CN111004727A - Endophytic fungus Z1 for increasing biomass of casuarina equisetifolia in high-salt environment - Google Patents
Endophytic fungus Z1 for increasing biomass of casuarina equisetifolia in high-salt environment Download PDFInfo
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Abstract
The invention discloses an endophytic fungus Z1 for increasing the biomass of casuarina equisetifolia in a high-salt environment, and belongs to the field of microorganisms. The classification of the endophytic fungus Z1 is named as: staphylococus (VitaceaeBotryosphaeria sp.) And is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 11 and 20 months; the strain preservation number is: CGMCC NO. 18817. The bacterial liquid of endophytic fungi Z1 is used for planting casuarina equisetifolia seedlings in a high-salt environment in a mode of rhizosphere soil pouring or direct seedling inoculation, and the biomass of casuarina equisetifolia can be increased. The growth of the casuarina equisetifolia under high salt stress is improved by establishing a casuarina equisetifolia-endophytic fungi symbiotic system, so that the strain information of the endophytic fungi of woody plants is enrichedAnd provides data basis and reference basis for the operation and the manufacture of the biological bacterial manure of casuarina equisetifolia engineering forest.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to an endophytic fungus Z1 for increasing the biomass 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 equisetifoliaL.) 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 certain salt resistance, but the improvement of the salt resistance is beneficial to playing a larger 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 Z1 for increasing the biomass of casuarina equisetifolia in a high-salt environment.
In order to achieve the purpose, the invention adopts the following technical scheme:
an endophytic fungus Z1 capable of promoting the biomass increase of casuarina equisetifolia under a high-salt environment, wherein the classification of the endophytic fungus Z1 is named as: staphylococus (VitaceaeBotryosphaeria 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, the preservation number is CGMCC NO. 18817.
When the endophytic fungus Z1 is cultured on a potato glucose medium (PDA medium), the initial colony is white, the hypha grows in a spindle shape, and the color of the colony becomes dark and gradually becomes dark green to black at the later stage of culture. The endophytic fungus Z1 staphylococcus strain is obtained by separating and purifying seeds of casuarina equisetifolia, can be prepared into bacterial liquid, and is used for planting casuarina equisetifolia 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 endophytic fungus Z1 Staphylococus strain into liquid culture medium, culturing for 72h at constant temperature by using a shaking table, and diluting the obtained culture solution to 5.5 × 10 with sterile water6L-1And (5) obtaining the product.
The formula of the liquid culture medium is as follows: peptone 5.0g, Yeast extract powder 2.0g, glucose (C)6H12O6•H2O) 20.0g, potassium dihydrogen phosphate (KH)2PO4) 1.0g, magnesium sulfate (MgSO)4•7H2O) 0.5g, ultra pure water 1000ml, pH 6.2-6.6.
The invention has the advantages that:
the obtained strain can relieve the restriction of salt stress conditions on the biomass of the strain, and can promote the increase of the biomass of the casuarina equisetifolia plant in a high-salt environment.
Drawings
FIG. 1 is a morphological diagram of hypha and colony of endophytic fungus Z1.
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 Ephedra distachya
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, absolute ethyl alcohol (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 process comprises the following steps: 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. Cutting different tissue materials of the collected casuarina equisetifolia sample with a scalpel, placing the casuarina equisetifolia sample in a sterilized plate culture medium, and culturing for 5-7 days in a 28 ︒ C constant temperature incubator.
(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 strain is inoculated on potato glucose agar (PDA) culture media containing NaCl with different concentrations (1%, 3%, 5% and 10%) by adopting a three-point inoculation method, and the strain is cultured for 7 days at a constant temperature of 28 ︒ C after being inoculated, and the growth condition is observed. Each group of experiments are performed in parallel for three times, the growth state of the strain is observed, and the strain which grows well on a Potato Dextrose Agar (PDA) culture medium containing NaCl is screened to obtain the target strain.
6. DNA extraction and characterization of endophytic fungi
(1) Extraction of total DNA of bacterial strain
The culture was transferred from the slant medium for storing the target strain to the plate 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 ︒ C1min, annealing 55 ︒ C30 s, extension 72 ︒ C1min, 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 was subjected to 1% agarose gel electrophoresis and then submitted to DNA sequencing by the company. Obtaining an ITS sequence: after SEQ ID No.1, a homology search was performed by BLAST at NCBI to find a nucleic acid sequence having a high similarity to the sequence, and to determine which genus the strain belongs to.
(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. Preliminarily determining that the Z1 strain is Protozoa viticola (Botryosphaeria sp.）。
Example 2
Preparing bacterial liquid: inoculating the activated salt-tolerant gluconobacter endophytic fungus Z1 strain into 60mL of potato glucose liquid culture medium, and culturing for 72h (28 ℃, 160 r.min.) on a constant-temperature shaking table-1). Counting the number of spores by a blood counting method, diluting the cultured bacterial liquid into 5.5 × 10 by a tenfold dilution method by sterile water6L-1。
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, 3 replicates each, were designed with NaCl for normal stress (0), mild stress (5%), moderate stress (10%) and severe stress (15%). NaCl stress test was performed at 8 months and 18 days in 2017, and biomass of all casuarina equisetifolia seedlings was measured at the time of stress to 60 days. The results are shown in Table 2.
TABLE 2 Effect of NaCl stress on Biomass of Ephedra sinica Stapf and Ephedra sinica Stapf EI seedlings
As can be seen from table 2, when the salt stress was 0 after 60d stress, the fresh weight of the aerial parts of casuarina equisetifolia infected by the Z1 strain was significantly greater than that of the uninfected plants, and the dry weight of the underground parts of the plants infected by Z1 was also significantly greater than that of the uninfected plants (P < 0.05); under mild stress, the fresh weight and dry weight of the overground and underground parts of the Z1 infected casuarina equisetifolia plant are obviously greater than those of the uninfected plant (P is less than 0.05); under moderate stress, the dry weight of the overground and underground parts of the infected plant Z1 is obviously greater than that of the uninfected plant, and the other treatments have no obvious difference; under severe stress, the fresh weight and dry weight of the overground and underground parts of Z1 infected casuarina equisetifolia plants are obviously greater than those of uninfected plants (P < 0.05). Under NaCl treatment of different concentrations, after the Z1 strain infects plants, the biomass increases along with the increase of the salt stress concentration; while the biomass of uninfected plants fluctuates less. Infection with the Z1 strain can increase the root-crown ratio under a certain stress concentration (10%), thereby increasing the biomass of casuarina equisetifolia plants.
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 Z1 for increasing biomass of casuarina equisetifolia in high-salt environment
<130>3
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<170>PatentIn version 3.3
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Claims (3)
1. An endophytic fungus Z1 for increasing the biomass of casuarina equisetifolia in a high-salt environment, which is characterized in that: the classification of the endophytic fungus Z1 is named as: staphylococus (VitaceaeBotryosphaeria sp.) And is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 11 and 20 months; the strain preservation number is: CGMCC NO. 18817.
2. The use of the endophytic fungus Z1 for increasing the biomass of Ephedra sinica Stapf in a high-salt environment as claimed in claim 1 for increasing the biomass of Ephedra sinica Stapf in a high-salt environment.
3. Use according to claim 2, characterized in that: the bacterial liquid of endophytic fungi Z1 is used for planting casuarina equisetifolia seedlings in a high-salt environment in a mode of rhizosphere soil pouring or direct seedling inoculation.
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| CN114437945A (en) * | 2022-03-01 | 2022-05-06 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Hypocrea vinifera MB1 and application thereof |
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| CN114437945A (en) * | 2022-03-01 | 2022-05-06 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Hypocrea vinifera MB1 and application thereof |
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