CN115838639A

CN115838639A - Imperata cylindrical seed endophytic fungus DF101 and application thereof

Info

Publication number: CN115838639A
Application number: CN202211626667.3A
Authority: CN
Inventors: 李海燕; 韩雪; 汤雯婷; 毛文沁; 高馨竹; 龚伟军
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2022-12-17
Filing date: 2022-12-17
Publication date: 2023-03-24
Anticipated expiration: 2042-12-17
Also published as: CN115838639B

Abstract

The invention discloses a strain of cogongrass seed endophytic fungus (B)Septoriella phragmitis) DF101, the preservation number of which in China general microbiological culture Collection center is CGMCC No.40427; the strain has the capabilities of dissolving phosphorus, producing IAA, producing siderophore and resisting cadmium, has good capabilities of promoting seed germination, improving the survival rate of seedlings and enhancing cadmium resistance under the stress of cadmium, and has good application prospect in the plant-microorganism combined remediation of heavy metal contaminated soil.

Description

Imperata cylindrical seed endophytic fungus DF101 and application thereof

Technical Field

Hair brushBelongs to the technical field of microorganisms, and particularly relates to a seed endophytic fungusSeptoriella phragmitis) DF101 and its use in the bioremediation of heavy metal contamination.

Background

Due to human activities such as mining and smelting, large-scale use of chemical fertilizers and pesticides, farmland pollution irrigation, disposal of solid wastes and the like, the heavy metal pollution of soil is getting more and more serious. The total exceeding rate of soil in China is 16.1%, wherein the number of exceeding standard points of 8 heavy metals such as cadmium, mercury, arsenic and the like accounts for 82.8% of all exceeding standard points. Among the various heavy metal contaminants, cadmium (Cd) is identified as one of the most important contaminants due to its strong biotoxicity and high transfer risk. Excessive heavy metals in soil not only inhibit crop growth, but also produce toxicity amplification through the food chain, seriously threatening human health. Therefore, remediation of heavy metal contamination in soil is imminent.

In all the remediation methods, the phytoremediation shows a good application prospect in remediation of heavy metal contaminated soil due to in-situ performance, environmental protection, low cost and simple operation. However, in practical application, the plant repair still has the problems of low repair efficiency, time consumption and the like, and the plant-microorganism combined repair is expected to solve the problems. Because many microorganisms can promote the growth of plants in heavy metal polluted environments by producing auxin, improving the nutrient absorption of plants, changing the bioavailability of heavy metals in soil and the like, the biomass of the plants is increased, and the remediation efficiency is improved.

The seeds are reproductive organs of plants, and a certain number of endophytes are colonized in the seeds. Researches show that seeds of many crops, including rice, wheat, cotton, corn and the like, contain endophytes which can not only promote the growth and development of host plants but also protect the host plants from being damaged by pathogens. Since part of the seed endophytes are microorganisms that spread vertically through the seed and become the earliest colonization in the neonatal plant, the seed endophytes have more important ecological significance for the host plant. For example, mastretta et al demonstrated that tobacco seeds inoculated with cadmium-resistant endophytes that reduced cadmium can grow under cadmium stressHeavy metal toxicity to tobacco; johnston-Monje and the like find that endophytes derived from corn seeds can stimulate host plants through functions of nitrogen fixation, siderophore secretion and the like, so that the endophytes can be quickly adapted to severe environments including heavy metal stress; truyens et al showed that Arabidopsis thaliana (under cadmium stress)Arabidopsis thaliana) The continuous seed endophytes have more obvious heavy metal resistance than the seed endophytes without cadmium stress. The researches show that some endophytes can be vertically spread to the next generation and endow the next generation with heavy metal resistance, and the method has important significance for adapting to the heavy metal stress environment of plants. The seed endophyte has a great application prospect in the aspect of improving the efficiency of the plant-endophyte combined remediation of heavy metal soil pollution.

Disclosure of Invention

The invention provides a plant seed endophytic fungus DF101 which is classified and namedSeptoriella phragmitis，Which is isolated from Imperata cylindrica (B)Imperata cylindrica L.) seeds, which are preserved in China general microbiological culture Collection center (CGMCC) in 2022, 11 months and 14 days, with the preservation number of CGMCC No.40427, the preservation address: the institute of microbiology, national academy of sciences, no. 3, west Lu No. 1, beijing, chaoyang, beicheng, area, beichen.

The invention also aims to provide a new application of the endophytic fungus DF101 from the plant seeds, namely the endophytic fungus DF101 from the cogongrass seeds is applied to the bioremediation of cadmium pollution, has the capabilities of producing IAA and siderophores, has higher phosphate solubilizing capability and stronger cadmium tolerance, and can promote the germination of the seeds, improve the survival rate of seedlings and enhance the cadmium resistance of the seedlings under the cadmium stress.

In order to realize the purpose, the invention adopts the following technical scheme:

1. baimao collected from Puxiong province in Jianshui city of Yunnan province: (I. cylindrica) The plant sample is placed in the sun for airing and air drying until the seeds can naturally fall off, and then the seed sample is placed in a refrigerator at 4 ℃ for storage for later use;

2. randomly selecting 600 seeds from the plant sample, and performing surface disinfection according to the following procedures: ethanol with a volume concentration of 75% is surface-sterilized for 2 times, each time for 1min, then rinsed 5 times with sterile water, and placed on sterile filter paper to suck up water. Then sticking the seeds with the sterilized surfaces to a PDA flat plate, culturing at 25 ℃, observing every other day, and selecting and purifying if fungi grow out around the tissue blocks during the culture period; meanwhile, whether the seed surface is disinfected completely is checked by a rinsing liquid checking method;

3. preparation of a solution containing different Cd ²⁺ Subpackaging PDB culture medium with concentration into conical flasks, selecting mycelia with uniform size from the separated endophytic strains, repeating the step three times for each concentration, placing the mycelia in a constant-temperature horizontal shaker at 28 ℃ for shake culture at 135rpm for 6d, and observing growth vigor of colonies under different concentrations; selected pair Cd ²⁺ The strain with stronger resistance is preserved on the inclined plane of PDA, and the fungus is named as DF101;

4. identification of Strain DF101

(1) DF101 morphological features: the edges of the colonies are regular and white at the initial culture stage, dense gray hyphae grow on the colonies, the colonies are flocculent, nearly circular and opaque, and the colonies begin to turn yellow brown after 6 days;

(2) And (3) molecular identification: extracting total DNA of the strain by using a MoBio PowerSoil DNA kit, detecting the total DNA, sending the total DNA to a sequencing company for sequence determination, and comparing a sequencing result with a sequence on NCBI; combining the morphological characteristics and the molecular identification result, and finally identifying the strainSeptoriella phragmitis(ii) a The culture medium used for storing and activating the strain is a PDA culture medium;

5. the invention separates seed endophytic fungi from cogongrassS. phragmitisDF101 is subjected to seed germination experiments to investigate the influence on seed germination, namely, endophytic fungi of seeds is carried outS. phragmitisThe research on the influence of DF101 inoculation on the germination of seeds under the stress of heavy metal provides a fungus strain and a theoretical research basis for the combined restoration of plants and microorganisms.

Compared with the prior art, the invention has the following beneficial effects:

the seed endophytic fungi provided by the inventionS. phragmitisDF101 is derived from the seeds of Imperata cylindrical, has the ability of dissolving phosphorus, producing IAA and siderophore, has better cadmium-resistant ability, and can be obtained in large amount by simple liquid fermentationThe mycelium is easy to obtain, low in cost and has potential for commercial application; the method can promote the germination of seeds under the stress of cadmium, can obviously improve the germination rate of the seeds in a heavy metal environment, improves the viability of the seeds, enhances the cadmium resistance of the seeds, and has important significance for repairing the heavy metal polluted soil.

Drawings

FIG. 1 shows the colony morphology of endophytic fungus DF101 from seeds of Imperata cylindrical on PDA medium (panel A: front side, B: back side);

FIG. 2 is a standard working curve of a phosphorus standard solution;

FIG. 3 is a standard working curve of an IAA standard solution;

FIG. 4 is a drawing showingS. phragmitisThe result of the determination of the phosphate-dissolving and IAA-producing capacity of DF101;

FIG. 5 shows that under the stress of cadmium with different concentrations,S. phragmitisseed germination rates of chenopodium ambrosioides under DF101 inoculation (E +) and non-inoculation (CK).

Detailed Description

The technical solutions of the present invention will be described in further detail below with reference to specific embodiments and drawings, but the present invention is not limited to the following technical solutions. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The method can be implemented by those skilled in the art with reference to various common tool books, scientific and technical documents or relevant specifications, manuals and the like before the application date of the invention;

the following examples were prepared with the following Chromium Azure S (CAS) test solutions:

solution A: 60.5mg of chromium azure is weighed and dissolved in 50mL of deionized water, 10mL of Fe is added ³⁺ Stirring and mixing the solution (containing 1mM ferric chloride hexahydrate and 10mM dilute hydrochloric acid);

solution B: 72.9mg of hexadecyl trimethyl ammonium bromide was dissolved in 40mL of deionized water;

and slowly pouring the solution A into the solution B, and uniformly stirring to obtain the CAS blue detection solution.

Example 1: seed endophytic fungiS. phragmitisIsolation, screening and characterization of DF101

(1) Collecting a cogongrass sample of the general male county of Water City of Yunnan province, placing the plant sample in the sun for airing and air-drying until the seeds can naturally fall off, and then placing the seed sample in a refrigerator at 4 ℃ for storage for later use;

(2) And (3) separation of endophytes: randomly selecting 600 seeds from the seed sample, surface-sterilizing with 75 vol% ethanol for 2 times (each time for 1 min), washing with sterile water for 5 times, and placing on sterile filter paper for water absorption. Then sticking the seeds with the sterilized surfaces to a PDA flat plate (90 mm), culturing 6 seeds in each dish at 25 ℃ for 60d, observing every other day, and selecting and purifying if fungi grow out around tissue blocks during the culture; meanwhile, whether the seed surface is disinfected completely is checked by a rinsing liquid checking method;

(3) Screening of cadmium resistant strains: preparation of Cd ²⁺ 0. 100, 200 and 400mg/L PDB culture medium is subpackaged into 100mL conical flasks, the subpackaged volume is 50mL, mycelia with uniform size are selected from separated endophytic strains to be placed in the conical flasks, each concentration is repeated for three times, the mycelia are placed on a constant-temperature horizontal shaking table at 28 ℃ and subjected to shaking culture at 135rpm for 6 days, and the growth vigor of the colonies under different concentrations is observed; selection pair Cd ²⁺ The strain with stronger resistance is stored on a PDA inclined plane and named as DF101;

(4) Identification of Strain DF101

(1) DF101 morphological features: the colony edge is regular and white at the initial culture stage, dense gray hypha grows in the colony, the colony is cotton-like, nearly circular and opaque, and the colony begins to turn yellow brown after 6 days (figure 1A: front side, B: back side);

(2) and (3) molecular identification: extracting total DNA of the strain by using a MoBio PowerSoil DNA kit, detecting the total DNA, sending the DNA to a sequencing company for sequence determination, comparing a sequencing result with a sequence on NCBI (national center for Biotechnology information for Biotechnology) as shown in SEQ ID NO 1; combining morphological characteristics and molecular identification results, andS. phragmitisthe homology reaches 98.93 percent, and the strain is finally identified as the strainS. phragmitis。

Example 2: seed endophytic fungiS. phragmitisDF101 cadmium tolerance test

Inoculating the separated endophytic strain to a PDA plate to prepare the product containing Cd ²⁺ PDB culture medium with concentration of 0, 50, 100, 200, 400 and 600mg/L is subpackaged into 100mL conical flasks, subpackaged volume is 50mL, bacterial blocks with uniform size are selected from strains with good growth vigor in the conical flasks, each concentration is repeated for three times, the conical flasks are placed on a constant-temperature horizontal shaking table at 28 ℃ and subjected to shaking culture at 135rpm for 6 days, growth vigor of the bacterial blocks is observed, and dry weight of mycelia is determined by a gravimetric method, wherein Cd is ²⁺ The culture solution with the concentration of 0mg/L is used as a control group, and the inhibition efficiency of the heavy metal on the strain can be calculated according to a formula;

growth inhibition rate = (control hypha dry mass-treated hypha dry mass)/control hypha dry mass × 100%

The results are shown in Table 1, strainsS. phragmitisDF101 growth in PDB Medium with higher cadmium concentrations indicatedS. phragmitisDF101 has better cadmium tolerance;

TABLE 1 Strain DF101 heavy Metal tolerance

Note: in the above table, "+" is well resistant; "+ -" is resistant but in poor growth conditions; "-" no resistance.

Example 3: seed endophytic fungiS. phragmitisDetermination of DF101 phosphate solubilizing ability

Phosphorus dissolving capacity: will be provided withS. phragmitisAfter DF101 is cultured in a PDB culture medium for 4 days at 28 ℃ and 125rpm in a shaking way, bacterial strains with good growth vigor are picked, bacterial blocks with uniform size are inoculated into an inorganic phosphorus liquid fermentation culture medium (containing 10g of glucose, 0.5g of ammonium sulfate, 0.3g of sodium chloride, 0.3g of potassium chloride, 0.3g of magnesium sulfate heptahydrate, 0.03g of ferrous sulfate heptahydrate, 1g of manganese sulfate tetrahydrate, 5g of tricalcium phosphate, 1L of distilled water and pH 7.0-7.5), simultaneously, PDB liquid culture medium with the same amount of inoculation is used as a blank control, each treatment is repeated three times, and the bacterial strains are cultured on the shaking way for 10 days at 28 ℃ and 125 rpm; the content of soluble phosphorus (the wavelength of a spectrophotometer is 700 nm) is measured by adopting a molybdenum-antimony colorimetric method, and the method comprises the following specific steps:

(1) centrifuging the fermentation liquor at 10000rpm for 15min, putting 1mL of the supernatant into a 50mL volumetric flask, adding 2 drops of 2, 6-dinitrophenol indicator, adjusting the pH value with 10% sodium hydroxide or 5% dilute sulfuric acid until the solution is just yellowish, adding 5mL of molybdenum-antimony anti-color-developing agent, and adding deionized water to the volume of 50mL;

(2) standing for 30min, performing color comparison with a spectrophotometer at 700nm, and simultaneously determining a blank control group;

(3) drawing a phosphorus standard curve in an experiment at the same time, respectively sucking 0, 2, 4, 6, 8 and 10mL of 5mg/L phosphorus standard solution into a 50mL volumetric flask, adding 2 drops of 2, 6-dinitrophenol indicator, adjusting the pH value with 10% sodium hydroxide or 5% dilute sulfuric acid until the solution is just yellowish, adding 5mL of molybdenum-antimony anti-color-developing agent, adding deionized water to the solution until the volume is 50mL to obtain 0, 0.2, 0.4, 0.6, 0.8 and 1.0mg/L phosphorus standard series solutions, standing for 30min, carrying out color comparison in a spectrophotometer at 700nm, drawing a standard working curve (figure 2), substituting the absorbance value measured in the step (2) into the standard working curve to obtain a standard working curveS. phragmitisThe phosphorus-dissolving amount of DF101 in the inorganic phosphorus culture solution is 43.026 mg/L (figure 4), which shows thatS. phragmitisDF101 has the ability to convert inorganic phosphorus into organic phosphorus, which makes plants more easily absorb phosphorus, thereby promoting plant growth.

Example 4: seed endophytic fungiS. phragmitisDetermination of DF101 IAA-producing ability

S. phragmitisCulturing DF101 in PDB culture medium (containing L-tryptophan 0.5 mg/mL) in dark at 28 ℃ and 125rpm on a shaking table for 10 days, centrifuging 1mL bacterial solution at 12000rpm for 15min, removing precipitate, adding equivalent Salkowski's reaction solution (1mL 0.5M ferric chloride, 49mL 35% perchloric acid) into 0.5mL supernatant, reacting in dark for 30min, measuring absorbance at 530nm, repeating each group for three times, and adjusting to zero by using the non-inoculated culture medium and the same-phase treatment as a control; IAA standard solutions at concentrations of 0, 2.5, 5, 10, 20, 40, 60, 80, 100 mg/L were used to determine absorbance values and to plot standard curves (FIG. 3) as described above, and then inoculatedS. phragmitisSubstituting the absorbance value of the experimental group into the standard curve to obtainS. phragmitisThe amount of IAA secreted by DF101 was 17.637 mg/L (FIG. 4),S. phragmitisDF101 uses L-tryptophan as precursor to synthesize plant hormone IAA, stimulate plant cell growth and proliferation, effectively absorb water and nutrients, and regulate plant growthAnd (5) taking a life and activity.

Example 5: seed endophytic fungiS. phragmitisDetermination of DF101 siderophore production capacity

Will be provided withS. phragmitisDF101 was shake-cultured in PDB medium at 28 ℃ and 125rpm for 4 days, the well-grown bacterial strain was inoculated into a non-iron Kirch's medium (containing 30g of sucrose, 2g of sodium nitrate, 1g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate heptahydrate, 0.5g of potassium chloride, and 1L of distilled water) to prepare a mass of a well-grown bacterial strain, the mass was shake-cultured at 28 ℃ and 125rpm for 5 days, the supernatant was separated at 8000rpm for 10min, 1mL of the supernatant was mixed with 1mL of CAS test solution, the mixture was allowed to react in the dark for 15min, the absorbance was measured at 630nm, the value was designated As, and the OD was measured in the same manner As in the blank non-inoculated control ₆₃₀ The value is marked As Ar, deionized water is used As a contrast for zero adjustment, and the specific calculation formula is the activity of the siderophore = [ (Ar-As)/Ar]×100%；

The calculation result shows that the chelating siderophore capacity of the strain DF101 is 74.52 percent, and siderophore is a kind of Fe chelate which is synthesized and secreted by bacteria and fungi in a low-iron environment and has strong specificity ³⁺ Researches show that some bacteria producing siderophores can enrich the iron element in the plant rhizosphere, thereby improving the absorption of plant iron nutrition and achieving the effect of promoting plant growth.

Example 6: seed endophytic fungi under heavy metal stressS. phragmitisEffect of DF101 on Chenopodium ambrosioides seed Germination

This example is intended to demonstrateS. phragmitisThe DF101 has the function of promoting the germination of seeds; herba Chenopodii (A)Dysphania ambrosioidesL.) test plants were obtained by the following experimental procedure:

A、S. phragmitispreparation of DF101 suspension: selecting a DF101 strain purified before, inoculating the strain into a PDA flat plate for activation, culturing in a water-proof constant-temperature incubator at 28 ℃ for 7 days, selecting a flat plate with good growth vigor and no pollution, selecting mycelia, inoculating the mycelia into a PDB culture medium, culturing in a constant-temperature shaking table at 28 ℃ and 125rpm for 4 days, filtering out the mycelia under aseptic conditions, washing with sterile water for 3 times to avoid the mycelia from being stained with the culture medium, shearing with sterile scissors, and transferring the sheared myceliaDiluting to constant volume of 150mL in sterile water to obtain DF101 mycelium suspension;

B. selecting full Chenopodium ambrosioides seeds with uniform size for surface disinfection, rinsing with 75% ethanol for 2 times (each time for 2min30 s), and rinsing with sterile water for 5 times. Soaking the seeds with the disinfected surfaces in DF101 mycelium suspension for 2h for colonization (E +), soaking the seeds in sterile water with the same volume as a control group (CK), and soaking the soaked seeds in Cd with different concentrations ²⁺ In the Petri dishes of the solution (Petri dishes spread with two layers of sterilized filter paper, cd in each Petri dish ²⁺ The solution was 5 mL); cd [ Cd ] ²⁺ The concentration of the solution is 0, 30, 50, 100 and 200mg/L, three repeated groups are arranged at each concentration, 25 seeds are placed in each dish, the culture is carried out under natural illumination, the germination number of the seeds is recorded from the beginning of the germination of the seeds until no germination exists, the germination number of the seeds is counted, and the germination rate (%) is calculated to be (Gt/T) multiplied by 100, wherein Gt is the number of the germinated seeds, and T is the number of the seeds.

The results are shown in FIG. 5, and the results are displayedS. phragmitisDF101 has obvious effect of promoting germination of chenopodium ambrosioides seeds under the stress of heavy metal, improves the survival rate of seedlings and enhances the cadmium resistance of chenopodium ambrosioides seeds.

The results of the above examples illustrate the seed endophytic fungi isolated in the present inventionS. phragmitisDF101 has the capability of dissolving phosphorus, producing IAA and siderophores, and better cadmium resistance, and has better promotion effect on the germination of chenopodium ambrosioides seeds under the stress of heavy metals.

Claims

1. An endophytic fungus (B) from the seeds of Imperata cylindricalSeptoriella phragmitis) DF101, the preservation number of which in China general microbiological culture Collection center is CGMCC No.40427.

2. The use of the endophytic fungus DF101 from the seeds of Imperata cylindrical as claimed in claim 1 for bioremediation of heavy metal pollution.

3. Use according to claim 1, characterized in that: the endophytic fungus DF101 of the imperata cylindrica seeds can promote the germination of the plant seeds under the stress of heavy metals.