CN111040956B - Endophytic fungus Y6 for enhancing oxidation resistance of casuarina equisetifolia in high-salt environment - Google Patents
Endophytic fungus Y6 for enhancing oxidation resistance of casuarina equisetifolia in high-salt environment Download PDFInfo
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Abstract
The invention discloses endophytic fungus Y6 capable of increasing oxidation resistance of casuarina equisetifolia in a high-salt environment, and belongs to the technical field of microorganisms. The classification of the endophytic fungus Y6 is named as Xylaria (C.donovani) ((C.donovani))Hypoxylon sp.) And the strain is preserved in the China general microbiological culture Collection center on 20 th month 11 in 2019, and the preservation number is CGMCC NO. 18814. The endophytic fungus Y6 bacterial liquid is applied to planting of casuarina equisetifolia seedlings in a high-salt environment in a mode of rhizosphere soil pouring or direct seedling inoculation, and the oxidation resistance of casuarina equisetifolia can be improved.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to an endophytic fungus Y6 capable of increasing the oxidation resistance of casuarina equisetifolia in a high-salt environment.
Background
Salt stress seriously affects the growth and development of crops and forest land plants, and is a major abiotic stress worldwide. Excess salt segregants, e.g. Na+、K+、Cl-When taken by plants, the ion concentration in plant cells is increased, and various biological enzymes in plant cells only have activity in a narrow range of ion concentration, such as Na+Ions and Cl-Ion requirement less than 50mM, for K+The ion content is 0.1-0.2M. Thus, a change in ion concentration affects biological enzyme activity and thus plant growth. Salt stress can also damage the function of plant cell membranes by a series of changes such as influencing the components, permeability, transportation and the like of the cell plasma membranes, thereby destroying the metabolism and various physiological functions of cells to a certain extent.
Ephedra, shortstem Ephedra herbCasuarina equisetifolia L.) is the earliest tree species of the family of the woodephedra introduced in various countries of the world and has the largest artificial cultivation area, and is also the most main afforestation tree species in China south China and the south east coast at present. The existing research shows that the casuarina equisetifolia has oneThe salt resistance is determined, but the improvement of the salt resistance is beneficial to playing greater ecological role. Therefore, how to increase the salt tolerance of casuarina equisetifolia and improve the ecological adaptability is the key point of future research.
A series of previous researches show that the symbiont of the plant endophytic fungi and the host is beneficial to the host to deal with adverse environment and promote the growth and development of the plant, but most of the researches are focused on the gramineae plants, while the woody plant endophytic fungi are less researched. Separating and identifying endophytic fungi from casuarina equisetifolia tissues, searching for endophytic fungi with growth promoting effect and certain salt tolerance, establishing casuarina equisetifolia-endophytic fungi symbiotic system, improving the growth of casuarina equisetifolia under high salt stress, and providing data basis and reference basis for enriching strain information of endophytic fungi of woody plants and producing biological bacterial fertilizer for casuarina equisetifolia work forest management.
Disclosure of Invention
The invention aims to provide an endophytic fungus Y6 for enhancing the oxidation resistance of casuarina equisetifolia in a high-salt environment.
In order to achieve the purpose, the invention adopts the following technical scheme:
an endophytic fungus Y6 for enhancing the oxidation resistance of casuarina equisetifolia in a high-salt environment, and the classification of the endophytic fungus Y6 is named as Xylaria (C) (A)Hypoxylon sp.) The microbial culture has been preserved in China general microbiological culture Collection center in 2019, 11 and 20 months, and the preservation address is as follows: no. 3 of Xilu No.1 of Beijing, Chaoyang, and the preservation number is CGMCC NO. 18814.
When the endophytic fungus Y6 is cultured on a potato glucose culture medium (PDA culture medium), the initial bacterial colony is white, the aerial hyphae flourish, and the middle color of the bacterial colony gradually becomes dark green to black at the later culture stage.
The endophytic fungus Y6 strain is obtained by separating and purifying Ephedra sinica Stapf seeds, can be prepared into bacterial liquid, and is used for planting Ephedra sinica Stapf seedlings in a high-salt environment in a mode of rhizosphere soil pouring or direct seedling inoculation.
The preparation method of the bacterial liquid comprises the following steps: inoculating the strain into liquid culture medium, and keeping the temperature of a shaking table constantAfter culturing for 72h, the obtained culture solution is mixed and diluted with sterile water, and the concentration of the diluted bacterial solution is 8.75X 105 cfu/mL. The liquid culture medium is PDB.
The invention has the advantages that:
the obtained strain can relieve the restriction of salt stress conditions on the growth of the strain, and can promote the increase of casuarina equisetifolia antioxidase in a high-salt environment, so that the damage of the salt stress on the strain is relieved.
Description of the drawings:
FIG. 1 is a morphological diagram of hypha and colony of endophytic fungus Y6.
FIG. 2 shows the effect of NaCl stress at different concentrations on the content of SOD in E and EI branches of casuarina equisetifolia. Different capital letters indicate significant differences at different treatment levels for the same NaCl level (Duncane test, P < 0.05), and different lower case letters indicate significant differences at different NaCl levels for the same treatment level (Duncan test, P < 0.05).
FIG. 3 shows the effect of NaCl stress at different concentrations on the content of Ephedra sinica Stapf and Ephedra sinica Stapf EI sprigs POD. Different capital letters indicate significant differences at different treatment levels for the same NaCl level (Duncane test, P < 0.05), and different lower case letters indicate significant differences at different NaCl levels for the same treatment level (Duncan test, P < 0.05).
FIG. 4 shows the effect of NaCl stress at different concentrations on the MDA content of Ephedra sinica EF and Ephedra sinica EI twigs. Different capital letters indicate significant differences at different treatment levels for the same NaCl level (Duncane test, P < 0.05), and different lower case letters indicate significant differences at different NaCl levels for the same treatment level (Duncan test, P < 0.05).
FIG. 5 shows the effect of NaCl stress at different concentrations on the content of EF and EI twigs of Ephedra sinica Stapf and CAT. Different capital letters indicate significant differences at different treatment levels for the same NaCl level (Duncane test, P < 0.05), and different lower case letters indicate significant differences at different NaCl levels for the same treatment level (Duncan test, P < 0.05).
FIG. 6 shows that different concentrations of NaCl stress on Ephedra sinica Stapf and Ephedra sinica Stapf EI small branches H2O2Influence of the content. Different capital letters indicate differences in treatment levels at the same NaCl levelDifferences were significant (Duncane test, P < 0.05), and different lower case letters indicated significant differences in different NaCl levels at the same treatment level (Duncan test, P < 0.05).
Detailed Description
In order to make the content of the present invention easier to understand, the technical solution of the present invention is further described below with reference to the specific embodiments, but the present invention is not limited thereto
Example 1 isolation of an endophytic fungus from Schima superba
1. Main instrument equipment
AX224ZH electronic balance (OHAUS), LS-75HD high-pressure steam sterilization pot (Jinan Laibao medical instruments Co., Ltd.), JB-CJ-1500FX super clean bench (Suzhou Jiabao purification engineering equipment Co., Ltd.), DNP-9162 constant temperature incubator (Shanghai Jinghong), ZHHWY 2111B constant temperature shaking incubator (Shanghai Zhicheng), Beckman Ultracentrifuge (USA), etc. The main instrument for identifying the strain comprises: FQD-48APCR augmentor (BIOER), EPS-100 nucleic acid electrophoresis (Shanghai Nature technology), SeqStaudio DNA sequencer (USA), mortar, ice bag, foam cartridge, pipette (Thermo), 1.5ml and 2ml EP tube (Axygen).
2. Primary reagents and culture media
Reagent: 70% alcohol, sodium hypochlorite, Potato Dextrose Agar (PDA) culture medium, Potato Dextrose (PDB) culture medium, NaCl, Taq enzyme and PCR related reagents, primers (ITS 1/ITS 4), OMEGA fungus genome kit, liquid nitrogen, ethanol (Tianjin Mao chemical reagent factory) and deionized water.
3. Isolation of endophytic fungi
(1) The tissue separation method is adopted to divide the casuarina equisetifolia sample into four parts according to different tissues of roots, stems, leaves and seeds of the casuarina equisetifolia sample, and the four parts are washed clean by running water and dried in the shade to sterilize the tissue surface in a super clean bench. The operation flow is as follows: soaking in 70% alcohol for 30s → washing with sterile water for 3 times → soaking in 10% sodium hypochlorite for 7min → washing with sterile water for 3 times. The casuarina equisetifolia samples were cut open with a scalpel, placed flat in a sterilized plate medium, and cultured in a 28 ︒ C incubator for 5-7 days.
(2) And (3) verification of the disinfection effect: and (3) coating sterile water for cleaning the sample in the last step of disinfection on an unused PDA culture medium, and culturing at a constant temperature of 28 ℃ for 4-7 days, wherein if no thallus grows out, the sample is disinfected completely. And (3) adopting a tissue blotting method, slightly rolling the sterilized sample tissue on an unused PDA culture medium or tightly adhering to the culture medium for 5min, taking away the sample tissue for comparison, and culturing at the constant temperature of 28 ℃ for 4-7 d, wherein the sample tissue is sterilized if no thallus grows out. Each control was repeated 3 times.
4. Purification of endophytic fungi
After tissue materials on a plate culture medium are cultured for 3-5 days, hyphae with good growth of bacterial colonies around the tissues are picked by an inoculating needle, strain purification is carried out on new potato glucose agar (PDA) culture media respectively by adopting a scribing method, the mycelia are placed in a constant temperature incubator upside down, and the mycelia are cultured for 4-7 days at a constant temperature and in a dark place. And repeatedly purifying for 3-4 times to obtain the purified strain. Inoculating the purified strain into slant culture medium, and storing at 4 deg.C.
5. Screening for endophytic fungi
(1) Primary screening by a flat plate: the activated purified strain is inoculated on Potato Dextrose Agar (PDA) culture medium containing NaCl with different concentrations (1%, 3%, 5%, 10%) by adopting a three-point inoculation method, and the strain is inoculated and then cultured for 7 days at constant temperature of 28 ︒ C, and the growth condition is observed. Each group of experiments is performed in parallel for three times, and the growth state of the strain is observed to obtain a target strain (figure 1).
6. DNA extraction and characterization of endophytic fungi
(1) Extraction of total DNA of bacterial strain
The slant culture medium of the target strain to be preserved was transferred to a plate culture medium, and cultured at 28 ︒ C for one week. The extraction of genomic DNA was carried out using OMEGA Fungal genomic Kit (Fungal DNA Kit 50).
(2) PCR amplification of 18S rDNA of strain
The ITS sequences are amplified by using fungus 18S rDNA universal primers ITS1 (5 '-TCCGTAGGTGAACCTGCGG-3') and ITS4(5 '-TCCTCCGCTTATTGATATGC-3') as positive and negative primers.
ITS region amplification selects universal amplification primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3'), ITS4(5 '-TCCTCCGCTTATTGATATGC 3'), PCR conditions are pre-denaturation 94 ︒ C5 min, denaturation 94 ︒ C1 min, annealing 55 ︒ C30 s, extension 72 ︒ C1 min, 35 cycles in total, and finally extension 72 ︒ C10 min.
The PCR amplification reaction adopts 50ul of reaction system comprising ddH2O 40.5µl，PCR Buffer（10х，Mg+plus) 5 μ l, dNTP (2.5mM)1 μ l, ITS1(20 μ M)1 μ l, ITS4(20 μ M)1 μ l, DNA1 μ l, Taq polymerase (5U/μ l)0.5 μ l. The PCR amplification product is detected by 1% agarose gel electrophoresis and then sent to a company for DNA sequencing, and the nucleotide sequence of the PCR amplification product is shown as SEQ ID NO. 1.
(3) Strain 18S rDNA sequence analysis
After obtaining the sequence, a homology search was performed by BLAST at NCBI to find a nucleic acid sequence having a high similarity (99%) to the sequence, and to determine which genus the strain belongs to. The classification of the strains was initially determined and named: charred coal bacterium (C.) (Hypoxylon sp.）。
Example 2
Preparing bacterial liquid: the activated salt-tolerant strain is inoculated into 60mL of potato glucose medium (PDB) liquid medium and is placed on a constant temperature shaking table for culturing for 72h (28 ℃, 160 r.min < -1 >). The concentration of the bacterial liquid was calculated by a blood cell count method to obtain a concentration of 8.75X 105cfu/mL of bacterial liquid.
The test adopts a soil culture pot experiment, the casuarina equisetifolia selected in the test is an annual seedling, the average seedling height is 20cm, the average ground diameter is 3.0mm, and the test is provided by the forest farm of Huian red lake country in Fujian province. Selecting casuarina equisetifolia with consistent growth vigor for planting in a plastic basin with the diameter of 15cm and the height of 10cm in 7, 3 months in 2017. The soil required by the experiment is uniformly mixed and weighed, and equal amount (4 kg) of yellow core soil is put into each pot, and the nutrient content in the soil is shown in table 1. After one month of recovery growth, inoculation is started in 2017, 8 and 3 months, and 100mL of bacterial liquid is applied to the casuarina equisetifolia rhizosphere soil for 3 consecutive days. Each inoculum treatment was repeated 4 times and blanked with distilled water.
TABLE 1 soil nutrient base values
NaCl stress: based on preliminary experiments and related data, 4 salt stress treatments of normal stress CK (0%), mild stress (5%), moderate stress (10%) and severe stress (15%) were designed with NaCl, with 3 replicates of each treatment. Performing NaCl stress test at 18/8/2017, and performing all-herba Ephedrae seedling H at 20d, 40d, and 60d2O2Malondialdehyde (MDA), superoxide dismutase (SOD), Catalase (CAT), and Peroxidase (POD). The results are shown in FIGS. 2 to 6.
When the salt stress concentration is increased, the Y6 strain infects H of plants2O2And the MDA content is obviously increased, the SOD, CAT and POD activities show the trend of increasing firstly and then decreasing, and H of the plant is not infected2O2The content is obviously reduced, the content of Malondialdehyde (MDA) is obviously increased, and the activities of superoxide dismutase (SOD), Catalase (CAT) and Peroxidase (POD) show a trend of increasing firstly and then decreasing. When the stress time is prolonged, Y6 infected and uninfected plants H2O2The activity of SOD, CAT and POD shows the trend of increasing first and then decreasing. In combined comparison, Y6 infested plant H under mild stress (5%) and short term (0 and 20 d) stress2O2Significantly lower than that of the uninfected plant, and the protective enzyme (SOD, CAT, POD) activity is also significantly higher than that of the uninfected plant, indicating that under mild and short-term stress, the infection of endophytic fungi can increase the protective enzyme activity and scavenge more active oxygen, resulting in the active oxygen of the Y6 infected plant being lower than that of the uninfected plant H under moderate and severe (10% and 15%) and long-term (40 d and 60 d) stress, and Y6 infected plant H under moderate and severe (10% and 15%) and long-term (40 d and 60 d)2O2The activity of the protective enzyme is obviously higher than that of the protection enzyme without infection, which shows that although the active oxygen of the plant after Y6 infection is increased under high stress, the activity of the protective enzyme is also obviously increased, so that the endophytic fungi can influence the protective enzyme system of the ephedra brachypoda under NaCl stress and improve the resistance of the ephedra brachypoda.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Fujian agriculture and forestry university
<120> an endophytic fungus Y6 for enhancing oxidation resistance of casuarina equisetifolia in high-salt environment
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<170> PatentIn version 3.3
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<400> 2
tccgtaggtg aacctgcgg 19
<210> 3
<211> 20
<212> DNA
<213> ITS4
<400> 3
Claims (3)
1. An endophytic fungus Y6 for enhancing the oxidation resistance of casuarina equisetifolia in a high-salt environment, which is characterized in that: the classification of the endophytic fungus Y6 is named as Xylaria (C.donax)Hypoxylon sp.) And the strain is preserved in the China general microbiological culture Collection center on 20 th month 11 in 2019, and the preservation number is CGMCC NO. 18814.
2. The use of the endophytic fungus Y6 for enhancing the antioxidant ability of Ephedra sinica Stapf in a high-salt environment as claimed in claim 1 in the cultivation of Ephedra sinica Stapf in a high-salt environment.
3. Use according to claim 2, characterized in that: the endophytic fungus Y6 bacterial liquid is applied to planting of casuarina equisetifolia seedlings in a high-salt environment in a mode of rhizosphere soil pouring or direct seedling inoculation.
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