An inoculant containing arbuscular mycorrhizal fungi and its application in alleviating tobacco continuous cropping obstacles.

Abstract

This invention belongs to the field of soil conservation and tobacco cultivation technology, specifically relating to an inoculant containing arbuscular mycorrhizal fungi (AMF) and its application in alleviating tobacco continuous cropping obstacles. By inoculating the arbuscular mycorrhizal fungi in the inoculant into the roots of tobacco plants, this invention improves the agronomic traits of tobacco, enhances its photosynthetic capacity, and increases its biomass. Simultaneously, this invention improves the agrochemical properties of continuously cropped soils through the arbuscular mycorrhizal fungi, increasing the available phosphorus content and decreasing the soil organic matter and available potassium content; it also significantly increases soil enzyme activity, thereby regulating soil nutrients and promoting the absorption and utilization of nitrogen, phosphorus, and potassium nutrients by tobacco plants. This provides a theoretical basis and technical support for using mycorrhizal technology to promote tobacco growth and improve continuously cropped soils.

Description

Technical Field

[0001] This invention belongs to the field of soil conservation and tobacco cultivation technology, specifically relating to an inoculant containing arbuscular mycorrhizal fungi and its application in alleviating tobacco continuous cropping obstacles. Background Technology

[0002] Tobacco (Nicotiana tabacum) is a solanaceous crop. Long-term continuous cropping leads to reduced tobacco yield, increased pests and diseases, soil compaction, and soil microbial imbalance, ultimately resulting in lower tobacco quality and yield. The problem of continuous cropping is rampant in tobacco-growing areas, and soil environmental degradation is worsening. Furthermore, continuous cropping leads to the accumulation or deficiency of certain nutrients in the soil. To achieve normal tobacco yield and quality, tobacco farmers are willing to spend significant human, material, and financial resources on the heavy use of chemical fertilizers and pesticides to obtain stable economic income. However, this approach is highly destructive to the environment and severely restricts the sustainable development of the tobacco industry.

[0003] Arbuscular mycorrhizal fungi (AMF) are a class of beneficial soil microorganisms widely distributed in terrestrial ecosystems, capable of establishing mutualistic symbiotic relationships with over 80% of terrestrial plants. Currently, considerable research has been conducted on the effects of AMF on promoting seedling propagation, growth, and development of maize and tobacco, and enhancing tolerance to abiotic stresses. However, there are no systematic reports on the role of AMF in improving soil nutrients in continuously cropped soils and regulating tobacco growth in such soils. Summary of the Invention

[0004] The purpose of this invention is to provide an inoculant containing arbuscular mycorrhizal fungi and its application in alleviating tobacco continuous cropping obstacles. The inoculant containing arbuscular mycorrhizal fungi can effectively alleviate tobacco continuous cropping obstacles, promote the growth and development of tobacco, and has a good effect on the conservation and restoration of tobacco continuous cropping soil.

[0005] The present invention provides an inoculum containing arbuscular mycorrhizal fungi, the inoculum comprising plant roots, plant cultivation substrate and arbuscular mycorrhizal fungi; the arbuscular mycorrhizal fungi colonize the plant roots and plant cultivation substrate, and the colonization rate of the arbuscular mycorrhizal fungi is not less than 90%.

[0006] Preferably, the plant root system includes corn roots and / or tobacco roots.

[0007] Preferably, the arbuscular mycorrhizal fungus includes *Funneliformis mosseae*.

[0008] Preferably, the plant cultivation substrate includes farmland soil and vermiculite, and the volume ratio of farmland soil to vermiculite is (2-3):1.

[0009] The present invention also provides the application of the inoculant described in the above technical solution in alleviating tobacco continuous cropping obstacles.

[0010] Preferably, the mitigation of tobacco continuous cropping obstacles includes soil conservation and restoration for tobacco continuous cropping and / or promotion of tobacco growth and development.

[0011] Preferably, promoting tobacco growth and development includes one or more of the following: improving tobacco agronomic traits, enhancing tobacco photosynthetic capacity, increasing tobacco biomass, and promoting the absorption of nutrients by tobacco.

[0012] The soil conservation and remediation for continuous tobacco cropping includes one or more of the following: increasing the available phosphorus content in the soil, increasing the enzyme activity in the soil, and regulating the content of allelochemicals in the soil.

[0013] The present invention also provides a method for alleviating tobacco continuous cropping obstacles, comprising: planting tobacco in tobacco continuous cropping soil containing an inoculum of arbuscular mycorrhizal fungi; wherein the inoculum containing arbuscular mycorrhizal fungi is the inoculum described in the above technical solution.

[0014] Preferably, the weight ratio of the inoculum containing arbuscular mycorrhizal fungi to the tobacco continuous cropping soil is 1:40.

[0015] Preferably, the tobacco is a tobacco seedling with four leaves and two buds; the amount of the inoculum containing arbuscular mycorrhizal fungi is 200g / tobacco plant.

[0016] Beneficial effects:

[0017] This invention provides an inoculum containing arbuscular mycorrhizal fungi (AMF). The inoculum comprises plant roots, a plant cultivation substrate, and AMF. The AMF colonizes the plant roots and the plant cultivation substrate, with a colonization rate of not less than 90%. By inoculating the AMF-containing inoculum into tobacco roots, this invention improves the agronomic traits of tobacco, enhances its photosynthetic capacity, and increases its biomass. Simultaneously, this invention improves the agrochemical properties of continuously cropped soils through the AMF, increasing the available phosphorus content in tobacco soils while reducing soil organic matter and available potassium content. It also significantly increases soil enzyme activity, thereby regulating soil nutrients and promoting the absorption and utilization of nitrogen, phosphorus, and potassium nutrients by tobacco plants. Experimental results show that compared to the uninoculated AMF treatment group, the AMF-inoculated treatment group effectively promotes the absorption of nitrogen and phosphorus by tobacco plants, significantly increasing the nitrogen and phosphorus content in tobacco plants. This provides a theoretical basis and technical support for using mycorrhizal technology to promote tobacco growth and improve continuously cropped soils. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 The diagram shows the AMF structure in the root cells of potted tobacco in Example 1, where A, B, C and D represent tufts, hyphae, hyphal rings and vesicles, respectively, and the scale bar is 50 μm.

[0020] Figure 2 This is a comparison of potted tobacco plants with different treatments 45 days after transplanting in Example 1;

[0021] Figure 3 The results of biomass determination of tobacco in different treatment groups in Example 1 are shown; where A is the fresh biomass weight and B is the dry biomass weight.

[0022] Figure 4 The photosynthetic parameters of tobacco leaves in different treatment groups in Example 1 are: A = Pn, B = Gs, C = Tr, and D = Ci.

[0023] Figure 5 The agrochemical properties of tobacco rhizosphere soil in different treatment groups in Example 2;

[0024] Figure 6 The enzyme activities in the rhizosphere soil of tobacco in different treatment groups in Example 2; where A is S-CAT, B is S-APC, C is S-SC, and D is PPO;

[0025] Figure 7 The aboveground and underground nutrient contents of tobacco plants in different treatment groups in Example 3 are: A, B, and C.

[0026] exist Figures 2-6 In the text, C+M- indicates that AMF is not inoculated on soils where tobacco is continuously cropped; C+M+ indicates that AMF is inoculated on soils where tobacco is continuously cropped; CM- indicates that AMF is not inoculated on soils where tobacco is not continuously cropped; and C-M+ indicates that AMF is inoculated on soils where tobacco is not continuously cropped.

[0027] * indicates a significant difference between the two groups (*: 0.01 ≤ p < 0.05, **: 0.001 ≤ p < 0.01, ***: p < 0.001, t-test; n = 3–5). The bar chart shows the two-way analysis of soil continuous cropping status and AMF inoculation (*: 0.01 ≤ p < 0.05, **: 0.001 ≤ p < 0.01, ***: p < 0.001, two-way analysis; n = 3–5). Detailed Implementation

[0028] The present invention provides an inoculum containing arbuscular mycorrhizal fungi, the inoculum comprising plant roots, plant cultivation substrate and arbuscular mycorrhizal fungi; the arbuscular mycorrhizal fungi colonize the plant roots and plant cultivation substrate, and the colonization rate of the arbuscular mycorrhizal fungi is not less than 90%.

[0029] In one embodiment, the plant root system of the present invention can be a corn root system and / or a tobacco root system; in another embodiment, the plant root system can be a corn root system. In one embodiment, the arbuscular mycorrhizal fungus includes *Funneliformis mosseae*.

[0030] In one embodiment, the plant cultivation substrate can be farmland soil and vermiculite, with a volume ratio of farmland soil to vermiculite of (2-3):1; in another embodiment, the volume ratio of farmland soil to vermiculite can be 2:1 or 3:1.

[0031] As one embodiment, the preparation method of the inoculant containing arbuscular mycorrhizal fungi includes the following steps: mixing rhizosphere soil containing arbuscular mycorrhizal fungal hyphae and spores with sterile plant cultivation substrate to obtain a mixed cultivation substrate; planting sterile plant seedlings in the mixed cultivation substrate until the colonization rate of arbuscular mycorrhizal fungi is not less than 90%; collecting plant roots and rhizosphere cultivation substrate to obtain the inoculant containing arbuscular mycorrhizal fungi.

[0032] As one embodiment, the rhizosphere soil containing arbuscular mycorrhizal fungal hyphae and spores is containing hyphae and spores of *Cyclocarya mosie*. In a specific embodiment, rhizosphere soil containing hyphae and spores of *Cyclocarya mosie* from the Institute of Plant Nutrition and Resources, Beijing Academy of Agricultural and Forestry Sciences, with accession number BGCYN05,1511C0001BGCAM0013, is used, but it should not be considered as the entire scope of protection of this invention.

[0033] In one embodiment, the present invention places the rhizosphere soil containing arbuscular mycorrhizal fungal hyphae and spores in the middle layer of a sterile plant cultivation substrate, i.e., a "sandwich" structure. First, a layer of sterile plant cultivation substrate is laid flat. Then, the rhizosphere soil containing arbuscular mycorrhizal fungal hyphae and spores is placed on top of the flat, sterile plant cultivation substrate, and finally, another layer of sterile plant cultivation substrate is placed on top. In another embodiment, the plant seedling is a corn seedling. The reason for using corn seedlings as the inoculum to prepare the inoculum containing arbuscular mycorrhizal fungi is that corn seedlings grow quickly, have a short growth cycle, and the arbuscular mycorrhizal fungi colonize the corn roots relatively quickly.

[0034] In one embodiment, the weight ratio of the rhizosphere soil containing arbuscular mycorrhizal fungal hyphae and spores to the plant cultivation substrate is 700g:6300g.

[0035] The present invention also provides the application of the inoculant described in the above technical solution in alleviating tobacco continuous cropping obstacles.

[0036] In one implementation, alleviating tobacco continuous cropping obstacles can involve soil conservation and / or promotion of tobacco growth and development. In another implementation, alleviating tobacco continuous cropping obstacles can involve soil conservation and restoration and promotion of tobacco growth and development. In one implementation, promoting tobacco growth and development includes improving tobacco agronomic traits, enhancing tobacco photosynthetic capacity, increasing tobacco biomass, and promoting the absorption of nutrients by tobacco; in another implementation, promoting the absorption of nutrients by tobacco can involve promoting the absorption of one or more of nitrogen, phosphorus, and potassium by tobacco.

[0037] In one embodiment, the soil conservation and remediation for tobacco continuous cropping can increase the available phosphorus content, increase the enzyme activity, and regulate the allelochemical content in the tobacco continuous cropping soil, among one or more of these. In another embodiment, the soil enzyme activity can be one or more of catalase, acid phosphatase, and sucrase.

[0038] This invention also provides a method for alleviating tobacco continuous cropping obstacles, comprising: planting tobacco in tobacco continuous cropping soil containing an inoculum of arbuscular mycorrhizal fungi; wherein the inoculum containing arbuscular mycorrhizal fungi is the inoculum described in the above-mentioned technical solution. As one embodiment, the weight ratio of the inoculum containing arbuscular mycorrhizal fungi to the tobacco continuous cropping soil can be 1:40; as another embodiment, the weight ratio of the inoculum containing arbuscular mycorrhizal fungi to the tobacco continuous cropping soil is 1:40. The weight ratio of the inoculum containing arbuscular mycorrhizal fungi to the tobacco continuous cropping soil described in this invention can achieve an AMF colonization rate of over 75% in tobacco roots, resulting in a high colonization rate. As one embodiment, the tobacco is a tobacco seedling with four leaves and two buds; the amount of the inoculum containing arbuscular mycorrhizal fungi can be 200g / plant; as another embodiment, the amount of the inoculum containing arbuscular mycorrhizal fungi can be 200g / plant.

[0039] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0040] Material descriptions used in the following embodiments:

[0041] AMF (F. mosseae) was obtained from the Institute of Plant Nutrition and Resources, Beijing Academy of Agricultural and Forestry Sciences, with accession number BGCYN05, 1511C0001BGCAM0013;

[0042] The formula for Hoagland nutrient solution is as follows: calcium nitrate 94.5 g / L, ammonium nitrate 8 g / L, potassium nitrate 50.6 g / L, potassium dihydrogen phosphate 13.6 g / L, magnesium sulfate heptahydrate 100.9 g / L, Na2EDTA·2H2O 7.46 g / L, FeSO4·7H2O 5.56 g, MnSO4·4H2O 4.46 g / L, H3BO3 1.24 g / L, ZnSO4·7H2O 1.72 g / L, CuSO4·5H2O 0.005 g / L, CoCl2·6H2O 0.005 g, KI 0.166 g, and Na2MoO4·2H2O 0.05 g / L.

[0043] Example 1

[0044] An inoculant containing arbuscular mycorrhizal fungi and its method for regulating tobacco growth, the specific steps of which are as follows:

[0045] 1. Preparation of inoculum:

[0046] (1.1) Preparation of cultivation substrate:

[0047] Large clumps of farmland soil were removed by passing it through a 10-mesh sieve. The mixture was then mixed evenly with horticultural vermiculite at a volume ratio of 2:1. The mixture was placed in a high-pressure steam sterilizer and sterilized at 121°C for 2 hours. The sterilization was repeated every 24 hours for a total of 3 times. The mixture was then cooled to room temperature to obtain a sterile cultivation substrate.

[0048] (1.2) After disinfecting the surface of the corn seeds, they were cultured until the corn germinated to obtain sterile corn seedlings:

[0049] Select corn seeds of uniform size, shape and plumpness, disinfect them with 8% sodium hypochlorite solution for 8 minutes, rinse them 3 times with sterile water, place them in a petri dish with double-layer sterile filter paper, and incubate them in the dark until the corn germinates (temperature is 28℃).

[0050] Note: Corn was chosen as the inoculum because corn seedlings grow quickly, have a short growth cycle, and AMF colonizes the corn roots relatively quickly.

[0051] (1.3) Thoroughly wipe the flowerpot (round flowerpot with a mouth width of 35cm and a height of 25cm, i.e., 35cm×25cm) with 75% alcohol to sterilize it, let it dry, and write a label. Lay the sterile cultivation substrate prepared in step (1.1) at the bottom of the sterilized flowerpot to 2 / 3 of the empty flowerpot. The amount of sterile cultivation substrate used is about 5800g.

[0052] Each pot contains 700g of rhizosphere soil containing AMF (F. mosseae) mycelium and spores, purchased from the Institute of Plant Nutrition and Resources, Beijing Agricultural and Forestry College. The preservation number is BGCYN05, 1511C0001BGCAM0013, resulting in 2000 spores per 50g of soil containing the fungicide.

[0053] Spread the above-mentioned microbial agent soil on top of the cultivation substrate in the sterile flower pot, transplant the sterile corn seedlings obtained in step (1.2) into the sterile flower pot substrate, and then cover it with a 3-5cm thick layer of sterile cultivation substrate level with the top of the flower pot to form a "sandwich" method for propagating AMF; the total amount of sterile cultivation substrate used is about 7300g.

[0054] Water thoroughly with 1 / 2 Hoagland nutrient solution until the solution seeps out from the bottom of the pot. During the growing season, maintain regular management to ensure normal plant growth. Then, water with 300mL of 1 / 2 Hoagland nutrient solution every 15 days. After about 30 days of cultivation, randomly check the corn roots and observe the colonization of AMF under an optical microscope using the cross-cross method. If typical structures such as AMF hyphae, tufts, vesicles, and hyphal rings are observed, the colonization is considered successful.

[0055] After culturing for about 60 days until the total colonization rate of AMF at the corn roots is not less than 90%, the above-ground parts of the corn are cut off and the top 1-2 cm of soil is removed. The roots and culture medium are collected to obtain an inoculum containing AMF.

[0056] 2. Cultivation of tobacco transplanted seedlings:

[0057] (2.1) First, sterilize the tobacco floating seedling substrate (purchased from Yunnan Mile Tobacco Supplies Co., Ltd.) with high pressure steam (sterilization conditions: 121℃, 2h; sterilize once every 24h, for a total of 3 times). After sterilization, mix the substrate with water appropriately (moisture content: 50%-60%). The substrate should be able to be squeezed into a ball in your hand and crumble when touched. After placing the substrate into the holes of the seedling tray, shake the seedling tray evenly and fill it with substrate. Make a 0.5cm deep seed hole in each hole, and then put in 1-2 K326 coated tobacco seeds (provided by Yuxi China Tobacco Seed Co., Ltd.), and gently cover with substrate.

[0058] Weigh 25g of tobacco-specific seedling fertilizer (N:P2O4:K2O = 18:12:13; Honghe Henglin Chemical Co., Ltd.) and pour it into the seedling tank. Add water (approximately 25L) until the floating tray is level with the surface of the seedling tank. After about 45 days of cultivation, tobacco seedlings with four leaves and two buds will be obtained.

[0059] (2.2) Commercially available horticultural vermiculite and farmland soil were used as the basic cultivation substrate for tobacco in a volume ratio of 1:3: farmland soil was passed through a 10-mesh sieve to remove larger impurities and mixed evenly with commercially available vermiculite as the cultivation substrate.

[0060] The AMF-containing inoculum obtained in step (1.3) was divided into two equal parts. One part was used as the inoculation group, and the other part was sterilized by high-pressure steam (121℃, sterilization for 2 hours, sterilization once every 24 hours, for a total of 3 times) and air-dried to obtain inactivated AMF as the non-inoculation control group.

[0061] There are two levels of soil continuous cropping status: soil with 5 years of tobacco continuous cropping (C+) and soil without tobacco planting for 5 years (C-). The agrochemical properties of soil with 5 years of tobacco continuous cropping and soil without tobacco planting for 5 years are compared in Table 1.

[0062] Table 1. Agrochemical properties of basic soils for continuous cropping and non-continuous cropping.

[0063]

[0064]

[0065] Note: * indicates a significant difference between treatment groups under independent samples t-test (*p<0.05, **p<0.01).

[0066] As shown in Table 1, the contents of organic matter, available phosphorus, available potassium, total nitrogen, and total phosphorus in the soil samples collected for planting were 35.52 g / Kg, 105.96 mg / Kg, 672.47 mg / Kg, 0.2473%, and 2.18 g / Kg (p<0.05), respectively, which were significantly higher than those in the non-continuously cropped soil. The content of available nitrogen in the continuously cropped soil was higher than that in the non-continuously cropped soil, while the content of total potassium in the continuously cropped soil was lower than that in the non-continuously cropped soil (p>0.05).

[0067] (2.3) A completely randomized group design was adopted for the experiment, with AMF and soil type as the two variables.

[0068] There were two levels of AMF inoculation: AMF inoculation and no AMF inoculation; there were also two levels of soil continuous cropping status: soil with 5 years of tobacco continuous cropping and soil without tobacco planting for 5 years. A total of 4 different planting treatment groups were established, with each treatment replicated 12 times. After the culture period, plant and soil samples were collected to obtain 4 different sample groups: ① Continuous tobacco soil without AMF inoculation: C+M-; ② Continuous tobacco soil inoculated with AMF: C+M+; ③ Tobacco non-continuous cropping soil without AMF inoculation: CM-; ④ Tobacco non-continuous cropping soil inoculated with AMF: C-M+.

[0069] Thoroughly clean the flowerpots (35cm×25cm) with 75% alcohol and use them as cultivation containers. Let them dry and label them with numbers. Lay a layer of cultivation substrate at the bottom (to 2 / 3 of the pot's height), filling each pot with 8kg of soil substrate. For the inoculation group, add an inoculum containing AMF (200g / plant, added twice, before and after transplanting) on ​​top of the cultivation substrate. The mass ratio of the AMF-containing inoculum to the cultivation substrate is 1:40. The uninoculated control group received inactivated AMF (200g / plant).

[0070] For the tobacco seedlings transplanted in step (2.1), one tobacco plant was planted per pot. The seedlings were required to be uniformly grown, healthy, and disease-free. The cultivation substrate was spread evenly from the surface of the inoculum layer to the top of the cultivation container, forming a "sandwich" method to inoculate *F. mosseae* onto the tobacco roots. After culturing the tobacco seedlings under different treatments in the cultivation containers for 45 days, the agronomic traits, photosynthetic parameters, and other indicators of the plants were measured.

[0071] 3. Methods for harvesting tobacco plants

[0072] Samples were collected after 45 days of cultivation in a tobacco greenhouse. Figure 2 (Comparison of potted plants under different treatments 45 days after transplanting). Before sampling, crop agronomic traits were measured, including plant height, stem circumference, leaf length, leaf width, and number of leaves. A Li-6400 portable photosynthesis system (Li-Cor Inc., USA) was used to measure photosynthetic parameters such as transpiration rate (Tr), stomatal conductance (Gs), net photosynthetic rate (Pn), and intercellular CO2 concentration (Ci) of functional leaves at the same location on the fourth leaf (out of 12 leaves from top to bottom) of tobacco plants.

[0073] When harvesting the samples, carefully remove the plants, shake off the soil attached to the roots, wash the roots with clean water, absorb excess water with filter paper, and weigh the fresh weight of the above-ground and underground parts of the crop separately.

[0074] A small amount of fresh root samples were used to detect and statistically analyze the colonization rate of *F. mosseae* using acid fuchsin staining and the cross-staining method. For the colonization rate analysis of *F. mosseae*: Tobacco root segments were collected, washed with tap water, and then placed in test tubes. If the tobacco root segments were dark in color, they were first decolorized with a root sample decolorizing solution (3 mL ammonia + 10 mL 30% hydrogen peroxide to a final volume of 600 mL) for 2 hours before analysis. After decolorization, the roots were rinsed with tap water, and then 10% KOH solution was added until the roots were completely submerged. The samples were then heated in a 90℃ water bath until they became translucent. Afterward, the roots were rinsed 2-3 times with tap water, acidified with lactic acid, and soaked for 10-20 minutes. Cut root samples into segments approximately 1 cm long and place them on a glass slide. Add 0.5% acidic fuchsin staining solution to stain the tobacco plant root segments. During the staining process, the back of the glass slide can be ignited with an alcohol lamp to accelerate the staining process, until white smoke is emitted 2-3 times. Decolorize the stained root segments with lactic acid glycerol. Select root segments of moderate thickness and a length of approximately 1 cm and press them onto a glass slide. Observe the pressed slides under a regular optical microscope and use the cross-multiplication method to count the colonization rate of *F. mosseae*. At least 300 fields of view should be examined for each root sample. Calculate the total colonization rate of *F. mosseae* using the following formula:

[0075] Colony rate (%) of *Funneliformis mosseae* = (Total number of fields of view - Number of blank fields of view) / Total number of fields of view × 100%

[0076] The remaining samples were placed in kraft paper envelopes, placed in an oven at 105℃ for 30 minutes to fix the green color, and dried at 75℃ to constant weight. The dry weight of the samples was then measured.

[0077] 4. Conclusion

[0078] As shown in Table 2: The roots of both tobacco and maize inoculated with AMF were successfully colonized by various structures of AMF (F. mosseae) (hyphae, arbuscular masses, vesicles, and hyphal rings). Figure 1 The colonization rate was 77.8% to 84.4%, indicating that AMF can colonize the roots of tobacco. The colonization rate of AMF in the uninoculated group was significantly lower than that in the inoculated group. Since the planting soil came from the field, AMF spores or mycelia were also present in the soil of the uninoculated group. Therefore, regardless of whether AMF was inoculated, AMF colonized the roots of the planted tobacco.

[0079] Table 2. AMF colonization rate of tobacco roots in different groups (mean ± standard error, n=3)

[0080] Group colonization rate (%) C+M- 27.8±3.11 C+M+ 77.8±3.27*** CM- 32.4±2.07 C-M+ 84.4±1.34***

[0081] Note: * indicates the difference in AMF colonization rate between the AMF-inoculated and non-AMF-inoculated groups in the same soil type (independent samples t-test, ***: p<0.001)

[0082] Table 3. Effects of different inoculation treatments and soil on tobacco growth and development (mean ± standard error, n = 5)

[0083]

[0084] Note: In the same column, the left * indicates a significant difference between the tobacco treatment groups in continuously cropped soil and non-continuously cropped soil under the same AMF treatment; the right * indicates a significant difference between the AMF-inoculated and non-AMF-inoculated treatment groups under the same soil conditions (*: 0.01≤p<0.05, **: 0.001≤p<0.01, ***: p<0.001, t-test; n=3-5, independentT-test). The bottom three rows represent two-way analysis of soil continuous cropping status and AMF inoculation (*: 0.01≤p<0.05, **: 0.001≤p<0.01, ***: p<0.001, two-way analysis; n=3~5).

[0085] As shown in Table 3 and Figure 3 As shown, compared with no inoculation, AMF inoculation significantly increased tobacco plant height, leaf number, and biomass. Under AMF inoculation conditions, the AMF-inoculated group in non-continuously cropped soil had significantly higher agronomic traits and biomass than the AMF-inoculated group in continuously cropped soil. Under no AMF inoculation conditions, tobacco plant height, leaf number, and aboveground biomass in non-continuously cropped soil were significantly higher than those in continuously cropped soil. The two-factor results indicate that continuous cropping conditions have a significant impact on tobacco plant height, stem circumference, and leaf number, while AMF inoculation has a significant impact on tobacco plant height, stem circumference, leaf length, leaf width, and leaf number. The interaction between the two significantly affects leaf length and leaf width.

[0086] like Figure 4 The study found that, compared to the uninoculated AMF treatment group, AMF inoculation significantly increased the net photosynthetic rate and intercellular CO2 concentration of tobacco. Furthermore, under AMF inoculation conditions, all photosynthetic parameters of tobacco in non-continuously cropped soils were significantly higher than those in continuously cropped soils. Similarly, for the uninoculated AMF treatment, all photosynthetic parameters of tobacco in continuously cropped soils were significantly higher than those in non-continuously cropped soils. Two-factor analysis showed that soil and AMF significantly affected the net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate of tobacco, and their interaction significantly influenced the net photosynthetic rate and intercellular CO2 concentration.

[0087] Example 2

[0088] An inoculant containing arbuscular mycorrhizal fungi and its method for regulating rhizosphere nutrients in tobacco-continuous cropping soil, comprising the following steps:

[0089] 1. Preparation of inoculum:

[0090] The same method for preparing the inoculum as in Example 1.

[0091] 2. Cultivation of tobacco transplanted seedlings:

[0092] The same method of cultivating tobacco transplanted seedlings as in Example 1 was used. The treatment of the inoculated group and the non-inoculated control group was the same as in Example 1. Soil samples were collected after cultivating tobacco seedlings under different treatments in cultivation containers for 45 days, resulting in 4 groups of samples: C+M- indicates continuous cropping soil without AMF inoculation; C+M+ indicates continuous cropping soil inoculated with AMF; CM- indicates non-continuous cropping soil without AMF inoculation; and C-M+ indicates non-continuous cropping soil inoculated with AMF.

[0093] 3. Harvesting methods for tobacco root soil:

[0094] Tobacco plants were carefully removed from their pots, roots and soil intact. The topsoil was removed, and loose soil around the roots was gently shaken off. Soil tightly bound to the roots was brushed off and mixed thoroughly to form the rhizosphere soil. The collected rhizosphere soil samples were transferred to a cool, shaded area to air dry and then sieved through a 60-mesh sieve. Three replicates were selected for each treatment to determine soil agrochemical properties: pH, available nitrogen, available phosphorus, available potassium, organic matter, total nitrogen, total phosphorus, and total potassium content. Simultaneously, soil nutrient and oxidation-related enzyme activities were measured: sucrase, catalase, polyphenol oxidase, and acid phosphatase. Soil pH was determined using the water extraction method; available nitrogen content was determined using the alkaline diffusion method; available phosphorus content was determined using the NaHCO3 extraction-molybdenum antimony colorimetric method; available potassium content was determined using flame photometry; and organic matter content was determined using the potassium dichromate titration method (specific methods refer to "Soil Agrochemical Analysis"). Soil enzyme activity kits were purchased from Suzhou Grees Biotechnology Co., Ltd.

[0095] 4. Conclusion:

[0096] like Figure 5As shown, different continuous cropping and inoculation conditions had little effect on soil pH. Compared with the uninoculated group, the available phosphorus content in the tobacco rhizosphere soil increased after AMF inoculation, while the contents of organic matter, available potassium, total nitrogen, total phosphorus, and total potassium all decreased. Furthermore, the available phosphorus and available potassium contents in the continuously cropped soil inoculated group were higher than those in the non-continuously cropped soil inoculated with AMF. Under the uninoculated AMF condition, the contents of available nitrogen, available phosphorus, and available potassium in the tobacco rhizosphere soil under continuous cropping conditions were significantly higher than those under non-continuous cropping conditions. The two-factor analysis in Table 4 shows that soil significantly affected the contents of available phosphorus and available potassium, while AMF significantly affected the contents of organic matter, available phosphorus, and available potassium. The interaction between the two significantly affected the contents of organic matter, available nitrogen, and available phosphorus.

[0097] Table 4 Results of the two-factor analysis

[0098]

[0099] like Figure 6 As shown in Table 5, under continuous cropping conditions, AMF inoculation significantly increased soil catalase and sucrase activity by 50.19% and 63.36%, respectively. Under non-continuous cropping conditions, AMF inoculation significantly increased soil catalase, acid phosphatase, and sucrase activity by 49.31%, 57.75%, and 46.88%, respectively. Overall, AMF inoculation effectively increased enzyme activity in tobacco rhizosphere soil, thereby increasing the content of available nutrients in the soil. The results indicate that AMF inoculation reduced soil nutrient content and increased the activities of soil catalase, sucrase, and acid phosphatase.

[0100] Table 5 Enzyme activity in tobacco rhizosphere soil of different treatment groups

[0101] Group S-CAT (umol / h / g) S-ACP (umol / h / g) S-SC (mg / d / g) PPO (nmol / h / g) C+M- 224.98±24.34 868.63±7.36 4.23±0.05 443.57±13.83 C+M+ 337.89±9.85*** 954.43±63.87 6.91±0.06*** 429.79±24.54 CM- ***291.88±9.22 *885.72±3.65 ***7.38±0.06 ***735.09±37.16 C-M+ *435.82±49.37** **1397.2±8.22*** ***10.84±0.15*** ***753.99±30.43 soil *** *** *** *** AMF *** *** *** NS Soil *AMF NS *** *** NS

[0102] Example 3

[0103] An inoculant containing arbuscular mycorrhizal fungi and its method for regulating nutrients in tobacco plants, comprising the following steps:

[0104] 1. Preparation of inoculum:

[0105] The same method for preparing the inoculum as in Example 1.

[0106] 2. Cultivation of tobacco transplanted seedlings:

[0107] The same method of cultivating tobacco transplanted seedlings as in Example 1 was used. The treatment of the inoculated group and the non-inoculated control group was the same as in Example 1. Soil samples were collected after cultivating tobacco seedlings under different treatments in cultivation containers for 45 days, resulting in 4 groups of samples: C+M- indicates continuous cropping soil without AMF inoculation; C+M+ indicates continuous cropping soil inoculated with AMF; CM- indicates non-continuous cropping soil without AMF inoculation; and C-M+ indicates non-continuous cropping soil inoculated with AMF.

[0108] 3. Harvesting methods for tobacco plants:

[0109] Samples were collected after 45 days of cultivation in the tobacco greenhouse. The plants were carefully removed, the soil attached to the roots was shaken off, the roots were washed with clean water, and excess water was absorbed with filter paper. The fresh weight of the above-ground and underground parts of the crop was weighed separately. A small amount of fresh root samples were taken and acid fuchsin staining was used to observe whether F. mosseae had colonized and the colonization rate.

[0110] The remaining samples were placed in kraft paper envelopes and placed in an oven at 105℃ for 30 minutes to kill the green, then dried at 75℃ to constant weight. The dry weight of the samples was then measured. The samples were then pulverized and mixed using a micro pulverizer to measure the content of crop nutrients N, P, and K, according to the method in NY / T2017-2011. Three replicate samples were set up for each treatment group.

[0111] 4. Conclusion

[0112] like Figure 7 As shown in Table 6, compared with the uninoculated group, AMF inoculation significantly promoted the accumulation of nitrogen, phosphorus and potassium in the underground parts of the plant. In non-continuous cropping soil, the nutrient accumulation content (except underground phosphorus content) of each part of the AMF-inoculated group was significantly higher than that of the uninoculated group. Under the condition of no AMF inoculation, the aboveground and underground total nitrogen content of tobacco under the non-continuous cropping soil condition was significantly higher than that of the continuous cropping treatment group.

[0113] Table 6 Nutrient content of aboveground and underground parts of tobacco in different treatment groups

[0114]

[0115]

[0116] Two-factor analysis showed that soil significantly affected the accumulation of nitrogen in the aboveground parts of tobacco and phosphorus in the aboveground and underground parts. AMF (ammonia pilosa) significantly affected the accumulation of nitrogen, phosphorus, and potassium in tobacco plants, and the interaction between the two significantly affected the accumulation of phosphorus and nitrogen in the aboveground parts of the plants. The results indicate that AMF inoculation promotes the absorption of nutrients, especially phosphorus, by tobacco.

[0117] Example 4

[0118] A method for jointly regulating the content of allelochemicals in soil under different continuous cropping conditions using AMF and soil, the specific steps of which are as follows:

[0119] 1. Preparation of inoculum:

[0120] The same method for preparing the inoculum as in Example 1.

[0121] 2. Cultivation of tobacco transplanted seedlings:

[0122] The same method of cultivating tobacco transplanted seedlings as in Example 1 was used. The treatment of the inoculated group and the non-inoculated control group was the same as in Example 1. Soil samples were collected after cultivating tobacco seedlings under different treatments in cultivation containers for 45 days, resulting in 4 groups of samples: C+M- indicates continuous cropping soil without AMF inoculation; C+M+ indicates continuous cropping soil inoculated with AMF; CM- indicates non-continuous cropping soil without AMF inoculation; and C-M+ indicates non-continuous cropping soil inoculated with AMF.

[0123] 3. Harvesting methods for tobacco plants:

[0124] Before tobacco planting, the contents of allelochemicals such as p-hydroxybenzoic acid, vanillic acid, p-coumaric acid, benzoic acid, cinnamic acid, ferulic acid, and myristic acid in continuously cropped and non-continuously cropped soils were measured. After 45 days of cultivation, tobacco seedlings under different treatments were carefully removed from their pots along with their roots and soil. The topsoil was removed, and the loose soil bound to the roots was gently shaken off. The soil tightly bound to the roots was brushed off and mixed thoroughly to form the rhizosphere soil. The collected rhizosphere soil samples were stored at -80℃ and the contents of p-hydroxybenzoic acid, vanillic acid, p-coumaric acid, benzoic acid, cinnamic acid, ferulic acid, and myristic acid were measured as soon as possible. The instruments used for measurement were an Agilent 1260 Xenometer tandem with a 6420A mass spectrometer, and the measurement method was high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS). The specific measurement steps are as follows:

[0125] 3.1. Standard Products

[0126] Standard: p-Hydroxybenzoic acid (Shanghai Yuanye, CAS: 99-96-7)

[0127] 3.2. Plot the standard curve

[0128] Use a 1000 μg / mL phenolic acid component mixed standard solution to dilute with methanol to prepare a series of mixed standard solutions of 10 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL, 2000 ng / mL, 5000 ng / mL, 10000 ng / mL, 20000 ng / mL, and 50000 ng / mL. Prepare and use immediately.

[0129] 3.3. Sample Extraction

[0130] (1) Weigh 1g of sample, add 10mL of 1mol / L sodium hydroxide aqueous solution, shake and extract on a shaking shaker at 40℃ for 2h, centrifuge at 4℃ and 8000rpm for 5min, and take the supernatant.

[0131] (2) Add 10 mL of 1 mol / L potassium hydroxide methanol solution to the precipitate, and extract it by shaking on a shaker at 40°C for 2 h. Centrifuge at 4°C and 8000 rpm for 5 min, collect the supernatant, and combine the two supernatants.

[0132] (3) Adjust the pH of the supernatant to 2.0 by dissolving it in hydrochloric acid, and concentrate it to dryness using a rotary evaporator;

[0133] (4) Add 10 mL of ethyl acetate and 10 mL of water to the residue from step 3, shake for 2 min, take the upper ethyl acetate phase, and repeat the extraction 3 times.

[0134] (5) Combine the ethyl acetate phases, add 0.5g of anhydrous sodium sulfate to remove the residual water, centrifuge at 8000rpm for 2min at 4℃, take the supernatant, and concentrate to dryness at 30℃ using a vacuum freeze concentrator.

[0135] (6) Add 2.0 mL of methanol to redissolve, vortex mix, filter through a 0.22 μm organic filter membrane, and wait for the instrument to detect. The entire pretreatment process must be protected from light.

[0136] 3.4. Chromatographic conditions

[0137] Xenoco column: Agilent Poroshell 120EC-C18 (2.7μm, 3*100mm); column temperature: 35℃; mobile phase: A: water (0.1% formic acid); B: acetonitrile; flow rate: 0.3mL / min; injection volume: 2μL;

[0138] 3.5. Mass Spectrometry Parameters

[0139] Ionization mode: ESI positive ion mode

[0140] Scan type: Mass spectrometry multiple reaction monitoring (MRM) technology.

[0141] 4. Conclusion:

[0142] As shown in Table 7, the contents of p-hydroxybenzoic acid and vanillic acid in the continuously cropped soil collected before tobacco planting were significantly higher than those in the non-continuously cropped soil, reaching 1596.65 ng / g and 1329.31 ng / g, respectively, while the contents of coumaric acid, ferulic acid, and myristic acid were significantly lower than those in the non-continuously cropped soil. Benzoic acid and cinnamic acid were not detected.

[0143] Table 7. Differences in allelic substance content in basic soils between tobacco continuous cropping and non-continuous cropping.

[0144]

[0145]

[0146] Note: * indicates a significant difference between treatment groups under independent samples t-test (*p<0.05, **p<0.01, ***p<0.001), NS indicates that the substance was not detected.

[0147] As shown in Table 8, after planting tobacco, compared with the treatment group without AMF inoculation, the contents of p-hydroxybenzoic acid, vanillic acid, p-coumaric acid, ferulic acid and myristic acid in the soil treated with AMF inoculation were reduced, while the contents of benzoic acid and cinnamic acid were increased. The change trends of the contents of the seven phenolic acids were consistent under continuous cropping and non-continuous cropping soil conditions. For tobacco continuous cropping soil, after inoculation, the contents of p-hydroxybenzoic acid, vanillic acid, p-coumaric acid, ferulic acid, and myristic acid decreased by 25.88%, 5.67%, 50.04%, 16.38%, and 792.08%, respectively, while the contents of benzoic acid and cinnamic acid increased by 50.44% and 80.23%, respectively. For tobacco non-continuous cropping soil, after inoculation, the contents of p-hydroxybenzoic acid, vanillic acid, p-coumaric acid, ferulic acid, and myristic acid decreased by 19.16%, 13.47%, 37.33%, 19.33%, and 9.26%, respectively, while the contents of benzoic acid and cinnamic acid increased by 190.51% and 33.85%, respectively.

[0148] In summary, the results indicate that AMF inoculation can reduce the content of p-hydroxybenzoic acid, vanillic acid, p-coumaric acid, ferulic acid, and myristic acid in the soil, while increasing the content of benzoic acid and cinnamic acid. P-hydroxybenzoic acid inhibits tobacco root growth and reduces its physiological activity, suppressing root potassium absorption and the expression of related genes. Vanillic acid promotes the growth of p-hydroxybenzoic acid. P-coumaric acid can bind to phosphate anions in the DNA double helix structure, inserting into the grooves of the DNA double helix structure, inhibiting bacterial cell function during DNA replication, transcription, and expression. Myristic acid has a certain allelopathic promoting effect on the growth of Fusarium root rot mycelia. Furthermore, p-hydroxybenzoic acid, ferulic acid, and vanillic acid exhibit significant synergistic effects. Cinnamic acid and benzoic acid strongly inhibit the activity of cucumber dehydrogenase, ATP-binding enzyme, and nitrate reductase, thereby inhibiting the absorption of potassium by plant roots. + NO3 - and H2PO4 - The absorption of AMF. That is, AMF inoculation can reduce the content of many harmful allelopathic substances in tobacco-growing soil, while the content of a small number of harmful allelopathic substances increases.

[0149] Table 8. Effects of different inoculation treatments on the accumulation of phenolic acids in tobacco rhizosphere soil (mean ± standard error, n = 4)

[0150]

[0151]

[0152] Note: * indicates a significant difference between treatment groups under independent samples t-test (*p<0.05, **p<0.01, ***p<0.001). In two-way analysis, * indicates that the independent variable has a significant effect on the dependent variable under LSD and two-tailed tests (NS: p>0.05, *p<0.05, **p<0.01, ***p<0.001). The right-hand * indicates the difference between AMF-inoculated and non-AMF-inoculated treatment groups under the same soil conditions, and the left-hand * indicates the difference between continuously cropped soil and non-continuously cropped soil treatment groups under the same inoculation conditions.

[0153] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for alleviating tobacco continuous cropping obstacles, characterized in that, include: Tobacco is planted in tobacco-continuous-cropping soil containing an inoculum containing arbuscular mycorrhizal fungi; The inoculant containing arbuscular mycorrhizal fungi includes plant roots, plant cultivation substrate, and arbuscular mycorrhizal fungi; the arbuscular mycorrhizal fungi colonize the plant roots and plant cultivation substrate, and the colonization rate of the arbuscular mycorrhizal fungi is not less than 90%. The plant root system is a corn root system; The arbuscular mycorrhizal fungus is *B. mossiosus*, with preservation number BGCYN05, 1511C0001BGCAM0013. Funneliformis mosseae ); The preparation method of the inoculant containing arbuscular mycorrhizal fungi includes the following steps: mixing rhizosphere soil containing arbuscular mycorrhizal fungal hyphae and spores with sterile plant cultivation substrate to obtain a mixed cultivation substrate; planting sterile plant seedlings in the mixed cultivation substrate until the colonization rate of arbuscular mycorrhizal fungi is not less than 90%; collecting plant roots and rhizosphere cultivation substrate to obtain the inoculant containing arbuscular mycorrhizal fungi.

2. The method according to claim 1, characterized in that, The plant cultivation substrate includes farmland soil and vermiculite, and the volume ratio of farmland soil to vermiculite is (2~3):

1.

3. The method according to claim 1, characterized in that, The inoculum containing arbuscular mycorrhizal fungi is in a weight ratio of 1:40 to the tobacco continuous cropping soil.

4. The method according to claim 1, characterized in that, The tobacco plant is a tobacco seedling with four leaves and two buds; the amount of the inoculum containing arbuscular mycorrhizal fungi is 200g per tobacco plant.

5. The application of the method according to any one of claims 1 to 4 in alleviating tobacco continuous cropping obstacles, characterized in that, The method of alleviating tobacco continuous cropping obstacles is to conserve and remediate tobacco continuous cropping soil; the method of conserving and remediating tobacco continuous cropping soil is to regulate the content of allelochemicals in tobacco continuous cropping soil; the method of regulating the content of allelochemicals in tobacco continuous cropping soil is to reduce the content of p-hydroxybenzoic acid, vanillic acid, p-coumaric acid, ferulic acid and myristic acid in the soil.

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