Method for improving black shank resistance of tobacco

Abstract

The invention discloses a method for improving black shank resistance of tobacco, which comprises the following steps: S1, preparing boric acid with different concentrations, and respectively adding the boric acid into each part of soil dissolved by 100ml of ultrapure water; s2, the soil is placed in a shade place, and soil with different exogenous boron masses is obtained; s3, raising seedlings by adopting a soil culture method, S4, transferring tobacco seedlings into a soil culture box to grow, S5, putting the same quantity of fungus cakes with the same radius; in the in-vitro trypan blue dyeing process, tobacco leaves at the same part are taken and placed in plastic trays, wet gauze is padded below the tobacco leaves to preserve moisture, three leaves are placed in each tray, activated tobacco phytophthora cakes are inoculated on the leaves, two tobacco phytophthora cakes are inoculated on each leaf in a manner of avoiding a main vein, and the tobacco leaves are placed in a 28 DEG C incubator after being sealed by a preservative film; s6, taking a third true leaf for respectively measuring the scab area, the photosynthetic parameter and the malondialdehyde (MDA) content after 3 days of pathogen infection treatment; and S7, observing by using a transmission electron microscope and trypan blue dyeing.

Description

Technical Field

[0001] The invention belongs to the technical field of tobacco, and particularly relates to a method for improving the black shank resistance of tobacco. Background Art

[0002] Tobacco black shank is a soil-borne oomycete disease caused by Phytophthora nicotianae. It is extremely harmful to tobacco plants and seriously affects tobacco yield and quality. In the seedbed period, tobacco plants infected with black shank will develop black spots and white mold at the base of the stem. In the field period, tobacco plants infected with black shank will develop black necrosis at the roots and necrotic spots at the base of the stem. The pith of the diseased stem will turn black and shrink due to the action of toxins, separating into "disc" shapes, and the vascular bundles will be destroyed, causing the tobacco leaves to wilt and turn yellow. In severe cases, the tobacco plants will die. The humid environmental conditions that are prevalent during the growing season are conducive to the growth and reproduction of pathogens, making tobacco black shank one of the most difficult diseases to control.

[0003] Boron is one of the essential nutrients for higher plants. It promotes the carbon and nitrogen metabolism, cell wall formation, cell elongation and division, pollen tube germination and growth, etc. In terms of stress resistance, boron can improve plant resistance by regulating plant phenol metabolism, stabilizing chlorophyll structure, and controlling cell membrane permeability. In terms of disease prevention and control, studies have shown that the application of appropriate amounts of boron can prevent and control diseases such as beet heart rot, chestnut plaster disease, and kiwifruit vine swollen disease. At the same time, appropriate concentrations of boron can also inhibit the growth of pathogens such as sulfur-colored Fusarium and gray mold.

[0004] At present, tobacco black shank is mainly controlled by screening disease-resistant varieties, using microbial preparations and chemical pesticides and agricultural control measures. However, due to the degradation of disease-resistant varieties, the emergence of drug resistance in pathogens, the blind use of chemical fungicides and the easy environmental pollution, it is necessary to find a green, efficient and low-cost measure to control tobacco black shank. Summary of the invention

[0005] In order to solve the above technical problems existing in the prior art, the present invention provides a method for improving the ability of tobacco to resist black shank disease.

[0006] The technical solution adopted by the present invention is:

[0007] A method for improving tobacco's ability to resist black shank disease, characterized in that the method comprises the following steps:

[0008] S1. Take boric acid as the boron source, weigh 12 kg of soil and divide it into 6 equal portions, 2 kg each; then weigh 11.27, 22.55, 33.82, 56.36, and 112.72 mg of boric acid respectively, add them to each portion of soil dissolved in 100 ml of ultrapure water, and stir evenly;

[0009] S2. Place the soil in a cool place, stir and loosen the soil once a day, and after seven days, obtain soils with exogenous boron contents of 0, 1, 2, 3, 5, and 10 mg / kg, respectively;

[0010] S3. Soil culture was used to raise seedlings. In the early stage of the experiment, seedling trays and I-shaped sponges were used as the medium. The tobacco seeds were disinfected with 10% hydrogen peroxide, then cleaned with distilled water, soaked for 1 day at room temperature and protected from light, and then evenly placed in the sponge. After the tobacco seeds germinated, the seedlings with the same growth were selected and moved to vermiculite with tweezers for growth;

[0011] S4. When the tobacco seedlings grow to the four-leaf stage, they are moved from the sponge to the soil culture box for growth. Each soil culture box contains 200 g of soil. The tobacco seedlings are processed when they grow to six leaves and one heart in the soil culture box.

[0012] S5. Place equal amounts of bacterial cakes with the same radius on the same part of tobacco leaves, avoiding the main veins; in vitro trypan blue staining, place tobacco leaves at the same part in a plastic tray, place moist gauze under the leaves to keep them moist, place 3 leaves in each tray, inoculate the leaves with activated tobacco phytophthora cakes, inoculate two tobacco phytophthora cakes on each leaf, avoid the main veins, seal with plastic wrap and place in a 28°C incubator;

[0013] Each of the above treatments was repeated three times, with 4 tobacco seedlings in each repeat;

[0014] S6. After 3 days of pathogen infection, the third true leaf was taken to measure the lesion area, photosynthetic parameters and malondialdehyde (MDA) content;

[0015] S7, transmission electron microscopy and trypan blue staining observation

[0016] The upper leaves of tobacco seedlings without inoculation and 3 days after inoculation were collected and cut into several small pieces of 1 mm × 1 mm. The cut small tissue pieces were transferred to EP tubes filled with 2.5% glutaraldehyde for fixation, and vacuumed with a vacuum pump until they sink to the bottom. After being placed at room temperature for 2 hours, they were fixed and stored at 4°C for transportation.

[0017] Then rinse with 0.1M phosphate buffer PB (pH7.4) for 3 times, 15 minutes each time, and then move to 1% osmium acid for fixation at room temperature in the dark for 7 hours. Then dehydrate in 30%-50%-70%-80%-95%-100% alcohol in ascending order, 1 hour each time;

[0018] The tissues were then dehydrated with acetone, embedded in Epon812, polymerized at 60°C for 24 hours, sliced ​​on an ultrathin slicer, and double-stained with uranyl acetate and lead citrate before observation and photography under a transmission electron microscope.

[0019] Furthermore, in step S6, the method for determining the lesion area is as follows:

[0020] The diameter of the lesions was measured 3 days after the tobacco leaves were infected by Phytophthora. 2 Calculate the lesion area;

[0021] Further, in step S6, the method for measuring photosynthetic parameters is as follows:

[0022] Three days after tobacco leaves were infected with Phytophthora, the CI-340 portable photosynthetic instrument produced by CID Company of the United States was used to measure indicators at 09:00-11:00 in the morning on a sunny day. The photosynthetic gas exchange parameters of the leaves were measured avoiding the main veins. The readings were taken for 1 min to obtain the stomatal conductance (Gs), intercellular CO2 concentration (Ci), net photosynthetic rate (Pn), and transpiration rate (Tr) under natural light.

[0023] Further, in step S6, the method for determining the content of malondialdehyde (MDA) is as follows:

[0024] Three days after tobacco leaves were infected by Phytophthora, leaves around the lesions were collected and the malondialdehyde (MDA) content was determined by the thiobarbituric acid method.

[0025] Further, in step S4, the entire culture period is carried out in an artificial incubator, and the incubator environment is maintained at: 28°C ± 2°C / 18°C ± 2°C day and night, a photoperiod of 14 / 10h cycle, a relative humidity of 70%, and a light intensity of 440 μmol·m -2 ·s -1 .

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] 1. The present invention treats tobacco plants with exogenous boron at an appropriate concentration. After inoculation with tobacco phytophthora, the lesion area is reduced to varying degrees. The infection area of ​​leaves in vitro after inoculation is also reduced. When the exogenous boron concentration is 2 mg / kg, the lesion area is the smallest and the staining area is also the smallest, indicating that appropriate boron application can improve tobacco disease resistance.

[0028] 2. After treatment with exogenous boron, the net photosynthetic rate of tobacco plants increased to varying degrees. When the boron content was 2 mg / kg, the net photosynthetic rate of tobacco plants reached the highest, indicating that an appropriate amount of exogenous boron can increase the photosynthetic rate of leaves and alleviate the damage caused by pathogen infection.

[0029] 3. After tobacco plants were inoculated with tobacco blight after being treated with appropriate concentrations of exogenous boron, the malondialdehyde content in the leaves decreased. When the exogenous boron concentration was 2 mg / kg, the malondialdehyde content was the lowest, indicating that the tobacco plants suffered the least damage.

[0030] 4. Treatment with appropriate concentrations of exogenous boron can promote the development of tobacco cells, but when the boron is excessive, it will inhibit the development of tobacco cells. After inoculation with tobacco blight, tobacco cells treated with 2 mg / kg boron suffered the least damage, while tobacco cells treated with 10 mg / kg boron produced a large number of mitochondria and suffered the most serious damage. This shows that appropriate boron application can improve tobacco's ability to resist black shank disease, while excessive boron has the opposite effect.

[0031] 5. The present invention can improve the ability of tobacco to resist black shank disease, which is of great significance for preventing and treating tobacco black shank disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the area of ​​lesions on tobacco seedlings inoculated with different boron treatments.

[0033] Figure 2 These are the photosynthetic parameters of tobacco seedlings inoculated with different boron treatments.

[0034] Figure 3 It is the content of malondialdehyde in tobacco seedlings inoculated with different boron treatments.

[0035] Figure 4 This is the trypan blue staining of tobacco seedling leaves treated with different boron after inoculation in vitro.

[0036] Among them: A: CK; B: B1; C: B2; D: B3; E: B4; F: B5.

[0037] Figure 5 The ultrastructure of tobacco seedling leaves treated with different boron and after inoculation.

[0038] Among them: A: CK; B: B1; C: B2; D: B3; E: B4; F: B5. DETAILED DESCRIPTION

[0039] The specific implementation of the embodiment of the present invention is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described here is only used to illustrate and explain the embodiment of the present invention, and is not used to limit the embodiment of the present invention.

[0040] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0041] The present invention will be described in detail below in conjunction with exemplary embodiments with reference to the accompanying drawings.

[0042] The tobacco variety used for the test was K326, provided by the Tobacco Research Institute of Henan Academy of Agricultural Sciences. Phytophthora nicotianae was isolated from tobacco plants infected with Phytophthora nicotianae in the tobacco growing area of ​​Luoshan County, Xinyang City, Henan Province. Boric acid solid powder was purchased from Zhengzhou Aikemu Chemical Co., Ltd., with a content of 99% analytical pure reagent.

[0043] The experimental site is the Tobacco Quality Ecology Laboratory of the Tobacco College of Henan Agricultural University.

[0044] refer to Figures 1 to 5 A method for improving tobacco's ability to resist black shank disease of the present invention comprises the following steps:

[0045] S1. Take boric acid as the boron source, weigh 12 kg of soil and divide it into 6 equal parts, 2 kg each; then weigh 11.27, 22.55, 33.82, 56.36, and 112.72 mg of boric acid respectively, add them to each part of soil dissolved in 100 ml of ultrapure water, and stir evenly; among them, only 100 ml of ultrapure water is added to the soil without exogenous boron treatment (i.e., the mass of exogenous boron is 0);

[0046] S2. Place the soil in a cool place, stir and loosen the soil once a day, and after seven days, obtain soils with exogenous boron contents of 0, 1, 2, 3, 5, and 10 mg / kg, respectively;

[0047] S3. Soil culture was used to grow seedlings. In the early stage of the experiment, seedling trays and I-shaped sponges were used as media. Tobacco seeds were disinfected with 10% hydrogen peroxide and then cleaned with distilled water. They were soaked for 1 day at room temperature and away from light, and then evenly placed in the sponge. After the tobacco seeds turned white and germinated, seedlings with consistent growth were selected and moved to vermiculite with tweezers for growth. When the tobacco seedlings grew to the four-leaf stage, they were moved from the sponge to the soil culture box for growth. Each soil culture box contained 200g of soil.

[0048] S4. When the tobacco seedlings grow to the four-leaf stage, they are moved from the sponge to the soil culture box for growth. Each soil culture box contains 200 g of soil. The tobacco seedlings are processed when they grow to six leaves and one heart in the soil culture box.

[0049] S5. Place equal amounts of bacterial cakes (4 mm in diameter) of the same radius on tobacco leaves at the same position, avoiding the main veins; in vitro trypan blue staining, place tobacco leaves at the same position in a plastic tray, place moist gauze under the leaves to keep them moist, place 3 leaves in each tray, inoculate the leaves with activated tobacco phytophthora cakes (4 mm in diameter), inoculate two tobacco phytophthora cakes on each leaf, avoiding the main veins, seal with plastic wrap and place in a 28°C incubator;

[0050] Each of the above treatments was repeated three times, with 4 tobacco seedlings in each repeat;

[0051] S6. After 3 days of pathogen infection, the third true leaf was taken to measure the lesion area, photosynthetic parameters and malondialdehyde (MDA) content;

[0052] S7, transmission electron microscopy and trypan blue staining observation

[0053] The upper leaves of tobacco seedlings that were not inoculated with different exogenous boron treatments and those that were inoculated for 3 days (the 4th leaf from top to bottom) were collected and cut into several small pieces of 1 mm × 1 mm. The cut small tissue pieces were transferred to EP tubes filled with 2.5% glutaraldehyde for fixation, and vacuumed with a vacuum pump until they sank to the bottom. After being placed at room temperature for 2 hours, they were fixed and stored at 4°C for transportation.

[0054] Then rinse with 0.1M phosphate buffer PB (pH7.4) for 3 times, 15 minutes each time, and then move to 1% osmium acid for fixation at room temperature in the dark for 7 hours. Then dehydrate in 30%-50%-70%-80%-95%-100% alcohol in ascending order, 1 hour each time;

[0055] Subsequently, the samples were dehydrated with acetone, embedded in Epon812, polymerized at 60°C for 24 h, and then sliced ​​on an ultrathin microtome (Leica UC7, Germany). After double staining with uranyl acetate and lead citrate, the samples were observed and photographed under a transmission electron microscope (Hitachi-HT7800, Japan).

[0056] Trypan blue staining was conducted using the method of Zhang Yudan et al. (Study on the inhibitory effect of chlorogenic acid on tobacco phytophthora and its control effect on tobacco black shank disease [J]. Crop Journal, 2022, (02)).

[0057] Specifically, in step S6, the method for determining the lesion area is as follows:

[0058] The diameter of the lesions was measured 3 days after the tobacco leaves were infected by Phytophthora. 2 Calculate the lesion area;

[0059] Specifically, in step S6, the method for measuring photosynthetic parameters is as follows:

[0060] Three days after tobacco leaves were infected with Phytophthora, the CI-340 portable photosynthetic instrument produced by CID Company of the United States was used to measure indicators at 09:00-11:00 in the morning on a sunny day. The photosynthetic gas exchange parameters of the leaves were measured avoiding the main veins. The readings were taken for 1 min to obtain the stomatal conductance (Gs), intercellular CO2 concentration (Ci), net photosynthetic rate (Pn), and transpiration rate (Tr) under natural light.

[0061] Specifically, in step S6, the method for determining the content of malondialdehyde (MDA) is as follows:

[0062] Three days after tobacco leaves were infected by Phytophthora, leaves around the lesions were collected and the malondialdehyde (MDA) content was determined by the thiobarbituric acid method.

[0063] Specifically, in step S4, the entire culture period is carried out in an artificial incubator, and the incubator environment is maintained at: 28°C ± 2°C / 18°C ± 2°C day and night, a photoperiod of 14 / 10h cycle, a relative humidity of 70%, and a light intensity of 440 μmol·m -2 ·s -1 .

[0064] like Figure 1 As shown in the figure, the effect of different concentrations of boron treatment on the lesion area of ​​tobacco seedlings after infection. The lesion area can intuitively show the degree of infection of tobacco leaves by Phytophthora. The lesion area of ​​B2 in the treatment group was significantly smaller than that of the control group. There was no significant difference between the other exogenous boron treatments and the control group. Among them, the lesion area of ​​B3 treatment was reduced compared with the control group, indicating that appropriate boron treatment can improve the ability of tobacco to resist black shank.

[0065] like Figure 1 The figure shows the effect of different concentrations of boron treatment on the photosynthetic parameters of tobacco seedlings after infection. Photosynthesis is the main source of plant dry matter, and pathogen infection affects the photosynthetic rate of plants. Figure 2 It can be seen that the net photosynthetic rate (Pn), transpiration rate (Tr), and stomatal conductance (Gs) generally increase first and then decrease with the increase of boron concentration. The intercellular CO2 concentration (Ci) is the opposite. When the exogenous boron concentration is 2 mg / kg, Pn and Tr reach the maximum, which is significantly higher than CK, indicating that appropriate boron application can alleviate the stress of pathogens on tobacco. The reasons why external adversity inhibits plant photosynthesis are mainly divided into stomatal restriction and non-stomatal restriction. CK and B5 have lower Pn and Gs and relatively higher Ci, indicating that non-stomatal restriction causes photosynthesis to weaken, that is, the mesophyll cells are infected by viruses, and the photosynthetic capacity is inhibited, resulting in a large amount of CO2 accumulating in the intercellular space. Therefore, excessive boron concentration is not conducive to tobacco to alleviate pathogen stress.

[0066] like Figure 3 The figure shows the effect of different concentrations of boron treatment on the malondialdehyde (MDA) content of tobacco seedlings after infection. MDA content is an indicator of the degree of oxidative damage in plants, and an increase in its content indicates that the plants have been oxidatively damaged. Figure 3 It can be seen that the MDA content first decreased and then increased with the increase of boron concentration, and B2 and B3 were significantly lower than CK, indicating that appropriate application of boron can reduce the damage of tobacco plants caused by pathogens.

[0067] like Figure 4 The effect of exogenous boron treatment on trypan blue staining of infected tobacco seedlings is shown in Figure 2. Trypan blue is a cell viability dye commonly used to detect cell death. Dead cells will be stained with dye. Figure 4 It can be seen that the leaf staining area of ​​B2 treatment is significantly smaller than that of the other treatments, and the injured area is the smallest, indicating that appropriate amount of exogenous boron can improve tobacco disease resistance.

[0068] like Figure 5 The effect of exogenous boron treatment on the ultrastructure of tobacco seedlings after infection is shown in Figure 2. The development of leaves can be observed microscopically by transmission electron microscopy. Figure 5 It can be seen that in the leaves of uninoculated tobacco plants, the chloroplast structure of B2 treatment was complete, starch granules were well developed, and the grana lamellae were arranged in an orderly manner and clearly visible; the chloroplast membrane structure of CK treatment was slightly blurred, the starch granules were larger, and the grana lamellae were arranged in a more orderly manner; the chloroplast membrane structure of B5 treatment was broken, no starch granules were seen, the grana lamellae were blurred, and there were more mitochondria around the chloroplasts. Compared with the leaves of uninoculated tobacco plants, the chloroplast structure of the leaves of inoculated tobacco plants was damaged to varying degrees, among which the chloroplast membrane of B2 treatment was slightly degraded, and the grana lamellae were still clearly visible and arranged neatly; the chloroplast membrane of CK treatment was broken, the grana lamellae were slightly blurred, and mitochondria appeared around; the chloroplast membrane of B5 treatment was severely broken, the grana lamellae were more blurred, and the arrangement was disordered, and more mitochondria appeared around. It can be seen that appropriate amount of exogenous boron can promote the development of tobacco cells, while excessive boron is the opposite. The increase in mitochondria indicates that cells need to generate more energy to maintain their own activities and resist external stress, which means that the B5 treatment was most seriously infected by pathogens, and the B2 treatment was least infected. An appropriate amount of boron can improve the ability of tobacco plants to resist infection by external pathogens, while excessive boron will reduce the disease resistance of tobacco.

[0069] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for improving tobacco's resistance to black shank disease, characterized in that: The method comprises the following steps: S1. Take boric acid as the boron source, weigh 12 kg of soil and divide it into 6 equal portions, 2 kg each; then weigh 11.27, 22.55, 33.82, 56.36, and 112.72 mg of boric acid respectively, add them to each portion of soil dissolved in 100 ml of ultrapure water, and stir evenly; S2. Place the soil in a cool place, stir and loosen the soil once a day, and after seven days, obtain soils with exogenous boron contents of 0, 1, 2, 3, 5, and 10 mg / kg, respectively; S3. Soil culture was used to raise seedlings. In the early stage of the experiment, seedling trays and I-shaped sponges were used as the medium. The tobacco seeds were disinfected with 10% hydrogen peroxide, then cleaned with distilled water, soaked for 1 day at room temperature and protected from light, and then evenly placed in the sponge. After the tobacco seeds germinated, the seedlings with the same growth were selected and moved to vermiculite with tweezers for growth; S4. When the tobacco seedlings grow to the four-leaf stage, they are moved from the sponge to the soil culture box for growth. Each soil culture box contains 200 g of soil. The tobacco seedlings are processed when they grow to six leaves and one heart in the soil culture box. S5. Place the same amount of mushroom cakes with the same radius on the same part of the tobacco leaves, avoiding the main veins; In the in vitro trypan blue staining, take tobacco leaves from the same part and place them in a plastic tray, put moist gauze under the leaves to keep them moist, place 3 leaves in each tray, inoculate the leaves with activated tobacco phytophthora cakes, and inoculate two tobacco phytophthora cakes on each leaf, avoiding the main veins, seal with plastic wrap and place in a 28°C incubator; Each of the above treatments was repeated three times, with 4 tobacco seedlings in each repeat; S6. After 3 days of pathogen infection, the third true leaf was taken to measure the lesion area, photosynthetic parameters and malondialdehyde (MDA) content; S7, transmission electron microscopy and trypan blue staining observation The upper leaves of tobacco seedlings without inoculation and 3 days after inoculation were collected and cut into several small pieces of 1 mm × 1 mm. The cut small tissue pieces were transferred to EP tubes filled with 2.5% glutaraldehyde for fixation, and vacuumed with a vacuum pump until they sink to the bottom. After being placed at room temperature for 2 hours, they were fixed and stored at 4°C for transportation. Then rinse with 0.1M phosphate buffer PB (pH7.4) for 3 times, 15 minutes each time, and then move to 1% osmium acid for fixation at room temperature in the dark for 7 hours. Then dehydrate in 30%-50%-70%-80%-95%-100% alcohol in ascending order, 1 hour each time; The tissues were then dehydrated with acetone, embedded in Epon812, polymerized at 60°C for 24 hours, sliced ​​on an ultrathin slicer, and double-stained with uranyl acetate and lead citrate before observation and photography under a transmission electron microscope.

2. A method for improving tobacco's resistance to black shank disease as claimed in claim 1, characterized in that: In step S6, the method for determining the lesion area is as follows: The diameter of the lesions was measured 3 days after the tobacco leaves were infected by Phytophthora. 2 Calculate the lesion area.

3. A method for improving tobacco's resistance to black shank disease as claimed in claim 1, characterized in that: In step S6, the method for measuring photosynthetic parameters is as follows: Three days after tobacco leaves were infected with Phytophthora, the CI-340 portable photosynthetic instrument produced by CID Company of the United States was used to measure indicators at 09:00-11:00 in the morning on a sunny day. The photosynthetic gas exchange parameters of the leaves were measured avoiding the main veins. The readings were taken for 1 min to obtain the stomatal conductance (Gs), intercellular CO2 concentration (Ci), net photosynthetic rate (Pn), and transpiration rate (Tr) under natural light.

4. A method for improving tobacco's resistance to black shank disease as claimed in claim 1, characterized in that: In step S6, the method for determining the content of malondialdehyde (MDA) is as follows: Three days after tobacco leaves were infected by Phytophthora, leaves around the lesions were collected and the malondialdehyde (MDA) content was determined by the thiobarbituric acid method.

5. A method for improving tobacco's resistance to black shank disease as claimed in claim 1, characterized in that: In step S4, the entire culture period is carried out in an artificial incubator, and the incubator environment is maintained at: 28°C ± 2°C / 18°C ± 2°C day and night, a photoperiod of 14 / 10h cycle, a relative humidity of 70%, and a light intensity of 440 μmol·m -2 ·s -1 .

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