CN115820496A - Cadmium-resistant rice endophytic asprella fusca and application thereof - Google Patents
Cadmium-resistant rice endophytic asprella fusca and application thereof Download PDFInfo
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Abstract
The invention discloses cadmium-resistant rice endophytic spirogyra (Herbaspirillum sp.) YUC and application thereof, wherein the cadmium-resistant rice endophytic spirogyra YUC is preserved in China Center for Type Culture Collection (CCTCC) NO: m20221716, preservation date 2022, 11 months, 03 days. The strain has high tolerance and high adsorption capacity to cadmium, so that the concentration of cadmium ions in a solution can be greatly reduced, and the biological toxicity of the cadmium ions can be relieved. In addition, the strain YUC is a rice endophyte and has the capability of promoting the growth and development of rice by producing IAA, dissolving phosphorus, producing siderophore and the like. Compared with the existing reports, the strain has the advantages that the cadmium ion removal capability is remarkably improved, and the strain has certain growth promotion potential. The strain can reduce the accumulation of cadmium ions in rice, realize the production and repair of the rice and provide a safe, effective, green and environment-friendly bioremediation method.
Description
Technical Field
The invention relates to the field of environmental microbiology and agricultural microorganisms, in particular to cadmium-resistant rice endophytic glusula and application thereof in aspects of heavy metal pollution remediation, agriculture and the like.
Background
Cadmium (Cadmium, cd) is a heavy metal element that is highly toxic and not essential to humans. Compared with other heavy metal pollutants, cd has fluidity, is easily absorbed by plant roots and transferred to the ground part, and then enters a human body through a food chain, so that the Cd is harmful to human health and the whole ecological system. In recent years, researchers have found that: about 10 percent of rice sold in the whole country has the problem of cadmium exceeding. And after the rice with the cadmium content exceeding the standard is eaten for a long time, various diseases can be caused, such as osteoporosis, kidney injury, sexual dysfunction, cancer and the like. Therefore, it is urgent to limit the accumulation of cadmium in rice and reduce the damage of cadmium to human health through food chain.
Aiming at the cadmium pollution problem in rice, the remediation method comprises physical remediation (reducing or eliminating the cadmium content in soil by a soil leaching method, a soil turning method, a soil changing method and the like), chemical remediation (adding a chemical passivator to adsorb and chelate cadmium ions), and biological remediation (reducing or changing the form of cadmium into a form with lower risk by using the life activities of animals, plants and microorganisms). Compared with the traditional physical and chemical remediation, the green and safe bioremediation has the advantages of low cost, easy operation, sustainability, no secondary pollution and the like, and is more and more valued by people. In particular, the enhancement of heavy metal restoration ability of plants by utilizing the adsorption of heavy metals by plant endophytes developed in recent years has gradually become a research hotspot.
In the process of long-term symbiosis of the endophyte and the host plant, a relationship of co-evolution, mutual benefits and mutual benefits is established. At present, reports about rice endophytes focus on the aspects of growth promotion, bacteriostatic activity, rice blast resistance and the like of rice, and are mainly applied to the fields of extracting natural active substances, manufacturing biological fertilizers or biological pesticides and the like. Reports of screening and separating cadmium-tolerant endophytes in rice and helping the rice to reduce cadmium stress pressure are less. Zhou et al screened 58 endophytes from rice seeds, of which Stenotrophomonas maltophilia R5-5 was able to tolerate 4mM/L Cd 2+ And after the rice is planted in the field, the cadmium content of the rice root is obviously reduced (Environmental polarization, 2020). Isolating Bacillus HNR-4 from rice seed, containing 1mg/L CdCl 2 After 24h of culture in liquid medium, the removal rate of the HNR-4 to cadmium reaches 77.57% (microbiological report, 2022). Patent 202111452900.6 (China, chengdu et al) reports that a rice endophytic bacillus can reduce or reduce the harm of heavy metals including cadmium to rice. At present, the publicly reported cadmium-resistant rice endophytes have the problems of less varieties, poor cadmium resistance, poor cadmium removal capacity and the like.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a rice endophytic asplenium sp with high cadmium tolerance and high cadmium adsorption capacity.
The technical scheme of the invention is as follows: a cadmium-resistant rice endophytic asprella (Herbaspirillum sp.) YUC is preserved in China center for type culture collection with the preservation number of CCTCC NO: m20221716, preservation date 2022, 11 months, 03 days.
The 16s rDNA sequence of the grass spirillum (Herbaspirillum sp.) YUC is shown as SEQ ID No. 1.
The grass spirillum sp (Herbaspirillum sp.) YUC is an endophytic bacterium obtained by screening and separating cadmium-treated rice by the inventor, has higher cadmium tolerance and is safer when being applied to in-situ removal of cadmium pollution in the rice.
The ability of grass spirillum (Herbaspirillum sp.) YUC is 600mg/L CdCl 2 The strain can lead Cd to grow in NB liquid culture medium, has very excellent cadmium tolerance capacity, and can lead Cd to grow after culturing for 102h 2+ The concentration is reduced from the initial 300mg/L to 74.73mg/L, cd 2+ The adsorption capacity is 225.27mg/L, and the removal rate of Cd can reach 75%. Therefore, the strain can be used for cadmium reduction treatment of soil or wastewater with high cadmium content.
The grass spirillum (Herbasspirillum sp.) YUC has the capability of promoting plant growth and development such as IAA production, phosphorus dissolution and siderophore production, and has a certain growth promoting potential for rice growth.
Compared with the prior art, the invention has the following beneficial effects:
the strain YUC is a strain of Herbaspirillum sp, and has high tolerance and high adsorption capacity to cadmium, so that the concentration of cadmium ions in a solution can be greatly reduced, and the biological toxicity of the cadmium ions can be relieved. In addition, the strain YUC is a rice endophyte and has the capability of promoting the growth and development of rice by producing IAA, dissolving phosphorus, producing siderophore and the like. Compared with the existing reports, the strain has the advantages that the cadmium ion removal capability is remarkably improved, and the strain has certain growth promotion potential. The strain can reduce the accumulation of cadmium ions in rice, realize the production and repair of the rice and provide a safe, effective, green and environment-friendly bioremediation method.
Preservation information
The grass spirillum (Herbasspirillum sp.) YUC is preserved in China center for type culture Collection in 2022 at 11 months and 03 days, with the preservation number of CCTCC NO: m20221716, deposit address: eight-way No. 299 in Wuchang area of Wuhan city, hubei province.
Drawings
FIG. 1 colony morphology (A) and SEM image (B) of the strain Herbaspirillum sp.YUC.
FIG. 2 phylogenetic tree of 16S rDNA gene sequences of strain Herbaspirillum sp.YUC.
FIG. 3 Strain Herbaspirillum sp.YUC under Normal conditions and containing 300mg/L CdCl 2 A growth curve chart under the stress of concentration and the adsorption of Cd ions by the growth curve chart.
FIG. 4 Effect of pH, temperature and salt concentration on growth of Herbaspirillum sp
Detailed Description
The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were all commercially available unless otherwise specified.
The media components referred to in the examples:
(1) NB medium preparation, beef extract 3g, peptone 10g, naCl 5g, pH 7.2 adjustment, water to 1L.
(2) And preparing an NA culture medium, namely adding 17g of agar into the NB culture medium to obtain the NA culture medium.
(3) Preparing an LB culture medium: 10g of tryptone, 5g of yeast extract and 10g of NaCl, adjusting the pH to 7.2, and adding water to 1L.
(4) Preparation of a Pikovskaya solid culture medium: 1L culture medium contains yeast extract 0.5g, glucose 10g, and Ca 5g 3 PO 4 、0.5g(NH 4 ) 2 SO 4 、0.3g KCl、0.3g NaCl、0.03g MgSO 4 ·7H 2 O、0.03g MnSO 4 、0.003g FeSO 4 ·7H 2 O, 18g of agar, and adjusting the pH value to 7.0-7.5.
(5) Preparation of CAS solid culture medium: 0.0605g of Chromoazuron S (CAS), 0.0729g of hexadecyltrimethylammonium bromide (HDTMA), feCl 2 ·6H 2 O0.002645 g, agar 9g,0.1mol/L phosphate buffer 50mL (NaH) 2 PO 4 ·2H 2 O 0.296g，Na 2 HPO 4 ·12H 2 O 1.214g，NH 4 Cl 0.125g，KH 2 PO 4 0.0375g，NaCl 0.0625g，pH 6.6-7.0)
(6) Preparing an Artocarpus (Ashby) nitrogen-free culture medium: mannitol 10g, KH 2 PO 4 0.2g，MgSO 4 ·7H 2 O0.2g，NaC1 0.2g，CaSO 4 ·2H 2 O 0.1g，CaCO 3 5.0g, 15g of agar, pH 7.2 to 7.4, and waterTo 1L. Sterilizing with high pressure steam at 121 deg.C for 20min, and storing at 4 deg.C.
(7) Preparing a JNFb culture medium: 5g of L-malic acid, K 2 HPO 4 0.6g，KH 2 PO 4 1.8g，MgSO 4 ·7H 2 O0.2g，NaCl 10.1g，CaC1 2 ·2H 2 0.02g of O, 2.0mL of trace element solution, (Na) 2 MoO 4 ·2H 2 O 0.2g，MnSO 4 ·H 2 O 0.235g，H 3 BO 3 0.28g，CuSO 4 ·5H 2 O 0.008g，ZnSO 4 ·7H 2 O0.024 g, water 1000 mL), bromothymol blue 2.0mL (0.5% aqueous solution dissolved with 0.2mol/L KOH), fe-EDTA 4.0mL (1.64% aqueous solution) vitamin solution 1.0mL. (biotin 0.01g, VB 0.02g, water 1000 mL), KOH 4.5g, adjusted to pH 5.8 with KOH, and water to 1L. The JNFb solid culture medium is prepared by adding 1.5% agar into JNFb culture medium, sterilizing with high pressure steam at 121 deg.C for 20min, and storing at 4 deg.C.
Example 1 screening of cadmium-tolerant Rice endophyte YUC
According to the method, rice treated by cadmium is selected, endophytes are separated from the rice, and then the endophytes are cultured under the stress of high-concentration cadmium, so that a rice endophyte tolerant to the concentration of 600mg/L cadmium is screened out. The specific operation steps are as follows:
acquisition and rescreening of rice endophytes
Selecting 2mg/L CdCl 2 The roots, stems and leaves of the treated rice (variety: jinxiangyu No. 1) at the seedling stage were used as experimental materials: firstly, washing the surfaces of roots, stems and leaves of the rice with clean water until the surfaces are clean; sterilizing with 0.1% mercuric chloride solution, soaking the leaves for 1min, soaking the roots and stems for 1.5min, washing with sterile water for 3-4 times, and applying the final washing solution on NA plate at 37 deg.C for overnight culture. No colony grows, which indicates that the surface of the sample is sufficiently sterilized and can continue to be done. If the bacterial colony grows, the surface of the sample is insufficiently sterilized, and the step is repeated; cutting the middle part of the leaf into 0.5cm square, cutting the stem and root into thin pieces, placing the cut tissue pieces on the surface of a NA culture medium plate, and culturing in an inverted manner in an incubator at 37 ℃ for 3-7 days. Picking up bacterial colonies around the tissue block on the NA culture plate, and carrying out sorting and graftingThe seeds were inoculated into 50mL NB medium and were shake-cultured at 37 ℃ and 250rpm for 24h.
The cultured bacterial liquid is sequentially inoculated into NB culture media with different cadmium concentrations (300, 400, 500 and 600 mg/L) according to the proportion of 1. The selection energy is 600mg/L CdCl 2 The NB medium of (2) was grown and well grown bacteria within 48h were streaked for purification. The purified strain was mixed with sterilized glycerol (final concentration of glycerol was 30%) and stored in a-80 ℃ refrigerator.
And finally obtaining a cadmium-resistant rice endophyte with the serial number of YUC, namely the strain of the patent. Strain YUC can tolerate Cd in liquid NB culture medium 2+ The concentration of (2) was 600mg/L.
Example 2 identification of the endophytic Spirospirium sp.YUC of rice
(1) Morphological characteristics of bacterial colony
The bacterial strain YUC is cultured on NB solid medium at 37 ℃ for 48h, the bacterial colony is circular, the diameter is 0.5-1 mm, the surface is smooth and moist, the edge is regular, the bacterial colony is milky white and translucent, and the specific bacterial colony morphology is shown as A in figure 1.
(2) Observation by Scanning Electron Microscope (SEM)
Centrifuging appropriate amount of bacteria solution, discarding supernatant, adding PBS buffer solution with appropriate pH (7.2-7.4) for washing for three times, gently suspending thallus during washing, centrifuging, and discarding supernatant. Then the sample is fixed with 2.5% glutaraldehyde for 1-3h, centrifuged, and then washed three times with PBS buffer, centrifuged, and the supernatant is removed. Dehydrating the sample with ethanol water solution according to concentration gradient of 30%, 50%, 70%, 80%, 90%, each step for about 15min, centrifuging to remove supernatant, then dehydrating in 100% ethanol for 15min, repeating the operation for 2 times, centrifuging to remove supernatant, placing the sample in a mixture of ethanol and tert-butanol 1 for 15min, and centrifuging to remove supernatant; the alcohol was then replaced 2 times with pure tert-butanol, each time for 15min. And finally, putting the mixture into a freeze dryer for drying. And after the sample is fully dried, dissolving a small amount of powder in purified water or alcohol for ultrasonic dispersion, dripping a small amount of solution on a copper mesh, fully drying and adhering the solution to a sample table with a conductive adhesive tape, spraying gold to prepare a sample, and finally observing under an electron microscope.
The strain YUC is rod-shaped under scanning electron microscope (see B in FIG. 1), slightly bent, 0.6-1.2 μm in length and about 0.5 μm in diameter.
(3) Microbiological characterization
The YUC strain is sequentially subjected to gram staining, carbon source utilization, antibiotic sensitivity and catalase experiments, and the result shows that the strain is gram staining negative bacteria, 5 types of sugar alcohols such as salicin, L-rhamnose, D-mannose, sucrose, maltose, D-glucose and the like and malonate can be used as carbon sources, organic acids such as citrate, acetate and the like can not be used, and the strain is sensitive to tetracycline, spectinomycin, gentamicin and peroxidase experiments and is positive.
(4) 16S rDNA molecular identification
Extracting the genome DNA of the rice endophytic bacterium YUC, and performing 16S rDNA amplification and sequencing by using the genome DNA as a template by using a PCR (polymerase chain reaction) technology.
(1) The primer sequence is as follows: f27 '5' AGAGTTTGATCATGGCTCAG-3 'and R1492' 5 'ACGGTTACCTTGTTACGACTT-3';
(2) PCR system set-up (50. Mu.L): 2 μ L of genomic DNA, 2 μ L of Forward Primer (10 μ M), 2 μ L of Reverse Primer (10 μ M), 25 μ L of ExTaq DNA polymerase mixed reagent, and 19 μ L of sterile water.
(3) The PCR reaction set program parameters were: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 15s, renaturation at 55 ℃ for 15s, extension at 72 ℃ for 90s, 34 cycles in total, extension at 72 ℃ for 5min, and finally storage at 12 ℃.
(4) After the PCR was completed, 5. Mu.L of the PCR product was subjected to 0.7% agarose gel electrophoresis to detect the target fragment having a size of about 1.5kb. And recovering the PCR product, and sending the PCR product to Wuhan Jinrui biotechnology limited for sequencing. The sequence length was 1330bp (SEQ ID No. 1).
(5) The 16S rDNA sequence of the obtained strain is subjected to Blast comparison, and the result shows that the similarity of the sequence and the strain YUC reaches 99 percent and the strain belongs to the genus Eisenia. The 16S rDNA sequence with high correlation was downloaded, and then MEGA11 was used to construct phylogenetic trees by the NJ method, the results are shown in FIG. 2.
Finally determining that the rice endophyte YUC is a grass spirillum sp by combining strain morphology, physiological and biochemical characteristics and 16S rDNA sequence comparison analysis. Its 16S rDNA sequence was registered on the NCBI website under accession number OP735538. The grass spirillum (Herbasspirillum sp.) YUC is preserved in China center for type culture Collection in 2022 at 11 months and 03 days, with the preservation number of CCTCC NO: m20221716, deposit address: eight-way No. 299 in Wuchang area of Wuhan city, hubei province.
Example 3 measurement of growth curve and cadmium adsorption ratio of whelk bacterium (Herbaspirillum sp.) YUC
Inoculating Herbaspirillum sp YUC at a ratio of 1:100 to cadmium-free and CdCl 300mg/L 2 In 100mL of NB medium, the bacteria were cultured at 37 ℃ and 220 r/min. Sampling at regular time, and determining OD by use of microplate reader 600 The value is obtained. And (3) referring to the national people's republic of China standard GB/T20975.6-2020, and measuring the concentration of cadmium in the sample by using a flame atomic absorption spectrophotometer. According to the measurement result, the grass spirillum (Herbaspirillum sp.) YUC is drawn in CdCl 2 Growth curves with concentrations of 0mg/L and 300mg/L and the concentration change of Cd in the culture medium.
Removal rate = (C) 0 -C t )/C 0 ×100％
In the formula: c 0 Concentration as control (mg/L); c t The concentration of cadmium in the supernatant (mg/L)
As shown in FIG. 3, in NB medium (without cadmium), the grass spirillum (He rbaspirillum sp.) YUC was in the growth phase within 0-20h and grew faster; the culture medium is in a stationary growth period within 20-60h, the OD value reaches about 2.3 at most, and the content of thalli in the culture medium reaches the highest value at the moment; after 80h the strain entered the decline phase.
In CdCl 2 In an NB culture medium with the concentration of 300mg/L, the growth of the grass snail (Herbaspirillum sp.) YUC is slow within 0-20h and is in a lag phase, the growth speed of the strain in the growth phase is fast within 20-60h, and the OD value reaches about 1.5 at most; the strain enters a stationary growth period for 60 hours, and the thallus content in the culture solution reaches the highest value at the moment; after 70h, the strain enters the decline phase. Meanwhile, the cadmium content in the culture solution gradually decreases along with the growth of the grass snail (Herbaspirillum sp.) YUC, and the cadmium concentration in the culture medium is reduced from 300mg/L to about 74.73mg/L after 102 h.The removal rate of heavy metal cadmium (Cd) is about 75%.
Example 4 measurement of growth promoting ability of Aspirallus herbicola (Herbaspirillum sp.) YUC
(1) Ability to produce indole-3 acetic acid IAA
The OD of the bacterium concentration is quantitatively detected by a Salkowski colorimetric method and an enzyme labeling instrument 600 At a value of 1, the amount of IAA secreted by the bacteria per unit volume of bacterial suspension.
The results showed that the grass spirillum (Herbasspirillum sp.) YUC turned the Salkowski colorimetric solution pink and the IAA yield was 3.53mg/L.
(2) Phosphorus dissolving capacity
mu.L of the bacterial suspension was spotted on Pikovskaya solid medium, and cultured at 37 ℃ for 3 to 5 days to observe the growth of colonies.
The result shows that transparent halos are arranged around the colony of the glufosinate-conch bacteria, and the bacteria have the phosphorus dissolving capacity.
(3) Capability of producing ammonium nitrogen
Inoculating the grass spirillum into NB culture medium, culturing at 37 ℃ for 2 days, and adding naphthalene reagent to observe color change.
The results show that the whelk bacterium cannot redden the naphthalene reagent, indicating that the whelk bacterium cannot produce ammonium nitrogen.
(4) Siderophore production capacity
The grass spirillum is inoculated in a CAS solid culture medium and cultured for 3 days at 37 ℃, and the existence of yellow halos around colonies is observed, and the appearance of the yellow halos indicates the generation of siderophore.
The result shows that yellow halos are shown around the colonies of the glufosinate-concha, which indicates that the glufosinate-concha has siderophores.
(5) Azotase Activity
(1) Detection of nitrogenase genes nifH and nifD
According to the literature, the azotase complex consists of azotase and azotase, wherein the azotase is an alpha 2 beta 2 heterotetramer consisting of NifD and NifK proteins, the azotase is a homodimer consisting of NifH proteins, neither protein has azotase activity when being present alone, and the azotase has the capability of catalyzing nitrogen to be reduced into ammonia only after the two proteins form a complex. Therefore, in the experiment for identifying the activity of the azosin of the grass spirillum, only the NifD and NifH genes need to be detected.
NifD upstream primer 5'-TGGGGICCIRTIAARGAYATG-3' downstream primer 5 '-TCRTTIGCIAATRTGRTGNCC-3', nifH upstream primer 5 '-TGCGAYCCAARGCGCACTC-3' downstream primer 5'-ATSGCCATCATYTCRCCGGA-3' PCR amplified bacterial genomic DNA, and DNA bands of expected sizes were 360bp (nifH) and 390bp (nifD).
The PCR results showed that the expected DNA bands of 360bp and 390bp were not amplified, so the grass snail does not contain the azotobacter genes nifH and nifD.
(2) Azotase Activity detection
Inoculating the spirillum into Ashby and JNFb culture medium without a nitrogen source for culture, observing the growth condition of bacteria, and changing the bromothymol blue JNFb culture medium from green to blue to obtain the positive.
The results indicated that the asplenium serrulatum could not grow normally and could not change the bromothymol blue JNFb medium from green to blue, and thus it was presumed that it had no nitrogenase activity.
In summary, the rice endophytic asprella (Herbaspirillum sp.) YUC has the capability of promoting plant growth and development such as IAA production, phosphorus dissolution and siderophore production, but the strain has no azotase activity and does not produce ammonium nitrogen.
Example 5 Effect of pH, temperature and salt concentration growth of Rice Oncomelania Hupensis (Herbaspirillum sp.) YUC
(1) Optimum pH
Inoculating bacterial suspension of Herbaspirillum sp YUC into 5mL NB culture medium, culturing at different pH (5.0, 6.0, 7.0, 8.0, 9.0), culturing for 12 hr, and measuring bacterial OD 600 Values were plotted.
As a result, as shown in FIG. 4A, the grass spirillum YUC grew well under the condition of pH 7.0-8.0. Bacterial growth is significantly inhibited when the pH is < 7.0 or > 8.0.
(2) Optimum growth temperature
Inoculating Herbaspirillum sp YUC into 5mL NB culture medium, culturing at different temperatures (25 deg.C, 30 deg.C, 37 deg.C, 40 deg.C) for 12 hr, and measuringDetermination of the OD of the bacterium 600 Values were plotted.
As shown in B in FIG. 4, all the grass spirillum (Herbaspirillum sp.) YUC can grow at a temperature ranging from 25 to 40 deg.C, and the growth of the strain is more suitable when the temperature is 37 deg.C.
(3) Salt tolerance concentration
mu.L of the activated bacteria were inoculated into 5mL of NB medium containing NaCl at various concentrations to give final NaCl concentrations (0%, 0.5%,1.0%,1.5%,2.0%,2.5% and 3%), and after 12 hours of incubation at 37 ℃ the OD of the bacteria was determined 600 And plotted.
The results are shown in fig. 4C, whelk (Herbaspirillum sp.) YUC grew well at salt concentrations of 0% to 2.5%. Bacterial growth was inhibited when the NaCl concentration was > 2.5%.
Claims (4)
1. A cadmium-resistant rice endophytic asprella (Herbaspirillum sp.) YUC is preserved in China center for type culture collection with the preservation number of CCTCC NO: m20221716, preservation date 2022, 11 months, 03 days.
2. Use of the whelk bacterium (Herbaspirillum sp.) YUC of claim 1 for cadmium reduction.
3. The use of claim 2, wherein the cadmium reduction is to reduce the cadmium content in cadmium-containing soil or wastewater.
4. Use of the grass spirillum sp (Herbaspirillum sp.) YUC of claim 1 for reducing the stress of cadmium ions on rice growth or reducing the cadmium content of rice.
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| CN116478870A (en) * | 2023-03-27 | 2023-07-25 | 云南大学 | Maltophilous oligotrophic single spore fungus OLR3-17 strain and application thereof |
| CN116656539A (en) * | 2023-04-20 | 2023-08-29 | 安徽农业大学 | Carpesium V8 and application thereof |
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| CN116478870A (en) * | 2023-03-27 | 2023-07-25 | 云南大学 | Maltophilous oligotrophic single spore fungus OLR3-17 strain and application thereof |
| CN116656539A (en) * | 2023-04-20 | 2023-08-29 | 安徽农业大学 | Carpesium V8 and application thereof |
| CN116656539B (en) * | 2023-04-20 | 2024-03-22 | 安徽农业大学 | Carpesium V8 and application thereof |
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