A method for increasing the resistance of tobacco to black shank disease

Abstract

The application discloses a method for improving the black leg disease resistance of tobacco, and comprises the following steps: S1, different concentrations of boric acid are configured, and each part of soil is added with 100ml ultrapure water; S2, the soil is placed in a cool place to obtain soil with different exogenous boron contents; S3, soil culture is adopted to cultivate seedlings; S4, the tobacco seedlings are moved to the soil culture box to grow; S5, the same radius and the same amount of fungus cakes are taken, the same parts of tobacco leaves are taken out and placed in a plastic tray, wet gauze is placed under the leaves to keep the leaves moist, 3 leaves are placed in each tray, the activated tobacco pythium cake is inoculated on the leaves, two pieces of tobacco pythium cake are inoculated on each leaf to avoid the main vein, the leaves are sealed with preservative film and placed in a 28 DEG C incubator; S6, after the fungus is inoculated for 3 days, the third true leaf is taken out to measure the lesion area, photosynthetic parameters and malondialdehyde (MDA) content; and S7, transmission electron microscopy and trypan blue staining observation are conducted.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of tobacco, and particularly relates to a method for improving the black leg disease resistance of tobacco. BACKGROUND

[0002] Tobacco black leg disease is a soil-borne oomycete disease caused by Peronospora tabacina, which has strong harmfulness to tobacco plants and seriously affects the yield and quality of tobacco. Black spots and white mold-like substances appear at the stem base of tobacco plants infected with black leg disease at the seedling bed stage. After tobacco plants are infected with black leg disease at the field stage, black necrosis appears at the root, necrotic spots appear at the stem base, the stem pith becomes black and shrinks due to the action of toxins, and is separated into a "disc" shape, the vascular bundle is damaged, the tobacco leaves become wilted and yellow, and the tobacco plants may even die in severe cases. The humid environmental conditions that exist universally during the growth season are conducive to the growth and reproduction of the pathogen, so that tobacco black leg disease has become one of the most difficult diseases to control.

[0003] Boron is one of the essential nutrients for higher plants, which can promote 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 control, studies have shown that the application of appropriate amount of boron can prevent and control diseases such as sugar beet heart rot, chestnut canker, kiwi vine galls, etc. At the same time, appropriate concentration of boron can also inhibit the growth of Fusarium sulphureum, Botrytis cinerea and other pathogenic fungi.

[0004] At present, tobacco black leg disease is mainly prevented and controlled by screening disease-resistant varieties, using microbial preparations and chemical pesticides, and agricultural prevention and control measures. However, due to the problems of resistance degradation of disease-resistant varieties, the emergence of pathogen resistance, the blindness of chemical fungicide use, and the environmental pollution caused by chemical fungicide use, it is necessary to find a green, efficient and low-cost measure for preventing and controlling tobacco black leg disease. SUMMARY

[0005] To solve the above technical problems existing in the prior art, the application provides a method for improving the black leg disease resistance of tobacco.

[0006] The technical scheme adopted by the application is as follows:

[0007] A method for improving the black leg disease resistance of tobacco, characterized in that the method comprises the following steps:

[0008] S1, taking boric acid as the boron source, weighing 12 kg of soil and dividing it into 6 equal parts, each part weighing 2 kg; then weighing 11.27, 22.55, 33.82, 56.36 and 112.72 mg of boric acid respectively, and adding them into each part of the soil dissolved in 100 ml of ultrapure water, and stirring uniformly;

[0009] S2, the soil is placed in a cool place, daily stirring soil once, after seven days to get the exogenous boron mass is 0, 1, 2, 3, 5, 10 mg / kg of soil;

[0010] S3, using soil culture, the early test with seedling tray and H-shaped sponge as medium, after the seed is sterilized with 10% hydrogen peroxide, then washed with distilled water, soak in distilled water for 1 day under room temperature and lightless condition, then evenly place in the sponge, after the tobacco seed germination, select the uniform growth seedlings, move to the vermiculite with forceps and grow;

[0011] S4, when the tobacco seedlings grow to four-leaf stage, move from the sponge to the soil culture box, each soil culture box contains 200g soil, the seedlings grow to six-leaf stage in the soil culture box and then treated.

[0012] S5, place the same radius and equal amount of fungus cake in the same position on the tobacco leaf, in the in vitro trypan blue staining, take the same position of tobacco leaf and place in the plastic tray, pad the wet gauze under the leaf to keep moist, place 3 leaves in each tray, inoculate the activated tobacco blight fungus cake on the leaf, inoculate two pieces of tobacco blight fungus cake on each leaf avoiding the main vein, seal with preservative film and place in 28℃ incubator;

[0013] Each of the above treatment is set for 3 times, and each repeat has 4 seedlings;

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

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

[0016] Collect the upper leaves of uninfected tobacco seedlings and tobacco seedlings after 3 days of pathogen infection under different exogenous boron treatments, cut into 1mm x 1mm small pieces, transfer the cut small tissue blocks to EP tubes containing 2.5% glutaraldehyde, fix with vacuum pump until the bottom is settled, place at room temperature for 2h, then fix and store at 4℃ for transportation;

[0017] Rinse with 0.1M phosphate buffer PB (pH 7.4) for 3 times, 15min each time, then move to 1% osmium acid for 7h at room temperature. Then sequentially dehydrate in 30%-50%-70%-80%-95%-100% alcohol, 1h each time;

[0018] Subsequently, dehydrate with acetone, penetrate and embed with Epon812, polymerize at 60℃ for 24h, then slice on an ultramicrotome, double stain with uranyl acetate and lead citrate, and observe and photograph under a transmission electron microscope.

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

[0020] The diameter of the disease spot is measured after the tobacco leaf is infected by the pythium for 3 days, and the disease spot area is calculated according to S = Pi r 2 .

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

[0022] After the tobacco leaf is infected by the pythium for 3 days, the CI-340 portable photosynthetic instrument produced by the CID company in the United States is used to measure the indicators in the morning from 09:00 to 11:00 on a sunny day, the photosynthetic gas exchange parameters of the leaf are measured away from the main leaf vein, the reading is 1 min, and the stomatal conductance (Gs), intercellular space CO2 concentration (Ci), net photosynthetic rate (Pn) and transpiration rate (Tr) under natural light are obtained.

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

[0024] After the tobacco leaf is infected by the pythium for 3 days, the leaf around the disease spot is collected, and the malondialdehyde (MDA) content is measured by the thiobarbituric acid method.

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

[0026] Compared with the prior art, the beneficial effects of the present application are reflected in:

[0027] 1. The tobacco plant is treated with an appropriate concentration of exogenous boron, the disease spot area is reduced to different degrees after inoculation of the tobacco pythium, the infection area is also reduced after the leaf is inoculated with the bacteria, the disease spot area is smallest and the staining area is also smallest when the exogenous boron concentration is 2mg / kg, which shows that appropriate boron application can improve the disease resistance of tobacco.

[0028] 2. After the exogenous boron treatment, the net photosynthetic rate of the tobacco plant is improved to different degrees, and the net photosynthetic rate of the tobacco plant reaches the highest when the boron content is 2mg / kg, which shows that appropriate exogenous boron can improve the photosynthetic rate of the leaf and alleviate the damage caused by the pathogenic bacteria.

[0029] 3. After the tobacco plant treated with an appropriate concentration of exogenous boron is inoculated with the tobacco pythium, the malondialdehyde content in the leaf is reduced, and the malondialdehyde content is lowest when the exogenous boron concentration is 2mg / kg, which shows that the damage to the tobacco plant is lowest.

[0030] 4. Appropriate concentration of exogenous boron treatment can promote tobacco cell development, while excessive boron can inhibit tobacco cell development; after inoculation of tobacco Phytophthora, the tobacco cells treated with 2mg / kg boron suffer the lowest degree of damage, and the tobacco cells treated with 10mg / kg boron produce a large number of mitochondria and suffer the most serious damage, which shows that appropriate boron application can improve the ability of tobacco to resist Phytophthora, while excessive boron is on the contrary.

[0031] 5. The present application can improve the ability of tobacco to resist Phytophthora, which has great significance for preventing and treating tobacco Phytophthora. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 is the lesion area of tobacco seedlings treated with different boron after inoculation.

[0033] Figure 2 is the photosynthetic parameter of tobacco seedlings treated with different boron after inoculation.

[0034] Figure 3 is the content of malondialdehyde of tobacco seedlings treated with different boron after inoculation.

[0035] Figure 4 is the situation of 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 is the ultrastructure of tobacco seedling leaves treated with different boron and inoculated leaves.

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

[0039] The specific embodiments of the embodiments of the present application will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the embodiments of the present application, and are not used to limit the embodiments of the present application.

[0040] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

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

[0042] The tobacco test variety is K326, which is provided by Henan Agricultural University Tobacco Institute. The Phytophthora nicotianae is isolated from the tobacco plants infected by Phytophthora nicotianae in a tobacco planting area in Luoshan County, Xinyang City, Henan Province. The boric acid solid powder is purchased from Zhengzhou Aikemu Chemical Co., Ltd., and the content is 99% of the analytical pure reagent.

[0043] The test site is the tobacco quality ecology laboratory of the Tobacco Institute of Henan Agricultural University.

[0044] Reference Figures 1 to 5 The method for improving the resistance of tobacco to black shank disease comprises the following steps:

[0045] S1. Take 12 kg of soil and divide it into 6 equal parts, each part weighing 2 kg. Then, take 11.27, 22.55, 33.82, 56.36, and 112.72 mg of boric acid, respectively, and add it to each part of the soil dissolved in 100 ml of ultrapure water, and stir well. The soil without external boron treatment (i.e., the mass of external boron is 0) only adds 100 ml of ultrapure water.

[0046] S2. Place the above soil in a cool place and stir the soil once a day. After seven days, obtain soil with external boron masses of 0, 1, 2, 3, 5, and 10 mg / kg, respectively.

[0047] S3. Use soil culture to grow seedlings. In the early stage of the test, use a seedling tray and an I-shaped sponge as the medium. After disinfecting the tobacco seeds with 10% hydrogen peroxide and cleaning them with distilled water, immerse them in distilled water at room temperature in the dark for 1 day, then evenly place them in the sponge. After the tobacco seeds germinate, select seedlings with consistent growth and move them to the vermiculite with tweezers. When the tobacco seedlings grow to the four-leaf stage, move them from the sponge to the soil culture box, and each soil culture box contains 200g of soil.

[0048] S4. When the tobacco seedlings grow to the four-leaf stage, move them from the sponge to the soil culture box, and each soil culture box contains 200g of soil. When the tobacco seedlings grow to the six-leaf stage in the soil culture box, perform the treatment.

[0049] S5. Place the same amount of fungus cake (4mm in diameter) at the same position on the tobacco leaf, avoiding the main vein. In the in vitro trypan blue staining, place the same part of the tobacco leaf in a plastic tray, pad the leaf with wet gauze to keep it moist, and place 3 leaves in each tray. Inoculate the activated tobacco Phytophthora nicotianae fungus cake (4mm in diameter) on the leaves, inoculate two pieces of tobacco Phytophthora nicotianae fungus cake on each leaf, avoiding the main vein, seal them with plastic wrap, and place them in a 28℃ incubator.

[0050] Each of the above treatment methods is set up 3 times, and each repetition contains 4 tobacco seedlings.

[0051] S6, 3 days after the pathogen infection treatment, the third leaf was taken to measure the lesion area, photosynthetic parameters and malondialdehyde (MDA) content, respectively;

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

[0053] Collect the upper leaves (counted from the top, the 4th leaf position) of tobacco seedlings treated with different exogenous boron and infected with bacteria for 3 days, cut into 1mm x 1mm small pieces, and transfer the cut small tissue blocks to an EP tube containing 2.5% glutaraldehyde for fixation. Pump the air until it sinks to the bottom, and place it at room temperature for 2h, then store and transport at 4℃;

[0054] Then rinse with 0.1M phosphate buffer PB (pH 7.4) for 3 times, 15min each time, then move to 1% osmium acid for 7h at room temperature. Then sequentially dehydrate in 30%-50%-70%-80%-95%-100% alcohol, 1h each time;

[0055] Then dehydrate with acetone, penetrate and embed with Epon812, polymerize at 60℃ for 24h, then slice on an ultramicrotome (Leica UC7, Germany), double stain with uranyl acetate and lead citrate, and observe and photograph under a transmission electron microscope (hitachi-HT7800, Japan).

[0056] Trypan blue staining was performed according to the method of Zhang Yudan et al. (Chlorogenic acid on the inhibition of tobacco Phytophthora and the prevention and control effect of tobacco black shank disease [J]. Crop Journal, 2022, (02)).

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

[0058] Measure the lesion diameter after the tobacco leaf is infected with Phytophthora for 3 days, and calculate the lesion area according to S = Πr 2

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

[0060] After the tobacco leaf is infected with Phytophthora for 3 days, use the CI-340 portable photosynthetic instrument produced by CID Company of the United States to measure the index at 09:00-11:00 on a sunny day, avoid the main leaf vein, measure the photosynthetic gas exchange parameters of the leaf, read for 1min, and get 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 measuring the malondialdehyde (MDA) content 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 using the thiobarbituric acid method.

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

[0064] like Figure 1 The figure shows the effect of different boron concentrations on the area of ​​lesions in infected tobacco seedlings. The lesion area directly reflects the degree of infection by *Phytophthora infestans*. In the treatment group B2, the lesion area was significantly smaller than that in the control group. The other exogenous boron treatments showed no significant difference compared to the control group. However, the lesion area in the B3 treatment was lower than that in the control group, indicating that appropriate boron treatment can improve the resistance of tobacco to black shank disease.

[0065] like Figure 1 The image shows the effect of different boron concentrations on the photosynthetic parameters of infected tobacco seedlings. Photosynthesis is the main source of plant dry matter, and pathogen infection affects the photosynthetic rate. Figure 2 It was found that net photosynthetic rate (Pn), transpiration rate (Tr), and stomatal conductance (Gs) generally showed a trend of first increasing and then decreasing with increasing boron concentration, while intercellular CO2 concentration (Ci) showed the opposite trend. When the exogenous boron concentration was 2 mg / kg, Pn and Tr reached their maximum, significantly higher than CK, indicating that appropriate boron application can alleviate pathogen stress on tobacco. The reasons for the inhibition of plant photosynthesis by external abiotic stress can be mainly divided into two types: stomatal limitation and non-stomatal limitation. CK and B5 had lower Pn and Gs and relatively higher Ci deficiency, indicating that non-stomatal limitation caused weakened photosynthesis, that is, mesophyll cells were infected by viruses, and their photosynthetic capacity was inhibited, leading to a large accumulation of CO2 in the intercellular space. Therefore, excessively high boron concentrations are not conducive to tobacco alleviating pathogen stress.

[0066] like Figure 3 The image shows the effect of different boron concentrations on malondialdehyde (MDA) content in infected tobacco seedlings. MDA content is an indicator of the degree of oxidative damage in plants; an increase in MDA content indicates that the plants have suffered oxidative damage. Figure 3 It can be seen that the MDA content first decreases and then increases with the increase of boron concentration. B2 and B3 are significantly lower than CK, indicating that appropriate application of boron can reduce the damage of tobacco plants to pathogen infection.

[0067] like Figure 4 The image shows the effect of exogenous boron treatment on trypan blue staining of infected tobacco seedlings. Trypan blue is a cell-active dye commonly used to detect cell death; dead cells are stained by this dye.Figure 4 It can be seen that the B2 treatment resulted in a significantly smaller stained area on the leaves compared to the other treatments, and the smallest damaged area, indicating that an appropriate amount of exogenous boron can improve the disease resistance of tobacco.

[0068] like Figure 5 The image shows the effect of exogenous boron treatment on the ultrastructure of infected tobacco seedlings. Transmission electron microscopy allows for microscopic observation of leaf development. Figure 5 It can be seen that in the leaves of uninoculated tobacco plants, the chloroplast structure of B2 treatment was intact, starch grains were well developed, and grana lamellae were arranged in an orderly and clearly visible manner; the chloroplast membrane structure of CK treatment was slightly blurred, starch grains were larger, and grana lamellae were arranged in a relatively orderly manner; the chloroplast membrane structure of B5 treatment was ruptured, no starch grains were observed, grana lamellae were blurred, and there were many mitochondria around the chloroplasts. Compared with the leaves of uninoculated tobacco plants, the chloroplast structure of inoculated tobacco plants was damaged to varying degrees. Among them, the chloroplast membrane of B2 treatment was slightly degraded, but the grana lamellae were still clearly visible and neatly arranged; the chloroplast membrane of CK treatment was ruptured, the grana lamellae were slightly blurred, and mitochondria appeared around them; the chloroplast membrane of B5 treatment was severely ruptured, the grana lamellae were more blurred and disordered, and more mitochondria appeared around them. This shows that an appropriate amount of exogenous boron can promote tobacco cell development, while excessive boron has the opposite effect. The increase in mitochondria indicates that cells need to produce more energy to maintain their own activities and resist external stress. This suggests that the B5 treatment was most severely infected by pathogens, while the B2 treatment was the least infected. Appropriate amounts of boron can improve the tobacco plant's ability to resist external pathogen infection, while excessive boron will reduce the tobacco's disease resistance.

[0069] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method of increasing the resistance of tobacco to black shank, characterized in that, The method comprises the following steps: S1. Taking 12 kg of soil and dividing it into 6 equal parts, each part weighing 2 kg; then taking 11.27, 22.55, 33.82, 56.36, 112.72 mg of boric acid respectively and adding it to each part of the soil dissolved in 100 ml of ultrapure water, and stirring evenly; S2. Placing the above soil in a cool place, stirring and loosening the soil once a day, and obtaining soil with exogenous boron mass of 0, 1, 2, 3, 5, 10 mg / kg respectively after seven days; S3. Using soil culture method to grow seedlings, in the early stage of the test, using seedling trays and I-shaped sponge as medium, after disinfecting the tobacco seeds with 10% hydrogen peroxide and then cleaning them with distilled water, the seeds are soaked in distilled water under room temperature and in dark condition for 1 day, then evenly placed in the sponge, after the tobacco seeds germinate, the seedlings with consistent growth are selected and moved to the vermiculite with tweezers for growth; S4. When the tobacco seedlings grow to 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, and the seedlings grow to six-leaf stage in the soil culture box for treatment; S5. Placing the same radius and equal amount of fungus cake on the same part of the tobacco leaf, avoiding the main vein; In the in vitro trypan blue staining, the same part of the tobacco leaf is placed in a plastic tray, and wet gauze is placed under the leaf to keep it moist, 3 leaves are placed in each tray, and the activated tobacco blight fungus cake is inoculated on the leaves, avoiding the main vein, and the tray is sealed with plastic wrap and placed in a 28℃ incubator; Each of the above treatment methods is set up 3 times of repetition, and each repetition has 4 seedlings; S6. After 3 days of pathogen infection treatment, the third true leaf is taken to measure the lesion area, photosynthetic parameters and malondialdehyde (MDA) content; S7. Transmission electron microscopy and trypan blue staining observation Collecting the upper leaves of the uninfected tobacco seedlings with different exogenous boron treatments and the tobacco seedlings after 3 days of inoculation, cutting them into small pieces of 1 mm x 1 mm, transferring the cut small tissue pieces to EP tubes containing 2.5% glutaraldehyde for fixation, and using a vacuum pump to extract air until it sinks to the bottom, placing it at room temperature for 2 hours, and then storing and transporting it at 4℃; Then, using 0.1M phosphate buffer PB, pH 7.4, rinsing 3 times for 15 minutes each time, and then moving to 1% osmium acid for 7 hours of room temperature fixation. Then sequentially enter 30%-50%-70%-80%-95%-100% alcohol for dehydration, 1 hour each time; Then use acetone for dehydration, Epon812 for penetration and embedding, polymerize at 60℃ for 24 hours, then slice on an ultramicrotome, double stain with uranyl acetate and lead citrate, and then observe and photograph under a transmission electron microscope.

2. A method of increasing the resistance of tobacco to black shank according to claim 1, wherein, In step S6, the method for measuring the lesion area is as follows: After 3 days of infection of the tobacco leaf by the blight fungus, the lesion diameter is measured, and the lesion area is calculated according to S=Πr².

3. A method of increasing the resistance of tobacco to black shank according to claim 1, wherein, In step S6, the method for measuring the photosynthetic parameters is as follows: After 3 days of infection, the photosynthetic parameters were measured by using the portable photosynthesis system (CID, USA) from 9:00 to 11:00 am on sunny days. 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. The method of claim 1, wherein the tobacco plant is a flue-cured tobacco plant. The method for determining the content of MDA in step S6 is as follows: After 3 days of infection, the leaves around the lesions were collected, and the content of MDA was determined by using the thiobarbituric acid method.

5. The method for improving tobacco's resistance to black shank disease as described in claim 1, characterized in that, In step S4, the whole culture period was carried out in an artificial incubator, maintaining the incubator environment as: day and night 28℃±2℃ / 18℃±2℃, light cycle 14 / 10h cycle, relative humidity 70%, light intensity 440μmol·m-2s-1. .​

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