Method for jointly promoting potassium absorption of flue-cured tobacco seedlings by using arbuscular mycorrhizal fungi and bacillus subtilis

Abstract

The invention discloses a method for jointly promoting potassium absorption of flue-cured tobacco seedlings through arbuscular mycorrhizal fungi and bacillus subtilis, and belongs to the technical field of tobacco cultivation. According to the method, the arbuscular mycorrhizal fungi and the bacillus subtilis are combined for flue-cured tobacco cultivation, the arbuscular mycorrhizal fungi and the bacillus subtilis generate the synergistic effect of systematic promotion, the growth and development of the flue-cured tobacco are further promoted through the synergistic effect of the arbuscular mycorrhizal fungi and the bacillus subtilis, and more importantly, the growth and development of the flue-cured tobacco are promoted through the synergistic effect of the arbuscular mycorrhizal fungi and the bacillus subtilis. The bacillus subtilis can promote growth of arbuscular mycorrhizal fungus hyphae, so that the quality of flue-cured tobacco seedlings is improved, potassium absorption of flue-cured tobacco is promoted, and the potassium content of tobacco leaves is increased.

Description

Technical Field

[0001] The invention relates to the technical field of tobacco cultivation, and in particular to a method for promoting potassium absorption of flue-cured tobacco seedlings by combining arbuscular mycorrhizal fungi and bacillus subtilis. Background Art

[0002] Arbuscular mycorrhizal fungi (AMF) are microorganisms widely found in soil and closely related to many plants. About 80% of terrestrial plants can be infected by AMF. After forming a symbiotic relationship with plants, AMF can promote the host plant's absorption of mineral elements and improve the host plant's tolerance to environmental stresses such as drought and salinity.

[0003] Growth-promoting rhizobacteria (PGPR) are an important class of beneficial microorganisms in the rhizosphere soil microecology. Studies have shown that Bacillus subtilis, as a PGPR commonly used in microbial fertilizers, can promote crop growth and development and improve plant stress resistance. In recent years, research on improving plant nutrient absorption and reducing the application of chemical fertilizers has gradually become a hot topic in the development of sustainable agriculture.

[0004] For tobacco, potassium is the most absorbed mineral element, but the potassium ion concentration in the soil is relatively low, about 0.025-5mmol / L. In the rhizosphere of plants, the potassium ion concentration is even lower, usually below 0.3mmol / L. Even in soils with high potassium ion concentrations or high potassium fertilizer content, the plant's absorption and utilization rate of potassium is still not high. Internationally, tobacco leaves with a potassium content higher than 3% are generally regarded as the standard for judging high-quality tobacco leaves, while the average potassium content of tobacco leaves in my country is 1%-2%, and rarely exceeds 2%. Therefore, improving the potassium nutritional efficiency of flue-cured tobacco is of great significance to improving the efficient utilization of nutrients, alleviating the problem of potassium deficiency in tobacco production in my country, and ensuring high-quality and high-yield tobacco leaves. Summary of the invention

[0005] In order to solve the problems of low potassium nutritional efficiency of flue-cured tobacco and low potassium content in tobacco leaves in the aforementioned flue-cured tobacco cultivation, the present invention provides a method for promoting potassium absorption of flue-cured tobacco seedlings by combining arbuscular mycorrhizal fungi and Bacillus subtilis. The method is to cultivate flue-cured tobacco by combining arbuscular mycorrhizal fungi with Bacillus subtilis, thereby improving the quality of flue-cured tobacco seedlings, promoting potassium absorption of flue-cured tobacco, and increasing the potassium content of tobacco leaves.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for promoting potassium absorption of flue-cured tobacco seedlings by combining arbuscular mycorrhizal fungi and Bacillus subtilis comprises the following steps:

[0008] a. Sterilization of seedling medium and culture medium: Tobacco-specific seedling medium, vermiculite, river sand, and perlite are sterilized at high temperature and high pressure and then used for later use;

[0009] b. Floating seedling cultivation: Select full flue-cured tobacco seeds, sterilize them with 10% hydrogen peroxide for 10 minutes, rinse them with distilled water several times, place them in a tray with a moist cotton layer on the bottom, and culture them in an artificial climate box under light for germination; the germinated seeds are cultivated in a sterilized tobacco seedling medium using the floating seedling cultivation method, and when the tobacco seedlings grow to three leaves and one heart, they are ready for transplanting and inoculation;

[0010] c. Preparation of mixed matrix: Sterilized vermiculite, river sand and perlite are mixed in a mass ratio of 1:2:2 and set aside;

[0011] d. Transplantation and inoculation of arbuscular mycorrhizal fungi and Bacillus subtilis: First, 2 / 3 of the mixed matrix is ​​spread flat on the bottom of the potting barrel as the potting matrix, then the arbuscular mycorrhizal fungi and Bacillus subtilis are mixed and evenly spread on the mixed matrix, then a certain thickness of the mixed matrix is ​​evenly spread on the evenly spread bacterial agent, and finally the mixed matrix is ​​used to complete the transplanting of tobacco seedlings;

[0012] e. Fertilization: On the day of transplanting, only sterilized water is applied to ensure the survival of the tobacco seedlings. Fertilization is carried out every 4 days thereafter in the form of Hogland nutrient solution.

[0013] Furthermore, the high temperature and high pressure sterilization parameters in step a are: 121°C, 1×10 5 kpa, 2h.

[0014] Furthermore, the parameters of the artificial climate box in step b are: temperature (28±2)°C, light intensity 4000 Lx, photoperiod 24 h / d, and relative humidity 70%~80%.

[0015] Furthermore, in the step d, the arbuscular mycorrhizal fungus is Glomus versiforme, and the spore content is 20-30 spores / g.

[0016] Further, the total viable count of Bacillus subtilis in step d is 10 11 CFU / g.

[0017] Furthermore, the application amount of the arbuscular mycorrhizal fungi in the step d is 10.00 g per tobacco plant.

[0018] Furthermore, the application amount of Bacillus subtilis in step d is 0.05 g per tobacco plant.

[0019] Furthermore, in the step d, a mixed matrix with a thickness of 0.50±0.1 cm is evenly spread on the evenly spread bacterial agent.

[0020] Furthermore, the potted barrels after transplanting in step e are placed on a light culture rack to cultivate tobacco seedlings, and 100 mL / plant Hoagland nutrient solution is applied every 4 days. Sterile water is added from time to time during the culture period to replenish moisture to maintain a moisture content of 70% for the mixed matrix. Other cultivation and management measures are carried out in accordance with tobacco cultivation and management requirements.

[0021] Furthermore, the parameters of the light culture rack in step e are: daytime temperature (28±2)°C, nighttime temperature (18±2)°C, light intensity 4000 Lx, and light cycle 14 h / d.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention uses arbuscular mycorrhizal fungi and Bacillus subtilis in combination to produce a synergistic effect of system promotion. The arbuscular mycorrhizal fungi and Bacillus subtilis synergistically further promote the growth and development of flue-cured tobacco. More importantly, Bacillus subtilis can promote the growth of arbuscular mycorrhizal fungi hyphae, thereby improving the absorption and utilization of potassium by flue-cured tobacco. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The infection rate of arbuscular mycorrhizal fungi in flue-cured tobacco under different treatments.

[0025] Figure 2 The effects of different treatments on the aboveground and underground biomass of flue-cured tobacco.

[0026] Figure 3 Effects of different treatments on potassium content in aboveground and underground parts of flue-cured tobacco.

[0027] Figure 4 Effects of different treatments on the potassium absorption kinetic parameter Vmax of flue-cured tobacco roots.

[0028] Figure 5 Effects of different treatments on the cation exchange capacity of flue-cured tobacco roots.

[0029] Figure 6 Effects of different treatments on the relative expression levels of genes related to potassium ion absorption in flue-cured tobacco leaves. DETAILED DESCRIPTION

[0030] In order to better understand the present invention, the present invention is further described below in conjunction with specific embodiments.

[0031] The arbuscular mycorrhizal fungus used in the examples of the present invention is Glomus versiforme purchased from the Mycorrhizal Biotechnology Institute of Qingdao Agricultural University, and its spore content is 20-30 spores / g.

[0032] The Bacillus subtilis used in the present invention was purchased from Beihai Qiangxing Biotechnology Co., Ltd., and the total viable count was 10 11 CFU / g.

[0033] The tobacco seedling medium used in the embodiments of the present invention was purchased from Zhengzhou Jintu Science and Technology Trading Co., Ltd.

[0034] The specific ingredients of the Hoagland nutrient solution used in the embodiment of the present invention are:

[0035] Normal potassium: 505.55 mg / L KNO 3 、1180.75 mg / L Ca(NO 3 ) 2 ·4H 2 O, 492.94 mg / L MgSO 4 7H 2 O, 2.782 mg / LH 3 BO 3 、1.979 mg / L MnCl 2 ·4H 2 O, 0.23 mg / L ZnSO 4 7H 2 O, 0.075 mg / LCuSO 4 ·5H 2 O, 0.097 mg / L Na 2 MoO 4 ·2H 2 O, 5.561 mg / L FeSO 4 7H 2 O, 7.485 mg / L EDTA-Na 2 .

[0036] Low potassium: 15.15 mg / L KNO 3 、412.25 mg / L NaNO 3 、1180.75 mg / L Ca(NO 3 ) 2 ·4H 2 O, 492.94 mg / L MgSO 4 7H 2 O, 2.782 mg / LH 3 BO 3 、1.979 mg / L MnCl2 ·4H 2 O, 0.23 mg / LZnSO 4 7H 2 O, 0.075 mg / L CuSO 4 ·5H 2 O, 0.097 mg / L Na 2 MoO 4 ·2H 2 O, 5.561 mg / L FeSO 4 7H 2 O, 7.485 mg / L EDTA-Na 2 .

[0037] Example 1

[0038] The method of this embodiment of using arbuscular mycorrhizal fungi and Bacillus subtilis to promote potassium absorption in flue-cured tobacco seedlings is as follows.

[0039] 1. Sterilization of seedling medium and culture medium: Tobacco special seedling medium, vermiculite, river sand, perlite at 121℃, 1×10 5 Sterilize under kpa conditions for 2h and set aside.

[0040] 2. Floating seedling cultivation: Select K326 flue-cured tobacco seeds with full grains, disinfect the surface with 10% hydrogen peroxide for 10 minutes, rinse with distilled water several times, place them in a tray with a moist cotton layer on the bottom, and culture them for germination under light in an artificial climate box; the germinated seeds are cultivated by floating seedling cultivation in a sterilized tobacco-specific seedling cultivation matrix, and when the tobacco seedlings grow to three leaves and one heart, they are prepared for transplanting and inoculation; the conditions for light culture and germination in this step are: daytime temperature 28℃, nighttime temperature 18℃, photoperiod of 16 h / d, and light intensity of 4 000 Lx.

[0041] 3. Preparation of mixed matrix: Sterilized vermiculite, river sand and perlite are mixed in a mass ratio of 1:2:2. The mass of the mixed matrix in this embodiment is 750 g.

[0042] 4. Transplantation and inoculation of arbuscular mycorrhizal fungi and Bacillus subtilis: First, use 2 / 3 of the mixed matrix as potting matrix and spread it flat on the bottom of a potting barrel with a height of 7.9 cm, a diameter of 17 cm, and a bottom diameter of 13 cm. Then, mix the arbuscular mycorrhizal fungi and / or Bacillus subtilis and spread it evenly on the mixed matrix. Then, spread 0.5 cm thick mixed matrix evenly on the evenly spread bacterial agent. Finally, use the remaining mixed matrix to complete the transplanting of tobacco seedlings.

[0043] 5. Experimental treatment: The experiment was conducted in the Key Laboratory of Tobacco Cultivation of Tobacco Industry of Henan Agricultural University, using potted experiments. The experiment was a two-factor randomized experiment, with the factors being potassium level and microbial agent type, where the potassium level included two potassium levels: normal potassium (NK) and low potassium (LK), and the microbial agent types included no microbial agent application, single application of 10.00g of arbuscular mycorrhizal fungi, single application of 0.05g of Bacillus subtilis, and application of 10.00g of arbuscular mycorrhizal fungi and 0.05g of Bacillus subtilis. There were 8 treatments in total, with 16 strains cultured in each treatment, and 3 replicates; as shown in Table 1.

[0044] Table 1 Processing settings

[0045]

[0046] 6. Fertilization: Only sterilized water was applied on the day of transplanting to ensure the survival of the tobacco seedlings. After transplanting, the potted barrels were placed on a light culture rack to cultivate the tobacco seedlings (daytime temperature 28°C, nighttime temperature 18°C, light intensity 4000 Lx, photoperiod 14 h / d). Thereafter, 100 mL / plant of Hoagland nutrient solution was applied every 4 days, with a potassium concentration of 5 mmol / L for normal potassium treatment and 0.15 mmol / L for low potassium treatment. During the culture period, sterilized water was added from time to time to replenish moisture and maintain the moisture content of the substrate at 70%. Other cultivation and management measures were carried out in accordance with tobacco cultivation and management requirements.

[0047] 7. 60 days after transplanting, the mycorrhizal infection rate, biomass, potassium content, root potassium absorption kinetic parameter Vmax, root cation exchange capacity, and relative expression of genes related to leaf potassium ion absorption of each treatment were measured.

[0048] 8. Results Analysis

[0049] Figure 1 The results showed that potassium level and potassium level × bacterial agent type had no significant effect on the root infection rate of tobacco plants, but the bacterial agent type had a significant effect on the root infection rate of tobacco plants. The T1 and T3 treatments did not show significant differences due to changes in potassium levels, indicating that potassium levels had no effect on the infection rate of arbuscular mycorrhizal fungi. The root infection rate of the T3 treatment was significantly higher than that of the T1 treatment at two potassium levels. The root infection rate of the T3 treatment increased by 34.54% compared with that of the T1 treatment at normal potassium levels, and increased by 21.62% compared with that of the T1 treatment at low potassium levels, indicating that the dual inoculation of arbuscular mycorrhizal fungi and Bacillus subtilis can promote the symbiosis of arbuscular mycorrhizal fungi and tobacco roots.

[0050] Figure 2The results showed that potassium level, bacterial agent type and their interaction had significant effects on the aboveground dry matter accumulation of flue-cured tobacco. Potassium level and bacterial agent type had significant effects on the aboveground dry matter accumulation of flue-cured tobacco, except that potassium level × bacterial agent type had no significant effect on the aboveground dry matter accumulation of flue-cured tobacco. Under the same bacterial agent type, the aboveground and underground dry matter accumulation of tobacco plants at normal potassium level was generally higher than that at low potassium level. Single inoculation of arbuscular mycorrhizal fungi and Bacillus subtilis could increase the aboveground and underground dry matter accumulation of tobacco plants at two potassium levels, but the aboveground and underground dry matter accumulation of tobacco plants in double inoculation treatment was higher than that in single inoculation treatment. Under normal potassium level, the aboveground and underground dry matter accumulation of T3 treatment increased by 76.85% and 39.66% respectively compared with the control. Under low potassium level, the aboveground and underground dry matter accumulation of T3 treatment increased by 31.05% and 51.26% respectively compared with the control.

[0051] Figure 3 The results showed that potassium level and bacterial agent type had significant effects on the potassium content of the aboveground part of flue-cured tobacco, while potassium level × bacterial agent type had no significant effect on the potassium content of the aboveground part of flue-cured tobacco. Potassium level, bacterial agent type, and potassium level × bacterial agent type all had significant effects on the potassium content of the underground part of flue-cured tobacco. Under the same bacterial agent type, the potassium content of the aboveground and underground parts of tobacco plants at the normal potassium level was generally higher than that at the low potassium level. Under the normal potassium level, the potassium content of the aboveground and root parts of tobacco plants in the T1 and T3 treatments were higher than those in the CK treatment. There was no significant difference in the potassium content of the aboveground part of the T2 treatment compared with the CK treatment, and the potassium content of the root part was significantly lower than that of the CK treatment. Under the low potassium level, the potassium content of the aboveground and root parts of the T1 and T3 treatments were significantly higher than those in the CK treatment. The potassium content of the aboveground part of the T2 treatment was lower than that of the CK treatment, but the potassium content of the root part was higher than that of the CK treatment.

[0052] Figure 4 The results showed that potassium level, bacterial agent type, and potassium level × bacterial agent type all had significant effects on the root potassium absorption kinetic parameter Vmax of flue-cured tobacco. The root potassium absorption kinetic parameter Vmax of T1 and T3 treatments was significantly higher than that of CK treatment under two potassium levels. Under normal potassium level, the root potassium absorption kinetic parameter Vmax of T2 treatment had no significant difference with that of the control, and the root potassium absorption kinetic parameter Vmax of T1 and T3 treatments increased by 86.01% and 78.54% respectively compared with the control. Under low potassium level, the root potassium absorption kinetic parameter Vmax of the three inoculated bacterial treatments was significantly higher than that of the control, and the root potassium absorption kinetic parameter Vmax of T3 treatment was the highest, which was increased by 96.71% compared with the CK treatment.

[0053] Figure 5The results showed that potassium level and bacterial agent type had significant effects on the cation exchange capacity of flue-cured tobacco roots, but potassium level × bacterial agent type had no significant effect on the cation exchange capacity of flue-cured tobacco roots. Under the same bacterial agent type, the root cation exchange capacity of tobacco plants at normal potassium level was generally higher than that at low potassium level. Under normal potassium level, the root cation exchange capacity of flue-cured tobacco in T1 and T3 treatments was significantly higher than that in CK treatment, and the root cation exchange capacity of flue-cured tobacco in T2 treatment was significantly lower than that in CK treatment. Under low potassium level, the root cation exchange capacity of flue-cured tobacco in T1 and T3 treatments was significantly higher than that in CK treatment, and there was no significant difference in the root cation exchange capacity of flue-cured tobacco in T2 treatment and CK treatment.

[0054] Figure 6 The results showed that potassium level, bacterial agent type and their interaction had significant effects on tobacco potassium absorption-related genes NtORK1, NtHAK1, NKT1, NKT2, NtKT12, NtTPK1 and NKC1. Potassium level had no significant effect on tobacco potassium absorption-related gene NtHAK5, but bacterial agent type and their interaction had significant effects. Under normal potassium level, the average relative expression of NtORK1, NKT1, NKT2 and NtHAK5 in T3 treatment had no significant difference compared with CK treatment, while the average relative expression of NtHAK1 and NtTPK1 was significantly upregulated compared with the control treatment. Under low potassium levels, the average relative expression levels of NtORK1, NtHAK1, NKT1, NKT2, NtKT12, NtTPK1, NtHAK5, and NKC1 in T3 treatment were significantly upregulated compared with those in CK treatment, which were 243.56%, 288.12%, 249.18%, 796.75%, 169.70%, 81.41%, 249.33%, and 131.37% higher than those in the control treatment, respectively.

[0055] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for promoting potassium absorption of flue-cured tobacco seedlings by combining arbuscular mycorrhizal fungi and Bacillus subtilis, characterized in that: The steps include: a. Sterilization of seedling medium and culture medium: Tobacco-specific seedling medium, vermiculite, river sand, and perlite are sterilized at high temperature and high pressure and then used for later use; b. Floating seedling cultivation: Select full flue-cured tobacco seeds, sterilize them with 10% hydrogen peroxide for 10 minutes, rinse them with distilled water several times, place them in a tray with a moist cotton layer on the bottom, and culture them in an artificial climate box under light for germination; the germinated seeds are cultivated in a sterilized tobacco seedling medium using the floating seedling cultivation method, and when the tobacco seedlings grow to three leaves and one heart, they are ready for transplanting and inoculation; c. Preparation of mixed matrix: Sterilized vermiculite, river sand and perlite are mixed in a mass ratio of 1:2:2 and set aside; d. Transplantation and inoculation of arbuscular mycorrhizal fungi and Bacillus subtilis: First, 2 / 3 of the mixed matrix is ​​spread flat on the bottom of the potting barrel as the potting matrix, then the arbuscular mycorrhizal fungi and Bacillus subtilis are mixed and evenly spread on the mixed matrix, then a certain thickness of the mixed matrix is ​​evenly spread on the evenly spread bacterial agent, and finally the mixed matrix is ​​used to complete the transplanting of tobacco seedlings; e. Fertilization: On the day of transplanting, only sterilized water is applied to ensure the survival of the tobacco seedlings. Fertilization is carried out every 4 days thereafter in the form of Hogland nutrient solution.

2. The method for promoting potassium absorption of flue-cured tobacco seedlings by combining arbuscular mycorrhizal fungi and Bacillus subtilis according to claim 1, characterized in that: The high temperature and high pressure sterilization parameters in step a are: 121°C, 1×10 5 kpa, 2h.

3. The method for promoting potassium absorption of flue-cured tobacco seedlings by combining arbuscular mycorrhizal fungi and Bacillus subtilis according to claim 1, characterized in that: The parameters of the artificial climate box in step b are: temperature (28±2)°C, light intensity 4000 Lx, photoperiod 24 h / d, and relative humidity 70%~80%.

4. The method for promoting potassium absorption of flue-cured tobacco seedlings by combining arbuscular mycorrhizal fungi and Bacillus subtilis according to claim 1, characterized in that: In step d, the arbuscular mycorrhizal fungus is a glomerulase ( Glomus versiforme ), spore content 20~30 / g.

5. The method for promoting potassium absorption of flue-cured tobacco seedlings by combining arbuscular mycorrhizal fungi and Bacillus subtilis according to claim 1, characterized in that: The total viable count of Bacillus subtilis in step d is 10 11 CFU / g.

6. The method for promoting potassium absorption of flue-cured tobacco seedlings by combining arbuscular mycorrhizal fungi and Bacillus subtilis according to claim 1, characterized in that: The application amount of the arbuscular mycorrhizal fungi in the step d is 10.00 g per tobacco plant.

7. The method for promoting potassium absorption of flue-cured tobacco seedlings by combining arbuscular mycorrhizal fungi and Bacillus subtilis according to claim 1, characterized in that: The application amount of Bacillus subtilis in the step d is 0.05 g per tobacco plant.

8. The method for promoting potassium absorption of flue-cured tobacco seedlings by combining arbuscular mycorrhizal fungi and Bacillus subtilis according to claim 1, characterized in that: In the step d, the mixed matrix with a thickness of 0.50±0.1 cm is evenly spread on the evenly spread bacterial agent.

9. The method for promoting potassium absorption of flue-cured tobacco seedlings by combining arbuscular mycorrhizal fungi and Bacillus subtilis according to claim 1, characterized in that: The potted barrels after transplanting in the step e are placed on a light culture rack to cultivate tobacco seedlings, and 100 mL / plant Hoagland nutrient solution is applied every 4 days. Sterile water is added from time to time during the culture period to replenish moisture to maintain a moisture content of 70% for the mixed matrix. Other cultivation and management measures are carried out in accordance with tobacco cultivation and management requirements.

10. The method for promoting potassium absorption of flue-cured tobacco seedlings by using arbuscular mycorrhizal fungi and Bacillus subtilis according to claim 9, characterized in that: The parameters of the light culture rack in step e are: daytime temperature (28±2)°C, nighttime temperature (18±2)°C, light intensity 4000 Lx, and photoperiod 14 h / d.

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