CN115838639B - Endophytic fungi DF101 of cogongrass seed and application thereof - Google Patents

Abstract

The invention discloses a cogongrass seed endophytic fungusSeptoriella phragmitis) DF101 with the preservation number of CGMCC No.40427 in China general microbiological culture Collection center; the strain has the capabilities of dissolving phosphorus, producing IAA, producing siderophores and resisting cadmium, well promoting seed germination under cadmium stress, improving the survival rate of seedlings and enhancing the cadmium resistance, and has a good application prospect in the combined repair of heavy metal contaminated soil by plants and microorganisms.

Description

Imperata seed endophytic fungus DF101 and its application

Technical field

The invention belongs to the field of microbial technology, and specifically relates to a seed endophytic fungus ( Septoriella phragmitis ) DF101 and its application in bioremediation of heavy metal pollution.

Background technique

Due to human activities such as mining and smelting, extensive use of chemical fertilizers and pesticides, farmland sewage irrigation, and solid waste disposal, soil heavy metal pollution is becoming more and more serious. The total exceedance rate of soil in my country is 16.1%, among which the number of exceedance points for eight heavy metals such as cadmium, mercury, and arsenic accounts for 82.8% of all exceedance points. Among various heavy metal contaminants, cadmium (Cd) has been identified as one of the most important due to its strong biological toxicity and high transfer risk. Excessive heavy metals in the soil will not only inhibit crop growth, but also produce toxic amplification through the food chain, seriously threatening human health. Therefore, it is urgent to remediate heavy metal pollution in soil.

Among all remediation methods, phytoremediation shows good application prospects in the remediation of heavy metal contaminated soil because of its in-situ, environmental protection, low cost, and simple operation. However, in practical applications, phytoremediation still has problems such as low repair efficiency and time-consuming, and plant-microbial joint remediation is expected to solve these problems. Because many microorganisms can promote the growth of plants in heavy metal-polluted environments by producing auxins, improving plant nutrient absorption, changing the bioavailability of heavy metals in the soil, etc., thereby increasing their biomass and improving remediation efficiency.

Seeds are the reproductive organs of plants, and there are a certain number of endophytic bacteria colonizing the seeds. Research shows that the seeds of many crops, including rice, wheat, cotton, corn, etc., contain endophytes, which can not only promote the growth and development of host plants but also protect them from pathogens. Because some seed endophytes are vertically transmitted through seeds and become the earliest microorganisms to colonize new plants, 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 can grow under cadmium stress, and the seed endophytes reduce the heavy metal toxicity of cadmium to tobacco; Johnston-Monje et al. found that endophytes derived from corn seeds, It can stimulate the host plant through functions such as nitrogen fixation and secretion of siderophores, allowing it to quickly adapt to harsh environments including heavy metal stress; Truyens et al. have shown that under cadmium stress, Arabidopsis thaliana seed endophytes grow continuously over several generations. The seed endophytes had more significant heavy metal resistance than those without cadmium stress. These studies indicate that certain endophytes can be vertically transmitted to the next generation and confer heavy metal resistance, which is of great significance for plants to adapt to heavy metal stress environments. Seed endophytes show great application prospects in improving the efficiency of plant-endophyte joint remediation of heavy metal soil pollution.

Contents of the invention

The invention provides a plant seed endophytic fungus DF101, classified and named Septoriella phragmitis, which is isolated from the seeds of Imperata cylindrica L., and was deposited in the General Microorganisms of the China Microbial Culture Collection Committee on November 14, 2022. Center, the collection number is CGMCC No.40427, and the collection address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing.

Another object of the present invention is to provide a new use of the above-mentioned plant seed endophytic fungus DF101, that is, to apply it in the bioremediation of cadmium pollution. The whitegrass seed endophytic fungus DF101 of the present invention has the ability to produce IAA and siderophores, and has higher It has the ability to dissolve phosphorus and has strong cadmium tolerance. It can promote seed germination, improve seedling survival rate and enhance its cadmium resistance under cadmium stress.

In order to achieve the above objects, the present invention adopts the following technical solutions:

1. Place the I. cylindrica plant samples collected from Puxiong Township, Jianshui City, Yunnan Province in the sun to dry until the seeds can fall off naturally, and then store the seed samples in a 4°C refrigerator for later use;

2. Randomly select 600 seeds from the plant samples and conduct surface disinfection according to the following procedures: surface disinfection with ethanol with a volume concentration of 75% twice, each time for 1 minute, then rinse with sterile water 5 times, and place in a sterile place. Blot dry on filter paper. Then, stick the surface-sterilized seeds onto a PDA plate, culture them at 25°C, and observe them the next day. During the culture period, if fungi grow around the tissue blocks, pick them out and purify them. At the same time, use the rinse solution test method to check whether the seeds are surface-disinfected. thorough;

3. Prepare PDB culture medium containing different Cd ²⁺ concentrations and distribute them into Erlenmeyer flasks. Pick out the mycelium of uniform size from the isolated endophytic strains and put them into Erlenmeyer flasks. Repeat three times for each concentration and place them in the Erlenmeyer flasks. After culturing for 6 days on a constant-temperature horizontal shaker at 28°C and shaking at 135 rpm, observe the growth of the colonies at different concentrations; select a strain with strong resistance to Cd ²⁺ and store it on a PDA slant, and name the fungus DF101 ;

4. Identification of strain DF101

(1) Morphological characteristics of DF101: In the early stage of culture, the edges of the colonies are neatly white, with dense gray hyphae, cotton-like, nearly round, and opaque. After 6 days, the colonies begin to turn yellowish brown;

(2) Molecular identification: Use the MoBio PowerSoil® DNA kit to extract the total DNA of the strain. After detection, it is sent to a sequencing company for sequence determination. The sequencing results are compared with the sequence on NCBI; combined with the morphological characteristics and molecular identification results, the final The strain was identified as Septoriella phragmitis ; the culture medium used for preservation and activation of this strain was PDA culture medium;

5. The present invention conducts a seed germination experiment with the seed endophytic fungus S. phragmitis DF101 isolated from Cogongrass to explore its impact on seed germination. That is, the seed endophytic fungus S. phragmitis DF101 is inoculated to affect the effects of seed under heavy metal stress. Research on the effects of germination provides fungal strains and theoretical research basis for plant-microbial joint repair.

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

The seed endophytic fungus S. phragmitis DF101 provided by the present invention is derived from Imperata cognac seeds. It has the ability to dissolve phosphorus, produce IAA, and produce siderophores, has good cadmium resistance, and can obtain a large amount of mycelium through simple liquid fermentation. , the bacteria are easy to obtain, low cost, and have the potential for commercial application; they can promote the germination of seeds under cadmium stress, significantly improve the germination rate of seeds in a heavy metal environment, improve their viability, and enhance their cadmium resistance, which is beneficial to The remediation of heavy metal contaminated soil is of great significance.

Description of the drawings

Figure 1 shows the colony morphology of Imperata seed endophytic fungus DF101 on PDA medium (Figure A: front, B: back);

Figure 2 is the standard working curve of phosphorus standard solution;

Figure 3 is the standard working curve of IAA standard solution;

Figure 4 shows the results of measuring the ability of S. phragmitis DF101 to solubilize phosphorus and produce IAA;

Figure 5 shows the seed germination rate of S. phragmitis DF101 inoculated (E+) and uninoculated (CK) under different concentrations of cadmium stress.

Detailed ways

The technical solutions of the present invention will be further described in detail below with reference to specific embodiments and drawings, but the present invention is not limited to the following technical solutions. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field. Persons of ordinary skill in the art can implement the method by referring to various commonly used reference books, scientific and technological documents or related instructions, manuals, etc. before the filing date of the present invention;

The preparation of chromium azure S (CAS) detection solution in the following examples is:

Solution A: Weigh 60.5 mg of chromium azure and dissolve it in 50 mL of deionized water, add 10 mL of Fe ³⁺ solution (containing 1 mM ferric chloride hexahydrate, 10 mM dilute hydrochloric acid) and stir to mix;

Solution B: Dissolve 72.9mg cetyltrimethylamine bromide in 40mL deionized water;

Slowly pour solution A into solution B and stir evenly to obtain CAS blue detection solution.

Example 1: Isolation, screening and identification of seed endophytic fungus S. phragmitis DF101

(1) Collect Imperata samples from Puxiong Township, Jianshui City, Yunnan Province, dry the plant samples in the sun until the seeds can fall off naturally, and then store the seed samples in a 4°C refrigerator for later use;

(2) Isolation of endophytes: 600 seeds were randomly selected from the seed samples, surface sterilized twice with ethanol with a volume concentration of 75%, each time for 1 minute, then rinsed 5 times with sterile water, and placed in a sterile place. Absorb the water on bacterial filter paper. Then, stick the surface-sterilized seeds onto a PDA plate (90mm), 6 seeds per plate, and incubate at 25°C for 60 days. Observe every other day. If fungi grow around the tissue block during the culture period, pick and purify them; at the same time, The rinse liquid test method tests whether the seed surface is completely disinfected;

(3) Screening of cadmium-resistant strains: Prepare PDB culture medium containing Cd ²⁺ 0, 100, 200, and 400 mg/L, and aliquot into 100 mL Erlenmeyer flasks. The aliquot volumes are all 50 mL. The separated contents Pick mycelium of uniform size in a Erlenmeyer flask, repeat each concentration three times, place it on a constant-temperature horizontal shaker at 28°C and shake at 135 rpm for 6 days, and observe the growth of the colonies under different concentrations; select Cd ^{2 +} The strain with strong resistance was stored on the PDA slant and named DF101;

(4) Identification of strain DF101

① Morphological characteristics of DF101: In the early stage of culture, the edges of the colonies are neatly white, with dense gray hyphae, cotton-like, nearly round, and opaque. After 6 days, the colonies begin to turn yellowish brown (Figure 1 A: front, B: back) ;

② Molecular identification: Use the MoBio PowerSoil® DNA kit to extract the total DNA of the strain. After testing, it is sent to a sequencing company for sequence determination. The sequencing results are shown as SEQ ID NO: 1. Compare the sequencing results with the sequence on NCBI; combine According to the morphological characteristics and molecular identification results, it has 98.93% homology with S. phragmitis , and the strain was finally identified as S. phragmitis .

Example 2: Cadmium tolerance experiment of seed endophytic fungus S. phragmitis DF101

Inoculate the isolated endophytic strains onto PDA plates, prepare PDB culture media containing Cd ²⁺ concentrations of 0, 50, 100, 200, 400, and 600 mg/L, and dispense them into 100 mL Erlenmeyer flasks. When the concentration is 50 mL, pick out uniformly sized bacterial blocks from well-growing strains and place them in a conical flask. Repeat three times for each concentration. Place them on a constant-temperature horizontal shaker at 28°C and shake at 135 rpm for 6 days. After 6 days of culture, observe the growth of the bacterial blocks and weigh them. The dry weight of mycelium was determined by the method, in which the culture medium with a Cd ²⁺ concentration of 0 mg/L was the control group. The inhibitory efficiency of heavy metals on the bacterial strain can be calculated according to the formula;

Growth inhibition rate = (dry mass of control mycelium - dry mass of treated mycelium)/dry mass of control mycelium × 100%

The results are shown in Table 1. The strain S. phragmitis DF101 can grow in PDB medium with higher cadmium concentration, indicating that S. phragmitis DF101 has better cadmium tolerance;

Table 1 Heavy metal tolerance of strain DF101

Note: In the above table, "+" has good resistance; "+-" has resistance, but the growth condition is not good; "-" has no resistance.

Example 3: Determination of phosphate-solubilizing ability of seed endophytic fungus S. phragmitis DF101

Phosphorus solubilizing ability: After culturing S. phragmitis DF101 in PDB culture medium on a shaking table at 28°C and 125 rpm for 4 days, pick out evenly sized bacterial clumps from well-growing strains and insert them into inorganic phosphorus liquid fermentation medium (containing 10g of glucose, sulfuric acid Ammonia 0.5g, sodium chloride 0.3g, potassium chloride 0.3g, magnesium sulfate heptahydrate 0.3g, ferrous sulfate heptahydrate 0.03g, manganese sulfate tetrahydrate 1g, tricalcium phosphate 5g, distilled water 1L, pH 7.0-7.5) At the same time, the same amount of PDB liquid culture medium was added as a blank control. Three replicates were set up for each treatment, and the culture was carried out on a shaking table at 28°C and 125 rpm for 10 days; the molybdenum antimony colorimetric method was used to determine the soluble phosphorus content (spectrophotometer Wavelength 700nm), the specific steps are as follows:

① Take the fermentation broth and centrifuge it for 15 minutes at 10000 rpm. Take 1 mL of the supernatant and place it in a 50 mL volumetric flask. Add 2 drops of 2,6-dinitrophenol indicator and adjust the pH with 10% sodium hydroxide or 5% dilute sulfuric acid until the solution is just right. It turns slightly yellow, add 5mL of molybdenum antimony anti-chromogenic agent and then add deionized water to adjust the volume to 50mL;

② After standing for 30 minutes, measure the color with a spectrophotometer at 700nm and measure the blank control group at the same time;

③ Draw a phosphorus standard curve during the experiment. Take 0, 2, 4, 6, 8, and 10 mL of 5 mg/L phosphorus standard solution into a 50 mL volumetric flask, add 2 drops of 2,6-dinitrophenol indicator, and use 10% Adjust the pH with sodium hydroxide or 5% dilute sulfuric acid until the solution just turns slightly yellow. Add 5 mL of molybdenum antimony antichromogenic agent and then add deionized water to adjust the volume to 50 mL to obtain 0, 0.2, 0.4, 0.6, 0.8, 1.0 mg/L. For the phosphorus standard series solution, let it stand for 30 minutes and then measure the color with a spectrophotometer at 700nm. Draw a standard working curve (Figure 2). Then substitute the absorbance value measured in step ② into the standard working curve to obtain the results of S. phragmitis DF101 cultured with inorganic phosphorus. The amount of dissolved phosphorus in the solution was 43.026 mg/L (Figure 4), indicating that S. phragmitis DF101 has the ability to convert inorganic phosphorus into organic phosphorus, making it easier for plants to absorb phosphorus, thereby promoting plant growth.

Example 4: Determination of IAA-producing ability of seed endophytic fungus S. phragmitis DF101

S. phragmitis DF101 was cultured in PDB medium (containing 0.5 mg/mL L-tryptophan) in the dark on a shaker at 28°C and 125 rpm for 10 days. Centrifuge 1 mL of bacterial liquid at 12,000 rpm for 15 min, remove the precipitate, and take 0.5 mL of the supernatant. Add an equal amount of Salkowski's reaction solution (1mL 0.5M ferric chloride, 49mL 35% perchloric acid), react in the dark for 30 minutes, and measure the absorbance value at 530nm. Each group is repeated three times, and the non-inoculated culture medium is treated as above. The control is zeroed; the IAA standard solution with concentrations of 0, 2.5, 5, 10, 20, 40, 60, 80, and 100 mg/L is used to measure the absorbance value and draw a standard curve (Figure 3) using the above method, and then inoculate S. phragmitis The absorbance value of the experimental group was substituted into the standard curve, and the amount of IAA secreted by S. phragmitis DF101 was 17.637 mg/L (Figure 4). S. phragmitis DF101 uses L-tryptophan as the precursor to synthesize the plant hormone IAA and stimulate the growth of plant cells. and proliferation, effectively absorbing water and nutrients, and regulating the life activities of plants.

Example 5: Determination of siderophore-producing ability of seed endophytic fungus S. phragmitis DF101

After S. phragmitis DF101 was cultured in PDB culture medium at 28°C and 125 rpm shaker for 4 days, the well-growing strains were picked into uniformly sized bacterial clumps and inserted into iron-free Zarbanz medium (containing 30g of sucrose, 2g of sodium nitrate, and phosphoric acid). 1g dipotassium hydrogen, 0.5g magnesium sulfate heptahydrate, 0.5g potassium chloride, 1L distilled water), and incubate with shaking at 28°C, 125rpm for 5 days, then separate at 8000rpm for 10 minutes, collect the culture supernatant, and add 1 mL of the supernatant Mix with 1 mL CAS detection solution, react the mixture in the dark for 15 minutes, measure the absorbance value at 630 nm, and record the value as As. Use the same method to measure the OD ₆₃₀ value in the blank control without bacteria, record it as Ar, and remove Ionized water was used as a control for zero adjustment. The specific calculation formula is siderophore activity = [(Ar-As)/Ar] × 100%;

The calculated result is that the capacity of strain DF101 to chelate siderophores is 74.52%. Siderophores are a type of small molecule compounds that are synthesized and secreted by bacteria and fungi in low-iron environments and have strong specificity for chelating Fe ³⁺ . Studies have shown that some Siderophore-producing bacteria can enrich iron in the rhizosphere of plants, thereby improving the absorption of plant iron nutrients and promoting plant growth.

Example 6: Effect of seed endophytic fungus S. phragmitis DF101 on seed germination of Schizonepeta under heavy metal stress

This example aims to prove the promoting effect of S. phragmitis DF101 on seed germination; using Dysphania ambrosioides L. as the test plant, the experimental process is as follows:

A. Preparation of S. phragmitis DF101 suspension: Select the previously purified DF101 strain, inoculate it into a PDA plate for activation, place it in a 28°C water-isolated constant-temperature incubator and cultivate it for 7 days, select a plate with good growth and no pollution, Pick the mycelium and inoculate it into the PDB medium. After culturing for 4 days in a constant temperature shaker at 28°C and 125 rpm, filter out the mycelium under sterile conditions and rinse it with sterile water three times to avoid mycelium. The body is stained with culture medium, and then cut into pieces with sterile scissors. The chopped hyphae are transferred to sterile water and the volume is adjusted to 150mL to prepare a DF101 mycelium suspension;

B. Select evenly sized and plump Schizonepeta seeds for surface disinfection: rinse twice with ethanol with a volume fraction of 75%, each time for 2 minutes and 30 seconds, and rinse with sterile water five times. The surface-sterilized seeds were soaked in DF101 mycelium suspension for colonization (E+) and soaked for 2 hours for colonization. The seeds were soaked in an equal volume of sterile water to form the control group (CK). The soaked seeds were soaked in a solution containing different The concentration of Cd ²⁺ solution is in a petri dish (the petri dish is covered with two layers of sterilized filter paper, and the Cd ²⁺ solution in each petri dish is 5mL); the concentration of Cd ²⁺ solution is 0, 30, 50, 100, 200mg /L, set three replicate groups for each concentration, place 25 seeds in each dish, culture under natural light, record the number of seeds from the beginning of germination until no germination, count the number of seeds germinated, and calculate the germination rate (%) is (Gt/T) ×100, where Gt is the number of germinated seeds and T is the number of seeds.

The results are shown in Figure 5. The results show that S. phragmitis DF101 can significantly promote the germination of Schizonepeta seeds under heavy metal stress, improve the survival rate of seedlings, and enhance its cadmium resistance.

The results of the above examples illustrate that the seed endophytic fungus S. phragmitis DF101 isolated in the present invention has the ability to dissolve phosphorus, produce IAA and siderophores, as well as have good cadmium tolerance, and is resistant to Schizonepeta under heavy metal stress. It has a better promoting effect on seed germination.

Claims (2)

1. Endophytic fungi of cogongrass seedSeptoriella phragmitis) DF101 has the preservation number of CGMCC No.40427 in China general microbiological culture Collection center.

2. The application of the cogongrass seed endophytic fungus DF101 in promoting germination of chenopodium ambrosioides seeds under the stress of heavy metal cadmium, which is characterized in that: the endophytic fungi DF101 of the cogongrass seed has the capability of tolerating cadmium, dissolving phosphorus, producing IAA and producing siderophores.