) a monocot plant; c3) a gramineous plant; c4) a plant of the Solanaceae family; c5) a cruciferous plant; c6) corn; c7) maize inbred line B73; c8) tomatoes; c9) tomato variety M82; c10) arabidopsis thaliana; c11) the wild type Arabidopsis thaliana Columbia-0 subtype.
The invention also provides a microbial fertilizer which contains the Shewanella sp YJD-CS2CCTCC NO: m2019330 or any of the above microbial agents.
The invention also discloses a method for improving the stress resistance of plants, which can be used for treating the plants by using the Shewanella, thereby improving the stress resistance of the plants.
In the above method, the "treating a plant with Shewanella" may be performed by applying Shewanella to a root system of the plant.
In the above method, the "treating the plant with Shewanella" may be carried out by spraying Shewanella onto the aerial parts (e.g., leaves) of the plant.
In the above method, the step of treating the plant with Shewanella may be specifically a step of treating the plant with any of the above-mentioned microbial agents.
In the method, the step of treating the plant by using any of the microbial inoculum can be realized by applying any of the microbial inoculum to a plant root system.
In the method, the step of treating the plant by using any of the microbial inoculum can be realized by spraying any of the microbial inoculum to the overground part (such as leaves) of the plant.
In any of the above methods, the Shewanella bacteria can be the Shewanella sp YJD-CS2CCTCC NO: and M2019330.
In the above method, the step of "using said Shewanella sp (Shewanella sp.) YJD-CS2CCTCC NO: the M2019330 or the microbial inoculum for treating the plant can be applied to the root system of the plant or sprayed to the overground part (such as leaves) of the plant.
In the method, the stress resistance can be three, any two or any one of salt resistance, alkali resistance and salt and alkali resistance.
In the above method, the plant may be any one of the following c1) to c 11): c1) a dicotyledonous plant; c2) a monocot plant; c3) a gramineous plant; c4) a plant of the Solanaceae family; c5) a cruciferous plant; c6) corn; c7) maize inbred line B73; c8) tomatoes; c9) tomato variety M82; c10) arabidopsis thaliana; c11) the wild type Arabidopsis thaliana Columbia-0 subtype.
Any one of the microbial fertilizers can be a compound microbial fertilizer and/or a biological organic fertilizer. The compound microbial fertilizer can be a fertilizer compounded by microbial inoculum, nutrient substances and organic matters. The compound microbial fertilizer has the functions of both microbes and chemical fertilizers. The biological organic fertilizer can be a fertilizer formed by compounding a microbial inoculum and a decomposed organic fertilizer. The dosage form of the compound microbial fertilizer and/or the biological organic fertilizer can be granules.
Any of the above mentioned improvements in plant stress resistance may in particular be manifested in an increase in the dry weight and/or fresh weight and/or total root length and/or root surface area of the plant. Any of the above-described improvements in plant stress resistance may also be manifested by an increase in the height of the aerial parts (i.e., an increase in plant height).
Experiments prove that the Shewanella sp YJD-CS2CCTCC NO provided by the invention has the following characteristics: the M2019330 can improve the resistance of tomatoes, corns and arabidopsis thaliana to saline-alkali stress, and is specifically characterized in that the dry weight, the fresh weight, the total root length and the root surface area are obviously increased, and the height of the overground part is increased (namely the plant height is increased). The invention has important application value.
Drawings
FIG. 1 shows the morphology of the bacterium YJD-CS2 observed by high-resolution transmission electron microscopy.
FIG. 2 shows the effect of YJD-CS2 microbial inoculum on root growth of Arabidopsis seedlings under saline-alkali stress conditions.
FIG. 3 is a statistical result of root length of Arabidopsis seedlings treated by YJD-CS2 microbial inoculum under saline-alkali treatment conditions.
FIG. 4 shows the effect of YJD-CS2 inoculum on whole seedlings and aerial parts of maize seedlings under alkali stress conditions.
FIG. 5 is the effect of YJD-CS2 inoculum on root morphology of maize seedlings under alkaline stress conditions.
FIG. 6 is a statistical result of the average total root length, average root surface area, average fresh weight and average dry weight of maize seedlings treated by YJD-CS2 microbial inoculum under alkali stress conditions.
FIG. 7 shows the effect of YJD-CS2 inoculum on whole seedlings and aerial parts of tomato seedlings under alkali stress conditions.
FIG. 8 is the effect of YJD-CS2 inoculum on root morphology of tomato seedlings under alkaline stress conditions.
FIG. 9 is a graph showing the statistics of the average total root length, average root surface area, average fresh weight and average dry weight of tomato seedlings treated with YJD-CS2 microbial inoculum under alkali stress conditions.
Deposit description
The strain name is as follows: shewanella genus
Latin name: shewanella sp.
The strain number is as follows: YJD-CS2
The preservation organization: china center for type culture Collection
The preservation organization is abbreviated as: CCTCC (China center for cell communication)
Address: university of Wuhan, China
The preservation date is as follows: 5 and 7 months in 2019
Registration number of the preservation center: CCTCC NO: m2019330
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.
The experimental procedures in the following examples are conventional unless otherwise specified.
The experimental materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
In the quantitative experiments in the following examples, three replicates were set up and the results averaged.
LB liquid medium: adding distilled water into 10g tryptone, 5g yeast extract and 10g sodium chloride, adjusting pH to 7.0, adjusting volume to 1L, sterilizing at 121 deg.C for 20min, and cooling.
LB solid medium: adding agar into LB liquid culture medium to make its concentration be 15 g/L; then sterilized at 121 ℃ for 20 min. The medium cooled to about 55 ℃ was poured into a petri dish and allowed to cool naturally.
The Columbia-0 subtype of wild type Arabidopsis thaliana (Arabidopsis thaliana) is described in the following references: kim H, Hyun Y, Park J, Park M, Kim M, Kim H, Lee M, Moon J, Lee I, Kim J.A genetic link between colored responses and flowing time through FVE in Arabidopsis thaliana Nature genetics.2004, 36: 167-. The Columbia-0 subtype of wild type Arabidopsis (Arabidopsis thaliana) is hereinafter referred to as Arabidopsis thaliana for short.
Example 1, Shewanella sp (Shewanella sp.) YJD-CS2CCTCC NO: isolation, identification and preservation of M2019330
Isolation of bacterium YJD-CS2
1. Adding 5g of soil sample (rhizosphere soil of plant Artemisia salina in West beach saline-alkali soil in suburb of Yinchuan city of Hui nationality of Ningxia, China) into 45mL of sterile distilled water, stirring for 15min, standing for 10min, collecting 1mL of supernatant, adding into a sterile test tube containing 9mL of sterile water, and mixing completely (the dilution is 10)-1) Then 1mL of the mixture is sucked from the test tube and added into another sterile test tube containing 9mL of sterile water to be uniformly mixed, and the like is repeated to prepare 10-2、10-3、10-4、10-5、10-6、10-7Bacterial suspensions of various dilutions. 0.1mL of each dilution was uniformly applied to LB solid medium and incubated at 30 ℃ for 2-3 days.
2. After completing the step 1, picking a single colony on an LB solid culture medium, and repeatedly purifying for more than 3 times. One of the bacteria selected was designated as bacterium YJD-CS 2.
II, identification of bacterium YJD-CS2
1. Morphological identification
(1) The bacterium YJD-CS2 was inoculated onto LB solid medium, and the morphology of a single colony was observed after 3 days.
The result shows that bacterial colonies of the bacteria YJD-CS2 are circular, the diameter is 2.0-4.0 mm, the edges are neat, the surface is smooth and wet, the color is light yellow, and the bacterial colonies are opaque.
(2) The bacterium YJD-CS2 was stained and identified as a gram-negative bacterium.
(3) And observing the morphology of the bacteria YJD-CS2 by using a high-resolution transmission electron microscope.
The results are shown in FIG. 1 (10000 times for the left image and 20000 times for the right image). The size of the bacterium YJD-CS2 was 1.07. mu. m.times. 3.084. mu.m, short rod shape.
2. 16S rDNA sequence homology analysis
The 16S rDNA of the bacterium YJD-CS2 is shown as sequence 1 in the sequence table.
The double-stranded DNA molecule shown in the sequence 1 in the sequence table is compared with the sequence in GenBank by utilizing Clustal X software. The results showed that the homology of the bacterium YJD-CS2 with Shewanella strain FDAARGOS _354 was the highest, reaching 99.38%.
III, preservation
According to the above morphology and the results of 16S rDNA sequence homology analysis, the bacterium YJD-CS2 separated and purified in the step is identified as a Shewanella bacterium. The bacterium YJD-CS2 has been preserved in China center for type culture Collection (CCTCC for short, with the address: Wuhan university, China) in 2019, 5 months and 7 days, with the preservation number of CCTCC NO: and M2019330. The bacterium YJD-CS2 is designated generally as Shewanella (Shewanella sp.) YJD-CS2CCTCC NO: m2019330, abbreviated as Shewanella (Shewanella sp.) YJD-CS2 or Shewanella YJD-CS 2.
And (3) strain preservation: inoculating a single colony of Shewanella YJD-CS2 to an LB liquid culture medium, and culturing at 30 ℃ for 16h to obtain a culture solution. 1 part by volume of the culture broth and 1 part by volume of a 50% (v/v) aqueous solution of glycerol were mixed and stored at-80 ℃.
Example 2 preparation of YJD-CS2 bacterial preparation
1. The strain stored at-80 ℃ in example 1 was activated on LB solid medium.
2. Selecting LB solid medium single colony, inoculating into conical flask (500 mL) containing 100mL LB liquid medium, culturing at 30 deg.C and 130r/min for 48 hr to obtain OD600nmA bacterial solution with a value of about 1.0. The bacterial liquid is the prepared YJD-CS2 bacterial agent.
Example 3 application of Shewanella YJD-CS2 in improving saline-alkali resistance of Arabidopsis thaliana
The specifications of the culture dish are 10cm multiplied by 10 cm.
First, preparation of culture medium
MS solid medium 1: dissolving 2.37g MS basic culture medium powder and 13g agar in distilled water, then adding distilled water to a constant volume of 1L, adjusting pH to 5.8, sterilizing at 121 deg.C for 20min, and cooling for use.
MS solid medium 2: dissolving 2.37g MS basic culture medium powder and 13g agar in distilled water, then adding distilled water to a constant volume of 1L, adjusting pH to 8.5, sterilizing at 121 deg.C for 20min, and cooling for use.
Salt solid medium: dissolving 2.37g of MS basic culture medium powder, 13g of agar and 5.844g of NaCl in distilled water, then using the distilled water to fix the volume to 1L, and adjusting the pH value to 8.0; the concentration of NaCl in the system is 100 mM; then, the mixture was sterilized at 121 ℃ for 20min, and the medium at about 55 ℃ was poured into petri dishes (25 mL each), and allowed to cool.
Alkali solid medium: 2.37g MS minimal medium powder, 13g agar and 0.168g NaHCO3Dissolving in distilled water, then using distilled water to fix the volume to 1L, and adjusting the pH value to 8.0; NaHCO 23The concentration in the system was 2 mM; then, the mixture was sterilized at 121 ℃ for 20min, and the medium at about 55 ℃ was poured into petri dishes (25 mL each), and allowed to cool.
Saline-alkali solid culture medium: 2.37g MS minimal medium powder, 13g agar, 5.844g NaCl and 0.168g NaHCO3Dissolving in distilled water, then using distilled water to fix the volume to 1L, and adjusting the pH value to 8.0; NaHCO 23The concentration in the system was 2mM, and the concentration of NaCl in the system was 100 mM; then, the mixture was sterilized at 121 ℃ for 20min, and the medium at about 55 ℃ was poured into petri dishes (25 mL each), and allowed to cool.
MS1+ microbial inoculum solid medium: a culture dish containing the MS solid culture medium 1 is taken, 0.1mL of YJD-CS2 microbial inoculum is coated on the surface of the culture medium below one fourth of the culture dish, and the culture dish is naturally air-dried.
MS2+ microbial inoculum solid medium: a culture dish containing the MS solid culture medium 2 is taken, 0.1mL of YJD-CS2 microbial inoculum is coated on the surface of the culture medium below one fourth of the culture dish, and the culture dish is naturally air-dried.
Salt + microbial inoculum solid medium: taking a culture dish filled with a salt solid culture medium, coating 0.1mL of YJD-CS2 microbial inoculum on the surface of the culture medium below one fourth of the culture dish, and naturally drying.
Alkali + microbial inoculum solid medium: taking a culture dish filled with an alkali solid culture medium, coating 0.1mL of YJD-CS2 microbial inoculum on the surface of the culture medium below one fourth of the culture dish, and naturally drying.
Saline-alkali + microbial inoculum solid culture medium: taking a culture dish filled with a saline-alkali solid culture medium, coating 0.1mL of YJD-CS2 microbial inoculum on the surface of the culture medium below one fourth of the culture dish, and naturally drying.
Second, the influence of YJD-CS2 microbial inoculum on saline-alkali stress resistance of arabidopsis thaliana
The culture conditions were: 22 ℃; 12h light/12 h dark; the light intensity was 12000 Lx.
1. Treating Arabidopsis seeds with 70% (v/v) ethanol aqueous solution for 5min, sterilizing with 2.6% (v/v) sodium hypochlorite aqueous solution for 10min, and washing with sterilized Tween water for 5 times.
2. After the step 1 is completed, sowing arabidopsis thaliana seeds in a solid culture medium (salt solid culture medium, alkali solid culture medium, saline-alkali solid culture medium, salt + microbial inoculum solid culture medium, alkali + microbial inoculum solid culture medium, saline-alkali + microbial inoculum solid culture medium, MS solid culture medium 1, MS solid culture medium 2, MS1+ microbial inoculum solid culture medium or MS2+ microbial inoculum solid culture medium), and vernalizing at 4 ℃ for three days. It should be noted that the seeds are not in direct contact with the microbial inoculum during sowing, namely, the seeds are sown in more than one fourth of the culture dish filled with the solid culture medium, and after subsequent vertical culture, the root of the arabidopsis thaliana does not grow to the culture medium coated with the microbial inoculum (the experiment researches the influence of the secretion of the microbial inoculum on the root growth of the arabidopsis thaliana).
3. The solid medium which completed step 2 was vertically cultured for 6 days, and the growth state of Arabidopsis thaliana was observed.
The growth state of arabidopsis thaliana is shown in fig. 2(1 is an MS solid culture medium 1, 2 is an MS1+ microbial inoculum solid culture medium, 3 is an MS solid culture medium 2, 4 is an MS2+ microbial inoculum solid culture medium, 5 is an alkali solid culture medium, 6 is an alkali + microbial inoculum solid culture medium, 7 is a salt solid culture medium, 8 is a salt + microbial inoculum solid culture medium, 9 is a saline-alkali solid culture medium, and 10 is a saline-alkali + microbial inoculum solid culture medium).
4. And (4) counting the main root length of the arabidopsis seedlings in the step 3 (the average value is obtained by repeating the experiment for three times, and 80 arabidopsis seeds are used each time).
The statistical results are shown in table 1 and fig. 3(1 is MS solid medium 1, 2 is MS1+ microbial inoculum solid medium, 3 is MS solid medium 2, 4 is MS2+ microbial inoculum solid medium, 5 is alkali solid medium, 6 is alkali + microbial inoculum solid medium, 7 is salt solid medium, 8 is salt + microbial inoculum solid medium, 9 is saline-alkali solid medium, 10 is saline-alkali + microbial inoculum solid medium, blank is medium, S25-12 is medium containing YJD-CS2 microbial inoculum).
TABLE 1
| Type of solid Medium | Average major root length (cm) |
| MS |
28.54 |
| MS1+ microbial inoculum solid culture medium | 34.67 |
| MS |
8.70 |
| MS2+ microbial inoculum solidCulture medium | 31.96 |
| Alkali solid culture medium | 1.18 |
| Solid culture medium of alkali and microbial inoculum | 25.32 |
| Salt solid culture medium | 7.87 |
| Solid culture medium of salt and microbial inoculum | 12.64 |
| Saline-alkali solid culture medium | 1.01 |
| Saline-alkali + microbial inoculum solid culture medium | 6.74 |
The results show that under the condition of salt-free alkali stress, the average main root length of the arabidopsis seedlings on the MS solid culture medium 1 and the MS1+ microbial inoculum solid culture medium is obviously different, the average main root length of the arabidopsis seedlings on the MS solid culture medium 2 and the MS2+ microbial inoculum solid culture medium is obviously different, and the arabidopsis seedlings coated with the YJD-CS2 microbial inoculum grow better; under the condition of saline-alkali stress, the average main root length of an arabidopsis seedling on a solid culture medium coated with the YJD-CS2 microbial inoculum is remarkably higher than that of a corresponding solid culture medium not coated with the YJD-CS2 microbial inoculum. Therefore, the Shewanella YJD-CS2 can improve the saline-alkali resistance of Arabidopsis.
Example 4 application of Shewanella YJD-CS2 in improving alkali stress resistance of corn
Agar solid medium: 5g of agar powder was dissolved in 500mL of distilled water and sterilized at 121 ℃ for 20 min. Pouring the agar culture medium with the temperature of about 55 ℃ into a culture dish, and naturally cooling.
Alkali liquor: 1 part by volume of 500 Xcaustic soda liquor and 499 parts by volume of water are mixed. 500 Xthe mother liquor of alkali: 10.599g of Na2CO3And 8.4g NaHCO3Dissolved in 500mL of deionized water, and the pH value is adjusted to 10.0 for later use.
The culture conditions are as follows: 28 ℃; 12h light/12 h dark; the light intensity was 12000 Lx.
1. Taking 80 corn inbred line B73 seeds, sterilizing for 5min by using 70% (v/v) ethanol water solution, and washing for 1 time by using sterilized water; then sterilizing with 2.6% (v/v) sodium hypochlorite aqueous solution for 15min, and washing with sterilized water for 5 times.
2. Sowing corn seeds in an agar solid culture medium, and vertically culturing for 7 days to obtain seedlings.
3. Transplanting the seedlings into pots filled with nutrient soil (2 seedlings per pot) to obtain 80 seedlings of 40 pots of corn.
4. 20 pots of corn seedlings are watered with 200mL of alkali liquor every week. And (3) taking another 20 pots of corn seedlings, and irrigating the corn seedlings with deionized water once a week, wherein 200mL of the deionized water is irrigated each time. The mixture was irrigated for 5 weeks.
5. And randomly dividing 20 pots of corn seedlings poured with alkali liquor into an alkali liquor control group and an alkali liquor fungicide group, wherein each group comprises 10 pots. The 20 pots of corn seedlings watered with deionized water are randomly divided into two groups, namely a water control group and a water microbial inoculum group, and each group comprises 10 pots. The experiments were as follows:
dripping 200 μ L YJD-CS2 microbial inoculum to the root of each corn seedling every 3 days for the alkaline microbial inoculum group, and culturing for 35 days; and (4) culturing the alkali liquor control group for 35 days under the same condition without applying a microbial inoculum.
Dripping 200 mu L of YJD-CS2 microbial inoculum to the root of each corn seedling every 3 days for the water microbial inoculum group, and culturing for 42 days; the water control group was cultured under the same conditions for 35 days without applying a microbial inoculum.
6. And observing and analyzing the phenotype of the growth and development of each group of corn, and recording the root system morphology of each group of corn seedlings by using a WiRHIO root system scanning analyzer.
The phenotypes of the overground part and the whole seedling of the maize seedling are shown in figure 4(1 is the overground part of a water treatment group, the left side is a water control group, and the right side is a water fungicide group, 2 is the overground part of an alkali liquor treatment group, the left side is an alkali liquor control group and the right side is an alkali liquor fungicide group, 3 is the whole seedling of the water treatment group, the left side is a water control group, and the right side is an alkali liquor control group, and 4 is the whole seedling of the alkali liquor treatment group, and the left side is an alkali liquor control group and the right side is.
The shape of corn root is shown in figure 5(1 is water control group, 2 is water bacterial agent group, 3 is alkali solution control group, and 4 is alkali solution bacterial agent group).
7. And (3) carrying out statistics on the total root length, root surface area, fresh weight and dry weight of each corn seedling by treating the corn seedlings with YJD-CS2 microbial inoculum for 35 days under the alkali stress condition, and averaging.
The results are shown in Table 2 and FIG. 6. Under the condition of water treatment, the dry weight, the fresh weight, the total root length and the root surface area of the maize seedlings applied with the YJD-CS2 microbial inoculum are all obviously higher than those of the maize seedlings not applied with the YJD-CS2 microbial inoculum; under the condition of alkali stress, the dry weight, the fresh weight, the total root length and the root surface area of the maize seedlings applied with the YJD-CS2 microbial inoculum are all obviously higher than those of the maize seedlings not applied with the YJD-CS2 microbial inoculum. Therefore, the Shewanella YJD-CS2 can improve the alkali stress resistance of the corn.
TABLE 2
Example 5 application of Shewanella YJD-CS2 to improvement of tomato alkali stress resistance
The culture conditions are as follows: 28 ℃; 12h light/12 h dark; the light intensity was 5000 Lx.
Alkali liquor: 1 part by volume of 500 Xcaustic soda liquor and 499 parts by volume of water are mixed. 500 Xthe mother liquor of alkali: 10.599g of Na2CO3And 8.4g NaHCO3Dissolved in 500mL of deionized water, and the pH value is adjusted to 10.0 for later use.
1. Placing 80 seeds of tomato variety M82 into a culture dish, adding a small amount of water, and placing the culture dish in a constant-temperature incubator for culturing until the seeds germinate and have short roots.
2. Transplanting the germinated tomato seedlings into pots (2 in each pot) filled with nutrient soil to obtain 80 tomato seedlings in 40 pots.
3. And (3) taking 20 pots of tomato seedlings, and irrigating 200mL of alkali liquor once every week. And pouring deionized water once every week for another 20 pots of tomato seedlings, wherein the amount of the deionized water is 200mL every time. The mixture was irrigated for 6 weeks.
4. Randomly dividing 20 pots of tomato seedlings poured with alkali liquor into two groups, namely an alkali liquor control group and an alkali liquor fungicide group, and 10 pots of tomato seedlings are poured into each group. Randomly dividing 20 pots of tomato seedlings watered with deionized water into two groups, namely a water control group and a water microbial inoculum group, and 10 pots of tomato seedlings are respectively arranged in each group. The experiments were as follows:
dripping 60 mu L of YJD-CS2 microbial inoculum to the root of each tomato seedling every 3 days for the alkaline microbial inoculum group, and culturing for 42 days; the control group of alkali liquor was cultured for 42 days under the same conditions without applying any microbial inoculum.
Dripping 60 mu L of YJD-CS2 microbial inoculum to the root of each tomato seedling every 3 days for the aquatic microbial inoculum group, and culturing for 42 days; the water control group was cultured for 42 days under the same conditions without applying the microbial inoculum.
5. And observing and analyzing the phenotype of the growth and development of each group of tomatoes, and recording the root system morphology of each group of tomato seedlings by using a WiRHIO root system scanning analyzer.
The phenotypes of the overground part and the whole seedling of the tomato seedling are shown in figure 7(1 is the overground part of a water treatment group, the left side is a water control group, and the right side is a water fungicide group, 2 is the overground part of an alkali liquor treatment group, the left side is an alkali liquor control group and the right side is an alkali liquor fungicide group, 3 is the whole seedling of the water treatment group, the left side is a water control group, and the right side is an alkali liquor control group, and 4 is the whole seedling of the alkali liquor treatment group, and the left side is an alkali liquor control group and the right side is.
The tomato root morphology is shown in FIG. 8(1 is water control group, 2 is water bacterial agent group, 3 is lye control group, and 4 is lye bacterial agent group).
7. And (3) carrying out statistics on the total root length, the root surface area, the fresh weight and the dry weight of each tomato seedling after being treated by YJD-CS2 microbial inoculum for 42 days under the alkali stress condition, and taking an average value.
The results are shown in Table 3 and FIG. 9. Under the water treatment condition, the dry weight, the fresh weight, the total root length and the root surface area of the tomato seedlings applied with the YJD-CS2 microbial inoculum are all obviously higher than those of the tomato seedlings not applied with the YJD-CS2 microbial inoculum; under the alkali stress condition, the dry weight, the fresh weight, the total root length and the root surface area of the tomato seedlings applied with the YJD-CS2 microbial inoculum are all obviously higher than those of the tomato seedlings not applied with the YJD-CS2 microbial inoculum. Therefore, the Shewanella YJD-CS2 can improve the saline-alkali stress resistance of tomatoes.
TABLE 3
<110> northeast university of forestry
Shewanella and application thereof in improving stress resistance of plants
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1459
<212> DNA
<213> Shewanella sp.
<400> 1
ggggctgttg ggggcaggct aacacatgca agtcgagcgg cagcacaagg gagtttactc 60
ctgaggtggc gagcggcgga cgggtgagta atgcctaggg atctgcccag tcgaggggga 120
taacagttgg aaacgactgc taataccgca tacgccctac gggggaaaga aggggacctt 180
cgggccttcc gcgattggat gaacctaggt gggattagct agttggtgag gtaatggctc 240
accaaggcga cgatccctag ctgttctgag aggatgatca gccacactgg gactgagaca 300
cggcccagac tcctacggga ggcagcagtg gggaatattg cacaatgggg gaaaccctga 360
tgcagccatg ccgcgtgtgt gaagaaggcc ttcgggttgt aaagcacttt cagtagggag 420
gaaagggtga gtcttaatac ggctcatctg tgacgttacc tacagaagaa ggaccggcta 480
actccgtgcc agcagccgcg gtaatacgga gggtccgagc gttaatcgga attactgggc 540
gtaaagcgtg cgcaggcggt ttgttaagcg agatgtgaaa gccctgggct caacctagga 600
atagcatttc gaactggcga actagagtct tgtagagggg ggtagaattc caggtgtagc 660
ggtgaaatgc gtagagatct ggaggaatac cggtggcgaa ggcggccccc tggacaaaga 720
ctgacgctca tgcacgaaag cgtggggagc aaacaggatt agataccctg gtagtccacg 780
ccgtaaacga tgtctactcg gagtttggtg tcttgaacac tgggctctca agctaacgca 840
ttaagtagac cgcctgggga gtacggccgc aaggttaaaa ctcaaatgaa ttgacggggg 900
cccgcacaag cggtggagca tgtggtttaa ttcgatgcaa cgcgaagaac cttacctact 960
cttgacatcc acagaagagt gcagagatgc ggttgtgcct tcgggaaccg tgagacaggt 1020
gctgcatggc tgtcgtcagc tcgtgttgtg aaatgttggg ttaagtcccg caacgagcgc 1080
aacccctatc cttatttgcc agcacgtaat ggtgggaact ctagggagac tgccggtgat 1140
aaaccggagg aaggtgggga cgacgtcaag tcatcatggc ccttacgagt agggctacac 1200
acgtgctaca atggcgagta cagagggttg caaagccgcg aggtggagct aatctcacaa 1260
agctcgtcgt agtccggatt ggagtctgca actcgactcc atgaagtcgg aatcgctagt 1320
aatcgtggat cagaatgcca cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1380
caccatggga gtgggctgca aaagaagtgg gtagcttaac cttcgggggg gcgctcacca 1440
actttggttt cattggtgc 1459
Claims (10)
1. Shewanella sp (Shewanella sp.) YJD-CS2, which has a collection number of CCTCC NO: and M2019330.
2. A microbial inoculum, which is characterized in that: the microbial agent contains the Shewanella sp YJD-CS2CCTCC NO: and M2019330.
3. The Shewanella sp (Shewanella sp.) YJD-CS2CCTCC NO: the application of the microbial inoculum of M2019330 or claim 2 in regulating and controlling plant stress resistance.
4. Use according to claim 3, characterized in that: the regulation and control of the plant stress resistance is to improve the saline-alkali resistance of the plant.
5. The Shewanella sp (Shewanella sp.) YJD-CS2CCTCC NO: the application of the M2019330 or the microbial inoculum as claimed in claim 2 in preparing products for improving the stress resistance of plants.
6. A method for improving the stress resistance of plants adopts Shewanella to treat the plants so as to improve the stress resistance of the plants.
7. The method of claim 6, wherein: the Shewanella is the Shewanella sp YJD-CS2CCTCC NO: and M2019330.
8. The method of claim 6 or 7, wherein: the "treatment of a plant with Shewanella" is a treatment of a plant with the microbial agent according to claim 2.
9. The use according to any one of claims 3 to 5 or the method according to any one of claims 6 to 8, wherein: the stress resistance is three, any two or any one of salt resistance, alkali resistance and salt and alkali resistance.
10. The use according to any one of claims 3, 4, 5 or 9 or the method according to any one of claims 6 to 9, wherein: the plant is any one of the following c1) to c 11): c1) a dicotyledonous plant; c2) a monocot plant; c3) a gramineous plant; c4) a plant of the Solanaceae family; c5) a cruciferous plant; c6) corn; c7) maize inbred line B73; c8) tomatoes; c9) tomato variety M82; c10) arabidopsis thaliana; c11) the wild type Arabidopsis thaliana Columbia-0 subtype.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173383A (en) * | 2012-12-26 | 2013-06-26 | 清华大学深圳研究生院 | Shewanella piezotolerans 34# and application thereof to algae inhibition |
| CN104962494A (en) * | 2015-06-25 | 2015-10-07 | 东北农业大学 | Shewanella having good degradation effect on herbicide atrazine |
| JP2017176128A (en) * | 2016-03-31 | 2017-10-05 | 株式会社前川製作所 | Improvement in salt resistance and/or osmotic pressure resistance of plant using comamonas bacteria |
| US20190071335A1 (en) * | 2016-04-14 | 2019-03-07 | Shandong University | Highly efficient aerobic phosphorus-removing bacteria capable of synthesizing nanoparticles by microbial self-assembly using waste water |
| CN109593686A (en) * | 2019-01-18 | 2019-04-09 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | A kind of Shewanella and its application in control vegetable field soil nitrogen pollution |
-
2019
- 2019-05-15 CN CN201910401921.1A patent/CN111944710B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173383A (en) * | 2012-12-26 | 2013-06-26 | 清华大学深圳研究生院 | Shewanella piezotolerans 34# and application thereof to algae inhibition |
| CN104962494A (en) * | 2015-06-25 | 2015-10-07 | 东北农业大学 | Shewanella having good degradation effect on herbicide atrazine |
| JP2017176128A (en) * | 2016-03-31 | 2017-10-05 | 株式会社前川製作所 | Improvement in salt resistance and/or osmotic pressure resistance of plant using comamonas bacteria |
| US20190071335A1 (en) * | 2016-04-14 | 2019-03-07 | Shandong University | Highly efficient aerobic phosphorus-removing bacteria capable of synthesizing nanoparticles by microbial self-assembly using waste water |
| CN109593686A (en) * | 2019-01-18 | 2019-04-09 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | A kind of Shewanella and its application in control vegetable field soil nitrogen pollution |
Non-Patent Citations (2)
| Title |
|---|
| 石伟等: "一株耐碳酸盐Bacillus属细菌的鉴定与特性分析", 《基因组学与应用生物学》 * |
| 许玫英等: "一个降解染料的希瓦氏菌新种――中国希瓦氏菌", 《微生物学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114009307A (en) * | 2021-11-04 | 2022-02-08 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | Functional cucumber seedling-raising biological matrix containing Shewanella and preparation method thereof |
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