CN118222412A - Tobacco endophytic fungus with drought resistance effect and application thereof - Google Patents
Tobacco endophytic fungus with drought resistance effect and application thereof Download PDFInfo
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Abstract
The application relates to a tobacco endophytic fungus with drought resistance and application thereof, belonging to the technical field of tobacco endophytic fungi, wherein each part of a tobacco K326 plant is cleaned, disinfected, separated and purified to obtain the tobacco endophytic fungus with drought resistance, namely Curvularia (Curvularia SPICIFERA S-3), and the tobacco endophytic fungus is prepared to obtain a tobacco endophytic fungus liquid with drought resistance; the tobacco endophytic fungus liquid provided by the application can influence POD, SOD, GSH, CAT and MDA enzyme activity of tobacco seedlings, and the application provides a green and efficient biological control method, and provides a new thought and direction for controlling abiotic stress in tobacco growth and development.
Description
Technical Field
The application belongs to the technical field of tobacco endophytic fungi, and particularly relates to a tobacco endophytic fungus with drought resistance and application thereof.
Background
The tobacco has high requirement on water in the growth period, and the yield and quality of the tobacco can be obviously affected if the tobacco is subjected to drought stress. The water resources are in shortage in China, the water resources are unevenly distributed in time and space, part of tobacco areas in China continuously suffer serious drought in recent years, the frequency and influence range of drought occurrence are enlarged, the duration and the suffered loss are increased, and the tobacco leaf production in China is greatly influenced. The drought problem of tobacco in growing season is solved through irrigation of the water conservancy facilities, and although the drought problem has good effect, the cost is too high, and complete irrigation of all tobacco fields is difficult to realize in a short period.
Plant endophytes are a group of microorganisms that assist their host plants in combating severe environmental stresses, producing secondary metabolites similar to the host plants and inducing the accumulation of plant secondary metabolites, and generally include endophytes, endophytes and endophytes. Currently, over 130 endophytes have been found in a large number of crops, medicinal plants and commercial crops such as fruit trees, covering 60 genera. Common endophytes include Agrobacterium (Agrobacterium), enterobacter (Enterobacter), bacillus (Bacillus), pseudomonas (Pseudomonas), bacillus (Methylobacterium), pantoea (Pantoea) and Agrobacterium (Agrobacterium), most of which are Frankia (Frankia) and most of which are Gramineae, medicinal plants, marine plants, etc., but are less tobacco.
The endophytes live in tobacco bodies for a long time and co-evolve, and studies of Hui Feiqiong and the like show that under drought stress, the inoculation of Pityrosporum indicum can reduce the damage degree of tobacco cell membranes, accumulate more cell permeation regulating substances, and improve the expression of related drought resistance genes in tobacco, so that the drought resistance of the tobacco is enhanced.
Most of the traditional methods adopt irrigation by a hydraulic maintenance facility, but the cost is too high, and the tobacco endophytic fungi are new ways for improving the stress resistance of tobacco leaves and improving the yield and quality of the tobacco leaves, so that the tobacco endophytic fungi with drought resistance are necessary to be provided.
Disclosure of Invention
The application aims to provide tobacco endophytic fungus strain with drought resistance and endophytic fungus liquid.
In order to achieve the above purpose, the application is realized by the following technical scheme:
In a first aspect, the present application provides a tobacco endophytic fungus, which is Curvularia (Curvularia SPICIFERA S-3), and Curvularia spica has drought resistance.
Alternatively, in one implementation of the first aspect, the identification of Curvularia (Curvularia SPICIFERA S-3) is: performing rDNA ITS PCR amplification on tobacco endophytic fungi by using fungus universal primers ITS1/ITS4, sequencing the ITS PCR amplification products, performing BLAST search on the obtained sequences in a GenBank accounting sequence database, finding that the ITS region of the tobacco endophytic fungi is highest in homology with reported regions, and determining that the strain of the tobacco endophytic fungi is Curvularia (Curvularia SPICIFERA S-3) by combining morphological identification; curvularia (Curvularia SPICIFERA S-3) is in the form of dark green colony, velvet, radial expansion, black after aging, and flat surface.
In a second aspect, the application also provides a preparation method of the endophytic fungi of tobacco, comprising the following steps:
Grinding tobacco leaves, cutting tobacco stems, cutting coarse roots, grinding fibrous roots, respectively laying on the surface of a first solid culture medium, and culturing the first solid culture medium in a constant-temperature incubator;
all tobacco endophytic fungi obtained through separation are inoculated into a second solid culture medium for purification, and after 2-3 times of spot grafting purification until colony is single, tobacco endophytic fungi, namely Curvularia (Curvularia SPICIFERA S-3) which has drought resistance effect, are obtained through screening;
The first solid culture medium and the second solid culture medium are prepared from the following components: potato extract powder 5g/L, glucose 20g/L, agar 14g/L and streptomycin 10mg/L.
Alternatively, in one implementation of the second aspect, the incubation temperature of the incubator is 28 ℃ and the incubation time is 5-7 days.
In a third aspect, the application also provides a preparation method of the endophytic fungi liquid of tobacco, comprising the following steps:
inoculating tobacco endophytic fungi into a liquid culture medium, and performing shake culture to obtain tobacco endophytic fungi liquid;
Wherein, the formula of the liquid culture medium is as follows: 5g/L of potato extract powder, 20g/L of glucose and 10mg/L of streptomycin; the shake culture conditions are as follows: the culture temperature is 30 ℃, the rotation speed is 220rpm, and the culture time is 48-72 hours.
In a fourth aspect, the application also provides an application method of the tobacco endophytic fungi in drought resistance of tobacco seedlings, comprising the following steps:
Preparing tobacco endophytic fungi bacterial liquid by using tobacco endophytic fungi;
And in the growth process of the tobacco seedlings, the tobacco seedlings are subjected to watering treatment by using tobacco endophytic fungus liquid so as to improve the drought resistance of the tobacco seedlings.
Optionally, in an implementation manner of the first aspect, the applying treatment to the tobacco seedlings with the endophytic fungus solution of tobacco includes:
When 5-6 true leaves grow out of the tobacco seedling, the tobacco endophytic fungus liquid is used for being poured into the soil of the ground part and root matrix of the tobacco seedling.
Optionally, in an implementation manner of the first aspect, the variety of the tobacco seedling is red flower dajinyuan, cloud tobacco 87, K326 or cloud tobacco 97.
The application has the beneficial effects that:
1. the tobacco endophytic fungus with drought resistance is obtained by separating and purifying tobacco K326 plants, the endophytic fungus is Curvularia spica (Curvularia spicifera), the tobacco endophytic fungus liquid is prepared from the tobacco endophytic fungus, the tobacco endophytic fungus liquid has obvious drought resistance, the activity of enzymes in tobacco seedlings can be influenced, and the influence in the whole growth and development process of tobacco is in a beneficial direction.
2. The application provides a green and efficient biological control method in drought stress resistance of tobacco, and provides a new thought and direction for controlling abiotic stress in growth and development of tobacco.
Drawings
FIG. 1 shows the morphology of Curvularia (Curvularia SPICIFERA S-3);
FIG. 2 is a diagram of Curvularia (Curvularia SPICIFERA S-3) fermentation;
FIG. 3 is a view of a tobacco seedling tray and planting pattern;
FIG. 4 is a comparison of the vigor of tobacco seedlings inoculated with Curvularia (Curvularia SPICIFERA S-3) versus tobacco seedlings not inoculated with Curvularia (Curvularia SPICIFERA S-3);
FIG. 5 shows the relative moisture content of different varieties of seedlings of Curvularia (Curvularia SPICIFERA S-3) after treatment;
FIG. 6 is a graph showing the effect of Curvularia (Curvularia SPICIFERA S-3) on POD enzyme activity of different varieties of seedlings;
FIG. 7 shows the effect of Curvularia (Curvularia SPICIFERA S-3) on SOD enzyme activity of different varieties of tobacco seedlings;
FIG. 8 is a graph showing the effect of Curvularia (Curvularia SPICIFERA S-3) on GSH enzyme activity of different varieties of seedlings;
FIG. 9 is a graph showing the effect of Curvularia (Curvularia SPICIFERA S-3) on CAT enzyme activity of various tobacco seedlings;
FIG. 10 shows the effect of Curvularia (Curvularia SPICIFERA S-3) on MDA enzyme activity of different varieties of seedlings.
Detailed Description
In order to make the objects, technical solutions and advantageous effects of the present application more apparent, preferred embodiments of the present application will be described in detail below to facilitate understanding by the skilled person.
The embodiment of the application provides a tobacco endophytic fungus, which comprises the following components: the tobacco endophytic fungus is Curvularia (Curvularia SPICIFERA S-3) which has drought resistance.
Alternatively, in one specific embodiment, the identification of Curvularia (Curvularia SPICIFERA S-3) is: performing rDNA ITS PCR amplification on tobacco endophytic fungi by using fungus universal primers ITS1/ITS4, sequencing an ITS PCR amplification product, performing BLAST search on the obtained sequence in a GenBank accounting sequence database, finding that the ITS region of the tobacco endophytic fungi is highest in homology with reported products, and determining that the strain of the tobacco endophytic fungi is Curvularia (Curvularia SPICIFERA S-3) by combining morphological identification; the Curvularia (Curvularia SPICIFERA S-3) is in the form of dark green colonies, velvet, radial expansion, black after aging and flat surface.
The technical effects are as follows: the application carries out PCR amplification of rDNA ITS of tobacco endophytic fungi, and sequencing of ITS PCR amplification products, and combines morphological identification to determine that the tobacco endophytic fungi is Curvularia (Curvularia SPICIFERA S-3).
In addition, the embodiment of the application also provides a preparation method of the endophytic fungi of tobacco, which comprises the following steps:
Grinding tobacco leaves, cutting tobacco stems, cutting coarse roots, grinding fibrous roots, respectively laying on the surface of a first solid culture medium, and culturing the first solid culture medium in a constant-temperature incubator;
all tobacco endophytic fungi obtained through separation are inoculated into a second solid culture medium for purification, and after 2-3 times of spot grafting purification until colony is single, tobacco endophytic fungi, namely Curvularia (Curvularia SPICIFERA S-3) which has drought resistance effect, are obtained through screening;
The first solid culture medium and the second solid culture medium are prepared from the following components: potato extract powder 5g/L, glucose 20g/L, agar 14g/L and streptomycin 10mg/L.
Alternatively, in a specific embodiment, the incubation temperature in the incubator is 28℃and the incubation time is 5-7 days.
The technical effects are as follows: the application adopts a solid culture medium formula as follows: potato leaching powder 5g/L, glucose 20g/L, agar 14g/L and streptomycin 10mg/L, and Curvularia spica (Curvularia SPICIFERA S-3) with the effect of promoting tobacco seedling growth can be screened out.
To further illustrate the method of preparing the endophytic fungi of tobacco, the following examples are set forth in more detail as follows:
example 1: isolation and purification of Curvularia (Curvularia SPICIFERA S-3)
1. Test medium
The solid culture medium adopts potato dextrose agar culture medium, and the liquid culture medium adopts potato dextrose broth culture medium. The specific culture medium formula is as follows:
Solid medium: potato extract powder 5g/L, glucose 20g/L, agar 14g/L and streptomycin 10mg/L.
Liquid medium: potato extract powder 5g/L, glucose 20g/L and streptomycin 10mg/L.
2. Isolation and purification of Curvularia (Curvularia SPICIFERA S-3)
Dividing tobacco K326 plants harvested from the field into tobacco leaves, tobacco stems, thick roots and fibrous roots; soaking tobacco leaf part in 75% alcohol for 30s, soaking in 0.1% mercuric chloride for 15s, and washing with sterile water for 3 times; wiping the skin of the stem part of the cigarette by adopting 75% alcohol, and reserving the skin and the middle column; soaking the crude root and fibrous root with 75% alcohol for 60s, soaking with 0.1% mercuric chloride for 20s, washing with sterile water for 3 times, and streaking the sterile water after the last washing on the flat plate as a control to check whether the sample is thoroughly disinfected.
Grinding tobacco leaves, cutting tobacco stems, cutting coarse roots, grinding fibrous roots, respectively laying on the surface of a first solid culture medium, and culturing the first solid culture medium in a constant-temperature incubator; adding sterile water into tobacco leaves and fibrous roots for grinding treatment, and uniformly laying grinding liquid and residues on the surface of a solid culture medium; dividing the tobacco stem part into small blocks with the length of 5mm-8mm by an inoculating knife, dividing the thick root part into small sections with the length of 3mm-5mm by an inoculating knife, and laying the section on the surface of a solid culture medium downwards; all the materials are placed in a constant temperature incubator at 28 ℃ for 5-7 days, and the growth condition of the bacterial colony is observed. As different fungi have different propagation speeds, the strain with faster growth can be separated and purified firstly, and the strain with slower growth is separated and purified after the strain is stabilized.
Purifying: and (3) inoculating all the separated tobacco endophytic fungi into a solid culture medium for purification, performing spot connection purification for 2-3 times until colonies are single, and screening to obtain the functional endophytic fungus strain with drought resistance.
Identification of the tobacco endophytic fungi: PCR amplification of rDNA ITS is carried out on tobacco endophytic fungi by using fungus universal primers ITS1/ITS4, sequencing is carried out on the ITS PCR amplification products, BLAST search is carried out on the obtained sequences in a GenBank accounting sequence database, the highest homology between ITS regions of the endophytic fungi to be tested and reported is found, morphological identification is combined, and the strain of the endophytic fungi strain is determined to be Curvularia (Curvularia SPICIFERA S-3).
As shown in FIG. 1, curvularia (Curvularia SPICIFERA S-3) has the morphology of: the colony is dark green, velvet-shaped, radially expanded, black after aging and flat in surface.
Based on the tobacco endophytic fungi and the preparation method thereof, the tobacco endophytic fungi can be used for preparing tobacco endophytic fungi liquid, and the tobacco endophytic fungi liquid can be used for pouring tobacco and has drought resistance.
Specifically, the embodiment of the application also provides a preparation method of the tobacco endophytic fungi bacterial liquid, which comprises the following steps:
inoculating tobacco endophytic fungi into a liquid culture medium, and performing shake culture to obtain tobacco endophytic fungi liquid;
Wherein, the formula of the liquid culture medium is as follows: 5g/L of potato extract powder, 20g/L of glucose and 10mg/L of streptomycin; the shake culture conditions are as follows: the culture temperature is 30 ℃, the rotation speed is 220rpm, and the culture time is 48-72 hours.
The technical effects are as follows: the tobacco endophytic fungus liquid is prepared by adopting a formula of a liquid culture medium, namely 5g/L of potato leaching powder, 20g/L of glucose and 10mg/L of streptomycin, and has the culture temperature of 30 ℃, the rotation speed of 220rpm and the culture time of 48-72 hours, and is used for watering tobacco seedlings and has drought resistance effect on the tobacco seedlings.
Likewise, the embodiment of the application also provides an application method of the tobacco endophytic fungi in drought resistance of tobacco seedlings, which comprises the following steps:
Preparing tobacco endophytic fungi bacterial liquid by using tobacco endophytic fungi;
And in the growth process of the tobacco seedlings, the tobacco seedlings are subjected to watering treatment by using tobacco endophytic fungus liquid so as to improve the drought resistance of the tobacco seedlings.
Optionally, in a specific embodiment, the tobacco seedling is irrigated with a tobacco endophytic fungus liquid, and when 5-6 true leaves grow out of the tobacco seedling, the tobacco endophytic fungus liquid is irrigated into the soil of the ground part and root matrix of the tobacco seedling.
Alternatively, in a specific embodiment, the tobacco seedling is of the variety Honghua Dajinyuan, yunyan 87, K326 or Yunyan 97.
The technical effects are as follows: when 5-6 true leaves grow out of tobacco seedlings, the tobacco endophytic fungus liquid is applied to the ground part and root matrix soil of the safflower macrogold, the cloud tobacco 87 and the cloud tobacco 326 or the cloud tobacco 97, and the tobacco endophytic fungus liquid can remarkably improve the water content of the safflower macrogold, the cloud tobacco 87 and the cloud tobacco 326 and the cloud tobacco 97, and particularly can remarkably improve the water content of the safflower macrogold; can also influence the enzyme activities of POD, SOD, GSH, CAT and MDA of Honghuadajinyuan, yunyan 87, K326 and Yunyan 97.
To further illustrate the drought-resistant effect of endophytic fungi in tobacco, a more detailed example is set forth below:
Example 2: drought-resistant effect of Curvularia (Curvularia SPICIFERA S-3) on tobacco seedlings of different varieties
1. Experimental method
Preparing a tobacco endophytic fungus liquid: inoculating fungus blocks with the size of about 1.5cm 2 to 100ml of liquid culture medium, filling the fungus blocks into a 250ml conical flask, sealing a bottle mouth, and shake culturing by a shaking table to obtain plant endophytic fungus liquid.
Wherein, the formula of the liquid culture medium is as follows: 5g/L of potato extract powder, 20g/L of glucose and 10mg/L of streptomycin; the shake culture conditions are as follows: the culture temperature is 30 ℃, the rotation speed is 220rpm, and the culture time is 48-72 hours. As shown in fig. 2.
Seedling raising: selecting four different varieties of tobacco coated seeds of safflower Dajinyuan, yunyan 87, K326 and Yunyan 97, sowing on a 3.4 small seedling tray, as shown in figure 3, sowing 3 seeds per bunch, sterilizing nutrient soil for seedling culture by high pressure, culturing alternately in an incubator in the dark under illumination for 16 hours under illumination, wherein the light intensity is 18000LX, and the temperature is 28 ℃; culturing in dark for 8h at 16 ℃; nutrient solution is added into the bottom of the seedling raising tray, and the nutrient solution needs to be supplemented every two days.
Application of tobacco endophytic fungus liquid: when the tobacco seedlings grow to 5-6 true leaves, the tobacco endophytic fungus liquid is taken to carry out watering treatment on the overground part and root soil of the tobacco seedlings, one tobacco seedling is reserved in each hole of a seedling raising tray before the watering treatment, the growth vigor of each tray of tobacco seedlings is ensured to be consistent, 3ml of endophytic fungus liquid is taken for each hole to be watered on the tobacco seedlings, and the tobacco endophytic fungus liquid is uniformly shaken before the taking; after watering, the upper part of the seedling raising tray is covered with a transparent isolation cover, and after three days, the transparent isolation cover is taken down for continuous culture in the incubator, namely, culture solution and water are not added in the whole process; and (3) setting a control group tobacco seedling (CK), wherein the control tobacco seedling is only poured with a solution in a liquid culture medium with the same volume, and is not poured with tobacco endophytic fungus bacterial liquid. And when the tobacco seedlings grow to 7 true leaves, comparing the growth vigor of the tobacco seedlings and measuring the relative water content.
The specific method for measuring the relative water content comprises the following steps: two 1g (W f) portions were weighed, one portion was baked in an oven at 70℃for 48h, and the dry weight (W d) was determined; soaking the other part in distilled water for 70min, wiping the leaf with absorbent paper when the weight reaches constant, measuring the weight (W t) at the moment, and measuring the relative water content of the tobacco leaf:
2. analysis of results
(1) Comparison of tobacco seedlings grown with and without tobacco endophytic fungus solution
As can be seen from FIG. 4, the left side of FIG. 4 is a control group tobacco seedling (CK), and the right side of FIG. 4 is a tobacco seedling poured with tobacco endophytic fungus liquid, the tobacco seedling poured with tobacco endophytic fungus liquid has good growth vigor, and most tobacco seedlings not poured with tobacco endophytic fungus liquid wither, which shows that the tobacco endophytic fungus liquid has good drought resistance.
(2) Testing the moisture content of tobacco seedlings of different varieties
As shown in fig. 5, the relative water content of tobacco K326 treated with curvularia spica was 15.12% higher than that of the control; the cloud tobacco 97 is 15.54% higher than the control after being treated by Curvularia spica; k326 is 14.63% higher after treatment; the treated golden element of safflower is 25.73 percent higher than the control after being treated by curvularia spica; wherein the relative water content of the safflower Dajinyuan is increased most.
Example 3: effect of Curvularia (Curvularia SPICIFERA S-3) on the enzymatic Activity of different tobacco varieties POD, SOD, GSH, CAT and MDA
FIG. 6 shows the effect of Curvularia (Curvularia SPICIFERA S-3) on the POD enzyme activity of different varieties of tobacco seedlings, as shown in FIG. 6, POD is an antidote for H 2O2, and can effectively control the accumulation of active oxygen in plant cells. The first row from the left in the different tobacco varieties in FIG. 6 is a control group, the second row is Curvularia (Curvularia SPICIFERA S-3), and the other rows are other strains except Curvularia (Curvularia SPICIFERA S-3), and as can be seen from FIG. 6, curvularia (Curvularia SPICIFERA S-3) can affect POD enzyme activity; the POD concentration activities of four kinds of tobacco treated by the curvularia aurea are improved, namely, the tobacco treated by the curvularia aurea are improved, and the POD concentration of the safflower brotica is higher than that of other tobacco varieties under the action of the curvularia aurea; the POD concentration of the safflower Dajinyuan under the action of Curvularia spica is higher than that of other strains.
FIG. 7 shows the effect of Curvularia (Curvularia SPICIFERA S-3) on the SOD enzyme activity of tobacco seedlings of different varieties, wherein under drought stress conditions, SOD is the first key enzyme for removing active oxygen in the tobacco in the whole defending system of resisting oxidative stress, the first column from left in different tobacco varieties in FIG. 7 is a control group, the second column is Curvularia (Curvularia SPICIFERA S-3), and other columns are other strains except Curvularia (Curvularia SPICIFERA S-3), and the effect of Curvularia (Curvularia SPICIFERA S-3) on POD enzyme activity can be seen from FIG. 7; curvularia spica can improve the SOD activity of four kinds of tobacco, namely Yunyan 87, yunyan 97, K326 and safflower Dajinyuan, can obviously improve the SOD concentration of the Yunyan 87, and the SOD concentration of the safflower Dajinyuan under the action of the Curvularia spica is higher than that of other kinds of four kinds of tobacco.
FIG. 8 shows the effect of Curvularia (Curvularia SPICIFERA S-3) on GSH enzyme activity of different tobacco seedlings, and as shown in FIG. 8, the increase of GSH content can participate in antioxidation on one hand and enhance stress resistance of plants; on the other hand, the active oxygen radicals in plant cells can be induced and eliminated through non-enzymatic reaction together with other antioxidants, the first row from left in different tobacco varieties in fig. 8 is used as a control group, the second row is used as Curvularia (Curvularia SPICIFERA S-3), and other rows are used as other strains besides Curvularia (Curvularia SPICIFERA S-3), and as can be seen from fig. 8, curvularia (Curvularia SPICIFERA S-3) can influence the POD enzyme activity; curvularia spica can increase GSH concentration of four kinds of tobacco including Yunyan 87, yunyan 97, K326 and safflower Dajinyuan, wherein Curvularia spica can significantly increase GSH concentration of Yunyan 97 and safflower Dajinyuan.
FIG. 9 shows the effect of Curvularia (Curvularia SPICIFERA S-3) on CAT enzyme activity of tobacco seedlings of different varieties, wherein the effect of CAT is similar to that of POD, and can reduce the accumulation of active oxygen, the first column from left in different tobacco varieties in FIG. 9 is a control group, the second column is Curvularia (Curvularia SPICIFERA S-3), and the other columns are other strains except Curvularia (Curvularia SPICIFERA S-3), and as can be seen from FIG. 9, curvularia SPICIFERA S-3 can have an effect on POD enzyme activity; CAT concentration of four kinds of tobacco treated by Curvularia spica is improved, namely, the CAT concentration of the four kinds of tobacco is improved.
FIG. 10 shows the effect of Curvularia (Curvularia SPICIFERA S-3) on MDA enzyme activity of tobacco seedlings of different varieties, wherein MDA is one of final products of peroxidation reaction of tobacco when the tobacco is subjected to drought stress, has great cytotoxicity on tobacco leaves, can cause dysfunction of tobacco plant cells, can damage nucleic acid, protein, enzyme and the like in the cells to a certain extent, the first column from left in different tobacco varieties in FIG. 10 is a control group, the second column is Curvularia (Curvularia SPICIFERA S-3), and the other columns are other strains except Curvularia (Curvularia SPICIFERA S-3), and the effect of Curvularia (Curvularia SPICIFERA S-3) on POD enzyme activity can be seen in FIG. 10; the MDA concentration of four kinds of tobacco of the cloud tobacco 87, the cloud tobacco 97, the K326 and the safflower Dajinyuan which are treated by the curvularia spica is obviously reduced, wherein the curvularia spica can obviously reduce the MDA concentration of the cloud tobacco 87, and the effect on the other three tobacco varieties is not obvious; curvularia spica can significantly reduce MDA concentration of cloud 87.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the application, and that, although the application has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the application as defined by the appended claims.
Claims (8)
1. A tobacco endophytic fungus, which is Curvularia (Curvularia SPICIFERA S-3) and has drought resistance.
2. The endophytic fungus of claim 1, wherein the identification of Curvularia (Curvularia SPICIFERA S-3) is: performing rDNA ITS PCR amplification on tobacco endophytic fungi by using fungus universal primers ITS1/ITS4, sequencing an ITS PCR amplification product, performing BLAST search on the obtained sequence in a GenBank accounting sequence database, finding that the ITS region of the tobacco endophytic fungi is highest in homology with reported products, and determining that the strain of the tobacco endophytic fungi is Curvularia (Curvularia SPICIFERA S-3) by combining morphological identification; the Curvularia (Curvularia SPICIFERA S-3) is in the form of dark green colonies, velvet, radial expansion, black after aging and flat surface.
3. A method for preparing endophytic fungi in tobacco, which is characterized by comprising the following steps:
Grinding tobacco leaves, cutting tobacco stems, cutting coarse roots, grinding fibrous roots, respectively laying on the surface of a first solid culture medium, and culturing the first solid culture medium in a constant-temperature incubator;
All tobacco endophytic fungi obtained through separation are inoculated into a second solid culture medium for purification, and after 2-3 times of spot grafting purification until colony is single, tobacco endophytic fungi, namely Curvularia (Curvularia SPICIFERA S-3) which has drought resistance effect, are obtained through screening;
the formulas of the first solid culture medium and the second solid culture medium are as follows: potato extract powder 5g/L, glucose 20g/L, agar 14g/L and streptomycin 10mg/L.
4. A tobacco endophytic fungus as claimed in claim 3, wherein the incubator is maintained at a temperature of 28 ℃ for a period of 5-7 days.
5. The preparation method of the endophytic fungus liquid of tobacco is characterized by comprising the following steps:
inoculating tobacco endophyte into a liquid culture medium, and performing shake cultivation to obtain tobacco endophyte bacterial liquid, wherein the tobacco endophyte is as described in claim 1 or 2;
Wherein, the formula of the liquid culture medium is as follows: 5g/L of potato extract powder, 20g/L of glucose and 10mg/L of streptomycin; the shake culture conditions are as follows: the culture temperature is 30 ℃, the rotation speed is 220rpm, and the culture time is 48-72 hours.
6. An application method of endophytic fungi in drought resistance of tobacco seedlings is characterized by comprising the following steps:
Preparing a tobacco endophyte bacterial liquid by using the tobacco endophyte, wherein the tobacco endophyte is as claimed in claim 1 or 2, and the tobacco endophyte bacterial liquid is prepared by the preparation method as claimed in claim 5;
And in the growth process of the tobacco seedlings, the tobacco endophytic fungus liquid is used for carrying out watering treatment on the tobacco seedlings so as to improve the drought resistance of the tobacco seedlings.
7. The application method according to claim 6, wherein the tobacco seedling is subjected to the watering treatment by using the endophytic fungi liquid of tobacco, comprising:
When 5-6 true leaves grow out of the tobacco seedling, the tobacco endophytic fungus liquid is used for being poured into the soil of the ground part and root matrix of the tobacco seedling.
8. The method according to any one of claims 6 or 7, wherein the tobacco seedling is of the variety red flower hoku yuan, yuanyan 87, K326 or yuanyan 97.
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