CN113940360A

CN113940360A - Application of sodium selenite in resisting phytophthora nicotianae

Info

Publication number: CN113940360A
Application number: CN202111201230.0A
Authority: CN
Inventors: 贾玮; 马晓寒; 张莉; 许嘉阳; 许自成
Original assignee: Henan Agricultural University; China Tobacco Jiangsu Industrial Co Ltd
Current assignee: Henan Agricultural University; China Tobacco Jiangsu Industrial Co Ltd
Priority date: 2021-10-15
Filing date: 2021-10-15
Publication date: 2022-01-18
Anticipated expiration: 2041-10-15
Also published as: CN113940360B

Abstract

The invention discloses application of sodium selenite in resisting phytophthora nicotianae, and belongs to the technical field of pesticides. According to the invention, sodium selenite can inhibit the growth of phytophthora nicotianae, the growth and development of tobacco seedlings can be improved and the invasion of phytophthora nicotianae can be resisted by sodium selenite treatment, the sodium selenite can be used for preparing preparations for resisting phytophthora nicotianae and preventing and treating tobacco black shank, and the black shank resistance of tobacco can be improved by applying the preparation containing exogenous selenium to tobacco. The invention can simultaneously promote the growth and development of tobacco plants and improve the black shank resistance of tobacco, and has great significance for preventing and controlling the black shank of the tobacco.

Description

Application of sodium selenite in resisting phytophthora nicotianae

Technical Field

The invention belongs to the technical field of pesticides, and particularly relates to application of sodium selenite in resisting phytophthora nicotianae.

Background

The tobacco black shank is also called black stalk crazy, aconite disease and the like, is a relatively serious soil-borne fungal disease caused by Phytophthora nicotianae, and due to the reasons of wide distribution of the tobacco black shank, long field latency and the like, tobacco plants are damaged by germs and then have the phenomena of withered and yellow leaves, rotting leaves, browning stems and even plant death, and the like.

Selenium (Se) is a necessary nutrient element for human beings and animals, is also a beneficial nutrient element for plants, and plays an important role in the aspects of human body antioxidant function, thyroid hormone metabolism, immune response, plant antioxidant system defense and the like. Although the essential role of selenium in higher plants is not clear, it has been found that all plants can absorb and accumulate selenium. Selenium is used As a beneficial element of plants and plays a positive role in the growth and development of higher plants, such As promoting growth, changing root system structures, repairing cell structures, improving quality and yield, and the selenium is proved to play a role in plant stress defense reaction and can relieve abiotic stress of drought, salinity and heavy metals (such As Cd, Pb, As and the like). Selenium is also researched on plant diseases and insect pests, wherein researches show that the growth of botrytis cinerea can be effectively prevented and inhibited by synthesizing mesoporous nano selenium (SenPs) with high loading efficiency, and obvious toxic and side effects on plants are not generated by multiple applications. In addition, exogenous selenium at a suitable concentration can inhibit the growth of sclerotinia by disrupting cellular structure.

At present, the main prevention and treatment means for the tobacco black shank comprise breeding disease-resistant varieties, chemical pesticide antibacterial agents, biological prevention and treatment and the like. However, because the screening degree of disease-resistant varieties is difficult at present, environmental pollution is easily caused by using chemical bactericides and the like, a new and environment-friendly prevention and treatment means needs to be found.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide the application of sodium selenite in resisting phytophthora nicotianae and preventing and treating tobacco black shank. The invention also aims to provide application of the sodium selenite in preparing a phytophthora nicotianae resistant preparation or a preparation for preventing and treating tobacco black shank. Still another object of the present invention is to provide a method for improving the black shank resistance of tobacco.

In the invention, in the first aspect, the culture dish in-vitro experiment proves that sodium selenite with different concentrations has different inhibiting effects on the growth of phytophthora nicotianae hyphae. When the concentration of the exogenous sodium selenite is less than 2mg/L, the inhibition effect on the growth of phytophthora nicotianae hyphae is small. When the concentration of the exogenous sodium selenite is more than 2mg/L, the exogenous sodium selenite has a remarkable inhibition effect on the growth of phytophthora nicotianae hyphae. When the concentration of the exogenous sodium selenite is 8mg/L, the inhibition effect on the growth of phytophthora nicotianae hyphae is maximum and higher than that of other concentrations.

In the invention, in the second aspect, through a pot experiment, the exogenous sodium selenite treatment is proved to be capable of improving the growth and development of the tobacco seedlings and the capability of resisting the invasion of phytophthora nicotianae, and meanwhile, different promotion effects on the growth of the tobacco seedlings under different concentrations of sodium selenite are proved. The concentration of the exogenous sodium selenite has the phenomenon of low promotion and high inhibition in the process of promoting the growth of tobacco plants, when the concentration of the exogenous sodium selenite is less than 8mg/L, the agronomic characters of the tobacco show the trend of gradually rising along with the rising of the concentration of the exogenous sodium selenite, and when the concentration of the exogenous sodium selenite is 8mg/L, the agronomic characters and physiological and biochemical indexes show the best; and when the concentration is 10mg/L, the agronomic characters and physiological and biochemical indexes of the tobacco seedlings are second to those of the sodium selenite with the concentration of 6-8 mg/L when the tobacco seedlings are treated.

Specifically, the invention provides the following technical scheme:

the application of exogenous selenium in inhibiting the growth of phytophthora nicotianae hyphae is provided.

The application of exogenous selenium in preventing and treating tobacco black shank is provided.

Application of exogenous selenium in preparation of preparation for resisting phytophthora nicotianae is provided.

The application of exogenous selenium in the preparation of a preparation for preventing and treating tobacco black shank is provided.

A method of improving the black shank resistance of tobacco comprising the step of applying to the tobacco a formulation comprising exogenous selenium.

In some embodiments, the exogenous selenium is tetravalent selenium (Se)⁴⁺) (ii) a Further, the exogenous selenium is sodium selenite.

In some embodiments, the formulation is a fertilizer, an additive, or a growth regulator.

In some embodiments, when the exogenous selenium is sodium selenite, the above-mentioned use or method uses sodium selenite at a concentration of 8 mg/L.

The invention has the following advantages and beneficial effects:

(1) the exogenous sodium selenite can inhibit the growth rate of phytophthora nicotianae hyphae, can increase the MDA content of the hyphae, increase the relative conductivity, slow down the pH value, can destroy the growth and development of the hyphae, and can ensure that the hyphae are shriveled and become thin and the density is obviously reduced.

(2) The exogenous sodium selenite treatment can promote the growth and development of tobacco seedlings, and improve the contents of plant hormones, MAPK kinase and other physiological and biochemical substances of tobacco plants, so that the self resistance of the tobacco plants is enhanced to cope with the invasion of biological stress.

(3) The relative conductivity of the tobacco plant which is treated by the exogenous sodium selenite and inoculated with the tobacco black shank bacteria is obviously lower than that of the tobacco plant which is not treated by the selenium-free inoculated bacteria, and the dying condition of the plant cells is judged by the trypan blue staining depth, so that the tobacco plant which is not treated by the selenium-free inoculated bacteria is deepest in color, the color depth is gradually reduced along with the increase of the exogenous sodium selenite concentration, and the color is shallowest when the exogenous sodium selenite concentration is 8mg/L, which indicates that the cell death degree is lowest.

(4) The invention can simultaneously promote the growth and development of tobacco plants and improve the black shank resistance of tobacco, and has great significance for preventing and controlling the black shank of the tobacco.

Drawings

FIG. 1 shows the bacteriostatic rate of tobacco black shank hyphae treated by different selenium sources.

FIG. 2 is the bacteriostatic rate of exogenous selenium treatment for four different species.

FIG. 3 is a graph of the effect of exogenous selenium treatment on the pH of tobacco black shank hyphae.

FIG. 4 is a graph of the effect of exogenous selenium treatment on the relative conductivity of tobacco black shank hyphae.

FIG. 5 is a graph of the effect of exogenous selenium treatment on the morphology of tobacco black shank hyphae.

FIG. 6 is a graph of the effect of exogenous selenium treatment on disease indices of different varieties of tobacco.

Figure 7 is a graph of the effect of selenium treatment on the relative conductivity of two varieties of tobacco.

FIG. 8 is a graph of the effect of selenium treatment on two varieties of tobacco Gibberellin (GA).

FIG. 9 is a graph of the effect of selenium treatment on abscisic acid (ABA) of two varieties of tobacco.

FIG. 10 is a graph of the effect of selenium treatment on two varieties of tobacco Jasmonic Acid (JA).

FIG. 11 is a graph of the effect of selenium treatment on two varieties of tobacco Salicylic Acid (SA).

FIG. 12 is a graph of the effect of selenium treatment on two varieties of tobacco auxin (IAA).

FIG. 13 is a graph of the effect of selenium treatment on MAPK content in two varieties of tobacco.

FIG. 14 is a trypan blue stain of selenium treated two varieties of tobacco, ABCD being PCK, PSe, ZPCK, ZPSE, and EFGH being the trypan blue stained and decolorized state corresponding to ABCD.

Detailed Description

In order to make the technical problems, technical solutions and advantages of the present invention more clear, the following embodiments are used for further detailed description, and are not to be construed as limiting the present invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. It will be apparent to those skilled in the art from this disclosure that many changes can be made in the specific embodiments disclosed herein, which will still achieve the same or similar results without departing from the spirit or scope of the invention.

Unless otherwise defined, technical means and scientific terms used in the examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and conventional means well known to those of ordinary skill in the art, the disclosures of which are incorporated herein by reference, and the materials to which they are incorporated. The experimental procedures in the following examples are conventional unless otherwise specified. The instruments used in the following examples are, unless otherwise specified, laboratory-standard instruments; the test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Example (b):

1. material

The tobacco test varieties are K326, Zhongyan 100, Honghuadajinyuan and Changjinhuang, which are widely planted in China. Phytophthora nicotianae (Phytophthora nicotianae), Alternaria alternata (Fries Keisslar), Fusarium solani (Fusarium solani), Nicotiana tabacum root rot (Thielaviopsis basicola) were provided by the Nicotiana tabacum research institute of agricultural sciences in Henan province. Sodium selenite and sodium selenate solid powder are purchased from Zhengzhou Akhm chemical Co., Ltd, and the content of the analytically pure reagent is 99%.

2. Cultivation method

The test site is a tobacco quality ecological laboratory of tobacco institute of agriculture university in Henan, sodium selenite and sodium selenate are used as selenium sources, water is used for preparing 0, 2, 4, 6, 8 and 10mg/L sodium selenite and sodium selenate solutions respectively for use, a soil culture method is adopted for seedling culture, a seedling culture disc and I-shaped lattice sponge are used as media in the early stage of the test, tobacco seeds are disinfected by 10% hydrogen peroxide and then are cleaned by distilled water, the tobacco seeds are uniformly placed in the sponge after being soaked for 1d under the condition of room temperature and light shielding, and seedlings with consistent growth are selected and transferred to vermiculite for growth by using tweezers after the tobacco seeds are exposed to the white and germinate. When the tobacco seedlings grow to the four-leaf stage, the tobacco seedlings are moved from the sponges to a soil culture box for growing. The whole culture period is carried out in an artificial incubator, and the environment of the incubator is maintained as follows: day and night at 28 + -2 deg.C/18 + -2 deg.C, light cycle of 14/10h, relative humidity of 70%, and illumination intensity of 440 μmol · m^-2·s^-1. Tobacco seedlings are treated when growing to six leaves and one core in a soil culture box. Selecting the seedlings with good growth and uniform growth, and adopting the external partTobacco seedlings are treated in a spraying mode, and the method specifically comprises the following steps: spraying 0, 2, 4, 6, 8, 10mg/L sodium selenite (about 5mL per plant) on the overground part of the tobacco seedling, treating 1 time every 1d, and continuing for 3 d. In the initial stage of the experiment (K326, Zhongyan 100, honghuadajinyuan and Changqiu yellow are the subjects to be tested, and the disease index is tested): control (CK) was normal growth, pathogen infestation (PCK) was inoculation with phytophthora nicotianae, and exogenous selenium treatments at different concentrations were T0, T2, T4, T6, T8, T10. In the further experiments (K326, zhongyan 100), the K326 variety: the Contrast (CK) is normal growth, the pathogen infection (PCK) is inoculated phytophthora nicotianae, the exogenous selenium treatment is Se, and the pathogen infection after the selenium treatment is PSe; the corresponding treatment of 100 varieties of cigarettes comprises the following steps: ZCK, ZPCK, ZSE, and ZPSE.

The hypha inoculation method is as follows: after the hyphae were overgrown in a solid culture dish, an equal number of fungus cakes (diameter 4mm) of the same radius were obliquely pricked into the base of the plant stem by means of an inoculating needle and fixed. In the trypan blue staining in vitro, the same tobacco leaves are taken and placed in plastic trays, moist gauze is arranged under the leaves to keep moisture, 3 leaves are placed in each tray, activated phytophthora nicotianae cakes (diameter is 4mm) are inoculated on the leaves, two leaves are inoculated on each leaf while avoiding the main vein, and the leaves are sealed by preservative films and then placed in an incubator at 28 ℃.

Setting each treatment for 3 times of repetition, repeating 4 tobacco seedlings each time, and taking a third true leaf to be tested in the germ infection treatment 4 d.

The hypha treatment was as follows: adding sodium selenite solutions with different concentrations into the solid culture medium, and adding phytophthora nicotianae cakes to complete the propagation of hyphae after the solid culture medium is solidified. The hyphae are set to be treated for 6 times according to different exogenous sodium selenite concentrations, which are respectively as follows: se0, Se2, Se4, Se6, Se8 and Se 10.

3. Measurement items and methods

3.1 measurement of hyphal growth inhibition Rate

The preparation method of the OA solid medium comprises the following steps: boiling 30g of oat in 1.5L of pure water, filtering warp cloth, adding 15g of agar into the solution, respectively pouring into 6 triangular flasks of 250mL, adding selenium solution according to test requirements, preparing 6 culture mediums into selenium-containing culture mediums of 0, 2, 4, 6, 8 and 10mg/L, uniformly mixing, sterilizing by a sterilizing pot, operating in a super clean bench, adding 10mL of each treatment OA culture medium with the temperature of about 40 ℃ into a culture dish of 90mm in diameter, and placing an activated phytophthora nicotianae cake (the diameter of 4mm) at the center of the OA culture medium after the culture mediums are solidified. The petri dish was sealed with a sealing film and placed upside down in an incubator at 20 ℃. After 6d, the hypha diameter was measured, and the hypha growth inhibition ratio was (control group-experimental group)/control group × 100%.

3.2 physiological and Biochemical assays

The mycelium is obtained by culturing in OA liquid culture medium without agar and under other conditions, placing 3 activated Phytophthora nicotianae cakes in each triangular flask, culturing for 6 days at 160rpm and 28 deg.C in a shaking table, filtering to obtain mycelium, and measuring relative conductivity, pH value and MDA content of 100 mg. Measuring at 10, 20, 30, 60, 90, 120, 180, 240 and 300min by using a pH meter, measuring at 10, 20, 30, 60, 90, 120, 180 and 240min by using a conductivity meter, and measuring the content of Malondialdehyde (MDA) by using a thiobarbituric acid method.

And when the infected tobacco plant has an obvious phenotype, taking the same leaf position to measure the contents of hormones such as gibberellin, abscisic acid, jasmonic acid, salicylic acid, auxin and the like and MAPK kinase, and measuring according to the kit instruction. The disease index is calculated according to the method of Zhang Cheng province and the like (Zhang Cheng province, Korea, Liu dynasty, and the like. the tobacco disease prevention and growth promotion effect of the volatile substance of the bacillus subtilis Tpb55 [ J ]. Chinese biological control, 2009,25(3): 245-.

3.3 scanning Electron microscope and Trypan blue staining Observation

Scanning electron microscopy photographs were taken on a scanning electron microscope (FEI QUANTA-200, the Netherlands) using a method such as Zhang S (Zhang S, Liu S, Zhang J, et al. synthetic anti-ocular effect of melatonin with a biological antibiotic against an organic granular substrate disease [ J ]. Journal of Pineal Research,2018: e 12492).

Trypan blue staining was tested using the method of tilling Li et al (Negative alignment of plant immunological derivative from cellulose alcohols products area target by multiple Phytophthora Avr3a-like effects [ J ]. New Phytologist, 2019.).

4. Measurement results

4.1 influence of exogenous selenium treatment with different concentrations and valence states on the inhibition rate of phytophthora nicotianae hyphae

The inhibition effect of selenium treatment with different concentrations and valence states on the growth of phytophthora nicotianae is measured in an in vitro contact experiment. The culture medium is Oat (OA) culture medium, and the test plant pathogenic fungus is Phytophthora nicotianae (Phytophthora nicotiana institute of agricultural science, Henan province). Exogenous sodium selenate and sodium selenite were added to the OA medium at 6 concentrations, such as 0, 2, 4, 6, 8, 10mg/L, to form different treatments. 10mL of OA medium (about 40 ℃ C.) treated differently was placed in a 90mm diameter dish, and after the medium solidified, the activated P.nicotianae cake (4 mm diameter) was placed in the center of the OA medium. The petri dish was sealed with a sealing film and placed upside down in a dark incubator at 28 ℃.

The diameter was measured by the cross method after 6 days of hypha growth, each treatment was repeated 3 times, and the growth inhibition rate of hypha was calculated, and the results are shown in FIG. 1, and the inhibition rates of sodium selenite at concentrations of 2, 4, 6, 8, and 10mg/L for Phytophthora nicotianae were: 19.22%, 38.24%, 44.12%, 55.88% and 50.56%. And the inhibition rates of sodium selenate with the concentrations of 2, 4, 6, 8 and 10mg/L on phytophthora nicotianae are respectively only as follows: 3.73%, 6.08%, 8.43%, 12.55% and 17.25%. It can be found that selenium treatment with different concentrations and valence states can inhibit the growth of hyphae, the inhibition rate of phytophthora nicotianae is increased along with the increase of the concentration, but the effect of sodium selenate is not obvious relative to sodium selenite, the inhibition effect of the whole sodium selenite is high, and the inhibition effect of the sodium selenate on the growth of phytophthora nicotianae hyphae is small when the concentration is lower than 2 mg/L. The sodium selenite has high inhibition rate in 8mg/L and 10mg/L and small difference, shows good effect of inhibiting hypha growth in the whole treatment, and is subsequently treated by taking the sodium selenite as a selenium source.

4.2 Effect of exogenous selenium treatment of different concentrations on inhibition rates of different pathogen hyphae

In order to measure the inhibition experiment of exogenous selenium treatment on other pathogenic bacteria, the invention adopts four different tobacco pathogenic bacteria to carry out parallel experiments, wherein exogenous selenium adopts sodium selenite as a selenium source, the test plant pathogenic fungi are Phytophthora nicotianae (Phytophthora nicotianae), Alternaria tabacum (Fries Keisslar), Fusarium solani (Fusarium solani) and moniliforme rhizogenes (Thielaviopsis basicola), and the culture mediums of the four different strains are respectively an OA oat culture medium, a PDA potato culture medium and a V8 culture medium.

Sodium selenite was added to the corresponding medium at 6 concentrations set at 0, 2, 4, 6, 8, 10mg/L, to form different treatments. 10mL of a culture medium having a different treatment temperature of about 40 ℃ was placed in a 90 mm-diameter dish, and after the culture medium solidified, each activated strain cake (diameter 4mm) was placed at the center of the culture medium. The culture dish is sealed by a sealing film and is placed in an incubator upside down for culture.

The diameter is measured by adopting a cross method after the hyphae grow for 6 days, each treatment is repeated for 3 times, and the growth inhibition rate of the hyphae is calculated, the result is shown in figure 2, different strains show higher inhibition rates along with the increase of the exogenous sodium selenite concentration, and the difference among the different strains is obvious, wherein the inhibition rate of the sodium selenite to the phytophthora nicotianae is the highest, the inhibition rate is 55.79% at the concentration of 8mg/L, and the inhibition rates are 21.94% and 19.92% at the concentration of 10mg/L respectively for the fusarium solani and the alternaria nicotianae. The lowest inhibition rate is the tobacco-based rhizobium rhizogenes, and the inhibition rate is 15.19% at the concentration of 10 mg/L. The exogenous sodium selenite has different inhibition effects on different tobacco diseases and has the best inhibition effect on phytophthora nicotianae.

4.3 Effect of exogenous selenium treatment at different concentrations on the pH of Phytophthora nicotianae mycelia

The method for culturing phytophthora nicotianae mycelia adopts a liquid culture medium, namely a method for adding no agar in an original OA solid culture medium, and other conditions are the same as 4.1. As shown in FIG. 3, it was found that the tendency of pH content change in 6 treatments decreased with time in a dose-and time-dependent manner as shown in FIG. 3. After 90-120 min, the pH values of 6, 8 and 10mg/L concentrations are obviously higher than those of other treatments.

4.4 Effect of exogenous selenium treatment of different concentrations on the relative conductivity of Phytophthora nicotianae mycelia

The relative conductivity was measured as in 4.3. The relative conductivity is an index representing the permeability of the cell membrane, the relative conductivity under normal treatment maintains a constant value and increases along with the growth and treatment time, and after the external stress, the permeability of the cell membrane increases, and the relative conductivity also increases. The results of the experiment are shown in fig. 4, and the trend of the change in relative conductivity increases with time in a dose and time dependent manner for all treatments. After 120min, the increase is obvious, and the relative conductivity of the concentrations of 8, 10 and 6mg/L is obviously higher than that of other treatments, which shows that the selenium treatment at the concentrations can obviously destroy the cell membrane of hyphae and limit the growth of the hyphae.

4.5 Effect of exogenous selenium treatment at different concentrations on Phytophthora nicotianae hypha Malondialdehyde (MDA)

The MDA content as a permeation regulating substance and a relative conductivity index show the state of cell membrane permeability, and the MDA content of hyphae is gradually increased along with the increase of exogenous selenium concentration, which indicates that the greater the membrane permeability is, as shown in Table 1, the MDA content is the highest under the concentration of 8mg/L, and is 10mg/L secondly. 0. No significant difference exists between the selenium concentrations of 2mg/L and 4mg/L, which shows that the membrane permeability of hypha is less influenced by low-content selenium.

TABLE 1 Effect of exogenous selenium treatment on tobacco Black shank hypha MDA

Note: different lower case letters indicate significant differences at a p <0.05 level.

4.6 Effect of exogenous selenium treatment at different concentrations on Phytophthora nicotianae hypha morphology

The shapes of hyphae in different treatment rooms are observed through comparison on microscopic and macroscopic phenotypes, and the fact that the growth rate of the hyphae after selenium treatment is slowed down, and the hypha diameter is reduced along with the increase of the exogenous selenium concentration can be found. It was found that in the micro-phenotype (FIG. 5), the hyphae that grew normally were dense and full. And hyphae treated by exogenous selenium are wrinkled and flat until withered, the density of the hyphae is obviously reduced, and the distortion and fracture traces at the tail ends of the hyphae are obvious, so that the hyphae have certain defects and are hindered from growing.

4.7 Effect of selenium treatment at different concentrations on disease index of infected tobacco seedlings

Four varieties of K326, Zhongyan 100, Honghuadajinyuan and Changjinhuang are selected for the experiment, and the K326 is found to have obviously reduced morbidity of each treatment compared with other three varieties. While the difference between the treatments of Zhongyan 100, honghuadajinyuan and Changqiu is not big. The disease index of the tobacco 100 and the long-neck yellow in the treatment group is higher, while the disease index is obviously reduced under the Se6 and Se8 treatments. And all four treatments achieved the minimum disease index under Se8 treatment. The following tests were performed with K326 and chinese tobacco 100 as both resistant and susceptible varieties. The specific results are shown in FIG. 6.

4.8 Effect of selenium treatment on the relative conductivity of infected tobacco seedling leaves

K326 and middle smoke 100 were selected on the basis of the previous experiments for subsequent experiments. The relative leaf conductivity was determined after the plants were infected with the pathogen. Sampling of K326: 0. the 0+ bacterium, 8mg/L Se and 8mg/L Se + bacterium are respectively marked as CK, PCK, Se and PSe. Sampling 100 of medium smoke: 0. the 0+ bacterium, 8mg/L Se and 8mg/L Se + bacterium are respectively marked as ZCK, ZPCK, ZSE and ZPSE. The results are shown in FIG. 7, and the change trends of the two varieties are consistent, namely the relative conductivity of the leaves is increased after the leaves are infected with pathogenic bacteria, and the relative conductivity is reduced under the treatment of exogenous selenium. The contrast treatment is not very different, and the relative conductivity of the medium tobacco 100 variety is slightly higher than that of the K326 variety.

4.9 Effect of selenium treatment on hormone content in leaves of infected tobacco seedlings

As can be seen from FIG. 8, the Gibberellin (GA) content of the two varieties of controls was low, 299.39 and 349.06ng/g, respectively, and by applying Se from an external source, the gibberellin content of both varieties of tobacco leaves was significantly increased, 14.64% and 21.09% higher than those of the controls, respectively. The invasion of phytophthora nicotianae also increases the GA content in the plant, which indicates that the gibberellin content is increased in vivo to cope with external stress after the plant is subjected to biotic stress. The GA content in the tobacco plant infected with phytophthora nicotianae after Se treatment is obviously increased, which shows that the disease resistance of the tobacco plant is improved. The change trends of the GA content of the K326 and the Chinese tobacco 100 are consistent.

The abscisic acid (ABA) content varied similarly in both varieties (fig. 9) and both reached a maximum at CK. After the phytophthora nicotianae is inoculated or selenium is applied exogenously, the ABA content of K326 is obviously reduced. After phytophthora nicotianae is inoculated, the ABA content of the two varieties without selenium treatment is respectively improved by 7.66 percent and 9.22 percent compared with the ABA content after selenium treatment.

The trend of the Jasmonic Acid (JA) content (FIG. 10) was the same as that of gibberellin, and both varieties showed the lowest control content when no infection occurred. The jasmonic acid content of tobacco treated by selenium is further improved. After the pathogen is infected, the jasmonic acid content continuously rises, and the content under Se treatment reaches the highest. Se treatment of the two varieties under pathogen infection was 29.70% and 25.51% higher than control jasmonic acid content, respectively.

The trend of the Salicylic Acid (SA) content (fig. 11) was consistent with gibberellin and jasmonic acid. The SA content represents the stress resistance of the plant. As can be seen from the figure, the SA content in the CK group was the lowest in the K326 cultivar, and increased to some extent after selenium treatment, indicating that selenium treatment promoted the production of tobacco hormones. Whereas medium smoke 100 has the highest SA content under germ and selenium treatment. Under the infection of pathogenic bacteria, the SA content of the selenium-applied K326 tobacco plant is 28.02 percent higher than that of the selenium-unapplied tobacco plant, and the SA content of the medium tobacco 100 is only 14.2 percent higher.

The content of auxin (IAA) is not much different between the two varieties (figure 12), and the content of auxin is consistent with the content of gibberellin, jasmonic acid and salicylic acid. After pathogen infestation, CK treatment of K326 and chinese smoke 100 was 34.30% and 24.22% higher than before non-infestation, respectively. The K326 strain produced more auxin to resist the invasion of the pathogen after the pathogen is infected. The Se-treated K326 and the medium smoke 100 had an infection 30.98% and 25.43% higher than the non-infection, respectively.

4.10 Effect of selenium treatment on MAPK content in infected tobacco seedlings

Mitogen-activated protein kinase/MAPK kinase (MAPK) is an important substance of the eukaryotic signaling network. From fig. 13, it is clear that MAPK levels also increased with pathogen infestation and exogenous selenium treatment. Consistent with the growth hormone change trend, the MAPK content of K326 and 100-Smoke after phytophthora nicotianae infection is respectively 28.50% higher and 25.06% higher than that before the phytophthora nicotianae infection. Under Se treatment, the MAPK content of K326 and 100 of the tobacco phytophthora nicotianae after infection is respectively 28.19 percent and 17.66 percent higher than that before infection.

4.11 Effect of selenium treatment on Trypan blue staining of tobacco seedlings after infection

When the cell is damaged or dead, trypan blue reagent can penetrate the denatured cell membrane to be combined with the disintegrated DNA, so that the trypan blue reagent can be used for detecting the integrity of the cell membrane, namely, the deeper the blue color, the more seriously the cell is damaged. As can be seen in FIG. 14, infection with pathogens in both varieties occurred more severely under normal growth conditions, and the conditions were significantly reduced after selenium treatment. The CG group is the darkest 100 color cigarette, which indicates poor disease resistance and large wound area, and the AE group is the second group. The selenium treated tobacco has obvious relieving effect on the two varieties, and the blue degree is lighter, which indicates that exogenous sodium selenite can improve the disease resistance of the tobacco.

The result shows that the exogenous sodium selenite with proper concentration can inhibit the growth of phytophthora nicotianae and reduce the pathogenicity of the phytophthora nicotianae, can improve the capability of resisting disease infection of tobacco plants, prevent the reduction of cell membrane permeability, promote the increase of hormone and MAPK kinase content, reduce disease indexes, and more intuitively discover that the resistance capability of the tobacco plants after selenium treatment to biological stress is obviously enhanced in trypan blue dyeing process. In a whole view, the exogenous sodium selenite concentration is firstly enhanced and then slowly reduced in the aspects of inhibiting the growth of hyphae and improving the disease resistance of tobacco, and the exogenous sodium selenite concentration is best in the aspect of 8mg/L concentration.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. The application of exogenous selenium in inhibiting the growth of phytophthora nicotianae hyphae is provided.

2. The application of exogenous selenium in preventing and treating tobacco black shank is provided.

3. Application of exogenous selenium in preparation of preparation for resisting phytophthora nicotianae is provided.

4. The application of exogenous selenium in the preparation of a preparation for preventing and treating tobacco black shank is provided.

5. A method for improving the black shank resistance of tobacco is characterized in that: comprising the step of administering to the tobacco a formulation comprising exogenous selenium.

6. The use according to any one of claims 1-4 or the method according to claim 5, wherein: the exogenous selenium is tetravalent selenium.

7. The use according to any one of claims 1-4 or the method according to claim 5, wherein: the exogenous selenium is sodium selenite.

8. The use according to any one of claims 1-4 or the method according to claim 5, wherein: the exogenous selenium is sodium selenite, and the use concentration of the sodium selenite is 8 mg/L.

9. The use of claim 3 or 4 or the method of claim 5, wherein: the preparation is a fertilizer, an additive or a growth regulator.