Method for regulating and controlling corn and tobacco growth and rhizosphere soil nutrients through combination of AMF and planting mode

Abstract

The invention belongs to the technical field of soil conservation, and particularly relates to a method for promoting plant growth and regulating soil nutrients. The invention discloses a method for regulating and controlling corn and tobacco growth and rhizosphere soil nutrients through combination of AMF and a planting mode. The method comprises the following steps: preparing an AMF-containing microbial inoculum by using M. mosseae (F. mosseae); cultivating flue-cured tobacco seedlings and corn seedlings; flue-cured tobacco seedlings and corn seedlings are jointly planted in an intercropping mode by utilizing an AMF-containing fungicide. The method can relieve continuous cropping obstacles of flue-cured tobaccos and promote growth of the flue-cured tobaccos and corn, and has a certain effect on soil conservation.

Description

Technical Field

[0001] The invention belongs to the technical field of soil conservation, and in particular relates to a method for promoting plant growth and regulating soil nutrients. Background Art

[0002] Tobacco (Nicotiana tabacum) belongs to the genus Nicotiana in the Solanaceae family. Continuous tobacco cropping will lead to a monotonous soil microbial community structure, resulting in a decline in soil quality in tobacco fields, which in turn affects tobacco plant growth and yield quality. The arable land area in Yunnan tobacco-growing areas is limited, and the problem of soil deterioration in tobacco-growing areas is becoming increasingly serious. Corn (Zea mays) is an important food crop and feed crop. Silage corn has the advantages of a short production cycle, high yield, and good green retention. Compared with flue-cured tobacco monoculture, intercropping food crops such as corn after tobacco can alleviate the problem of food and tobacco competing for land, improve the multiple cropping index, and increase farmers' income.

[0003] If grain crops are planted directly after tobacco, the low-lying corn will be affected by the high-lying tobacco during the symbiotic period. For example, shading will lead to insufficient light and temperature, the tobacco root system is much larger than the corn root system, and fertilizer cannot be applied during the symbiotic period, resulting in low nutrient absorption capacity of corn. The above factors will cause the corn to grow slowly. At the end of the symbiotic period, the corn root system is still small and cannot interact with the tobacco root system. It is difficult to explore the regulatory effect of the interaction between the roots of the two on soil nutrients after planting for one year.

[0004] Arbuscular mycorrhiza fungi (AMF) are a class of beneficial soil microorganisms widely distributed in terrestrial ecosystems. About 80% of terrestrial plants on Earth can form symbioses with them. At present, many studies have been conducted on AMF in promoting corn and tobacco seedling reproduction, growth and development, and enhancing abiotic stress tolerance. However, there is no systematic report on the comprehensive mechanism of AMF combined with different planting patterns on the rhizosphere soil nutrients of crops. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for jointly regulating the growth of corn and tobacco and rhizosphere soil nutrients by using AMF and planting patterns.

[0006] In order to solve the above technical problems, the present invention provides a method for jointly regulating the growth of corn and tobacco and rhizosphere soil nutrients by AMF and planting pattern, comprising the following steps:

[0007] 1) Using F. mosseae to prepare an AMF-containing bacterial agent;

[0008] 2) Cultivation of flue-cured tobacco seedlings and corn seedlings;

[0009] 3) Using the AMF-containing bacterial agent obtained in step 1), the flue-cured tobacco seedlings and corn seedlings obtained in step 2) are intercropped and planted together.

[0010] As an improvement of the method of the present invention, step 1) is:

[0011] The soil (farmland soil) and vermiculite (horticultural vermiculite) are mixed in a volume ratio of 2:1, and then sterilized to obtain a sterile cultivation medium 1;

[0012] First, a sterile cultivation substrate is added to the bottom of the sterile container (sterile cultivation container) as a bottom layer, and then the surface of the bottom layer is covered with rhizosphere soil containing hyphae and spores of F. mosseae, and the surface of the rhizosphere soil is covered with a sterile cultivation substrate as a top layer to form a "sandwich";

[0013] Transplant the sterile corn seedlings (three leaves and one heart) into the rhizosphere soil of the above-mentioned sterile container, irrigate with 1 / 2 Hoagland nutrient solution (i.e., irrigate with 1 / 2 Hoagland nutrient solution until the nutrient solution seeps out from the bottom of the container), irrigate with 1 / 2 Hoagland nutrient solution once every 20±1 days, and co-cultivate until the colonization rate of F. mosseae at the roots of corn is not less than 90%, cut off the above-ground part of the corn and remove the top 1-2 cm sterile cultivation substrate, and then collect the obtained contents in the sterile container (including the root system and the culture substrate, etc.) to obtain a bacterial agent containing AMF.

[0014] In the present invention, Huidan No. 4 corn is used as a host plant, and arbuscular mycorrhizal fungi (AMF) are inoculated into the roots of the corn, and the arbuscular mycorrhizal fungi is Funneliformis mosseae.

[0015] As a further improvement of the method of the present invention, in step 1): the soil (farmland soil) is sieved through a 10-mesh sieve; and the corn is Huidan No. 4.

[0016] As a further improvement of the method of the present invention, in step 1), the co-cultivation time is 60±5 days.

[0017] As a further improvement of the method of the present invention, the formula of the 1 / 2 Hoagland nutrient solution in the step 1) is: 94.5 g / L calcium nitrate, 8 g / L ammonium nitrate, 50.6 g / L potassium nitrate, 13.6 g / L potassium dihydrogen phosphate, 100.9 g / L magnesium sulfate heptahydrate, 7.46 g / L Na2EDTA·2H2O, 5.56 g FeSO4·7H2O, 4.46 g / L MnSO4·4H2O, 1.24 g / L H3BO3, 1.72 g / L ZnSO4·7H2O, 0.005 g / L CuSO4·5H2O, 0.005 g CoCl2·6H2O, 0.166 g KI, and 0.05 g / L Na2MoO4·2H2O.

[0018] As a further improvement of the method of the present invention, the step 2) is:

[0019] Flue-cured tobacco seeds (K326) were grown in a floating manner using a sterile substrate for 45±1 days to obtain flue-cured tobacco seedlings that could be transplanted;

[0020] Corn seeds (Yunrui 319) were grown with sterile vermiculite until they had three leaves and one heart, and then the corn seedlings were ready for transplanting.

[0021] As a further improvement of the method of the present invention, step 2) is:

[0022] 1. Preparation of flue-cured tobacco seedlings (floating seedling cultivation):

[0023] First, the tobacco floating seedling medium (special seedling medium for flue-cured tobacco) is sterilized to obtain a sterile medium; sterile water is added to the sterile medium and mixed evenly (the water content is controlled to be 50% to 60%, that is, the medium can be kneaded into a ball in the hand and falls apart when touched) to obtain a moistened medium;

[0024] Place the moistened substrate into the holes of the sterile seedling tray, make a seed hole with a depth of 0.5±0.1cm in each hole, put 1-2 flue-cured tobacco seeds (tobacco coated seeds), and then cover with the moistened substrate;

[0025] Add tobacco seedling fertilizer to the seedling pool, and then add water until the floating plate is flush with the surface of the seedling pool; cultivate for 45±2 days to obtain flue-cured tobacco seedlings that can be transplanted; the tobacco seedling fertilizer: water = 1±0.1g / 1L;

[0026] 2. Sterile corn seedlings:

[0027] After the corn seeds are disinfected, they are first cultured on sterile double-layer filter paper in the dark until the corn sprouts, and then the seedlings are grown on sterile vermiculite until they have three leaves and one heart, and the corn seedlings are ready for transplanting.

[0028] As a further improvement of the method of the present invention, the step 3) is:

[0029] The soil (farmland soil) and vermiculite (horticultural vermiculite) are mixed in a volume ratio of 3:1, and then sterilized to obtain a sterile cultivation medium 2;

[0030] A layer of sterile cultivation substrate 2 (accounting for about 2 / 3 of the volume of the container) is spread on the bottom of the container, and the inoculant containing AMF is added, and then the flue-cured tobacco seedlings and corn seedlings obtained in step 2) are transplanted in an intercropping manner (i.e., the flue-cured tobacco seedlings and sterile corn seedlings are placed on the surface of the inoculant layer), and then the inoculant containing AMF is added, and finally a layer of sterile cultivation substrate 2 is spread (flush with the top of the container);

[0031] The relationship between the total weight of the AMF-containing inoculants used twice and the total number of transplanted flue-cured tobacco and corn seedlings was 200 ± 20 g / plant;

[0032] That is, in the present invention: the inoculant containing AMF is added twice, before and after transplanting (the same amount can be added).

[0033] As a further improvement of the method of the present invention, in step 3), the spacing between the flue-cured tobacco seedlings and the corn seedlings is 30±3 cm.

[0034] As a further improvement of the method of the present invention, the planting time of step 3) is 45±2 days.

[0035] Note: After planting, the agronomic traits, photosynthetic characteristics and chlorophyll content of the crops were measured, and plant and soil samples were collected. That is, after planting, the samples were harvested and the indicators were measured to clarify the effects of AMF and different planting patterns on the growth of corn and tobacco and rhizosphere soil nutrients.

[0036] In the invention process, the present invention adopts a two-factor completely random group design experiment, and the variables are two factors: inoculation of AMF and planting mode.

[0037] There were two levels of AMF inoculation: AMF inoculation and no AMF inoculation; there were three planting patterns: maize monoculture, tobacco monoculture, and maize and tobacco intercropping. There were 6 different planting treatment groups, each treatment was repeated 12 times, and tobacco and maize samples were collected after the cultivation, and 8 different groups of samples were obtained, namely: ① Tobacco monoculture without AMF inoculation: KD M-; ② Tobacco monoculture with AMF inoculation: KDM+; ③ Maize monoculture without AMF inoculation: YD M-; ④ Maize monoculture with AMF inoculation: YD M+; ⑤ Tobacco intercropped with tobacco and maize without AMF inoculation: KJ M-; ⑥ Tobacco intercropped with tobacco and maize with AMF inoculation: KJ M+; ⑦ Maize intercropped with tobacco and maize without AMF inoculation: YJ M-; ⑧ Maize intercropped with tobacco and maize with AMF inoculation: YJ M+. The experimental cultivation container was a rectangular flower pot (50cm×20cm×18cm), each pot contained about 7-8kg of crop cultivation substrate.

[0038] The AMF (F. mosseae) of the present invention was purchased from the Institute of Plant Nutrition and Resources of Beijing Agricultural and Forestry College, with the deposit number of BGC YN05,1511C0001BGCAM0013.

[0039] Based on early research, the present invention uses pot experiments to achieve full interaction between the root systems of corn and flue-cured tobacco, and at the same time, through exogenous inoculation of AMF, explores the combined regulatory effect of AMF and planting pattern on the growth of corn and tobacco and rhizosphere soil nutrients.

[0040] The exogenous addition of F. mosseae in the present invention can expand the absorption area of ​​the host plant root system, promote plant growth and nutrient absorption and utilization, increase crop yield and improve soil physical and chemical properties. Taking the tobacco variety K326 widely planted in Yunnan, silage corn Yunrui 319 and AMF F. mosseae as research objects, the influence of the combined action of arbuscular mycorrhizal fungi and planting patterns on the growth of flue-cured tobacco and corn and the regulation of soil nutrients are explored.

[0041] The present invention has the following technical advantages:

[0042] (1) The present invention uses AMF (F. mosseae) to inoculate the roots of crops and utilizes different planting patterns to improve the agronomic traits of crops and increase the chlorophyll content and biomass of flue-cured tobacco and corn leaves.

[0043] (2) The present invention improves soil physical and chemical properties by inoculating AMF (F. mosseae), increases soil pH, alkaline nitrogen content, soil respiration rate and soil respiration rate of corn roots, and increases the effective phosphorus content of corn root soil under monoculture; AMF significantly increases the activities of acid phosphatase, urease, phytase in tobacco root soil and urease and sucrase in corn root soil, thereby regulating soil nutrients and promoting the absorption of total nitrogen, total phosphorus and total potassium nutrients by crops. Compared with monoculture, the interaction between the tobacco cluster stage and the corn small bell mouth stage significantly increases the stem girth and fresh weight of tobacco; increases the alkaline nitrogen, effective phosphorus and quick-acting potassium content of corn rhizosphere soil and the soil respiration rate of tobacco roots, and promotes the absorption of total potassium content by tobacco and corn roots. It provides a certain theoretical basis for interplanting corn after the second shed of flue-cured tobacco is harvested. Since corn is in the small bell mouth stage at the end of the symbiotic period of corn and flue-cured tobacco, which is consistent with the interaction time of corn and flue-cured tobacco in this experiment, it provides a theoretical research basis for the conservation of soil for planting corn after tobacco.

[0044] In summary, the present invention discloses a method for jointly regulating the growth of corn and tobacco and rhizosphere soil nutrients by arbuscular mycorrhizal fungi (AMF) and planting patterns. The present invention utilizes Funneliformis mosseae to be inoculated into the roots of corn and tobacco for colonization, and combines different planting patterns to regulate soil nutrients. Inoculation of AMF has a significant promoting effect on the agronomic traits of flue-cured tobacco, increases the soil enzyme activity of the flue-cured tobacco root system, and promotes the absorption and utilization of nutrients by crops. The present invention can alleviate the obstacles of continuous cropping of flue-cured tobacco, promote the growth of flue-cured tobacco and corn, and has a certain effect on soil conservation. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0046] Figure 1-Figure 6 In the data, KDM+ indicates that K326 was inoculated with AMF as a single crop; KDM- indicates that K326 was not inoculated with AMF as a single crop; KJM+ indicates that K326 was intercropped with tobacco inoculated with AMF and Yunrui 319; KJM- indicates that K326 was intercropped with tobacco inoculated with AMF and Yunrui 319 was not inoculated with AMF; YDM+ indicates that Yunrui 319 was inoculated with AMF as a single crop; YDM- indicates that Yunrui 319 was not inoculated with AMF as a single crop; YJM+ indicates that Yunrui 319 was intercropped with corn inoculated with AMF and K326; YJM- indicates that Yunrui 319 was intercropped with corn inoculated with AMF and K326 was not inoculated with AMF.

[0047] * indicates that the data between the two groups are significantly different (*: 0.01≤p<0.05, **: 0.001≤p<0.01, ***: p<0.001, t-test; n=3-4). The table in the bar graph represents the two-way anova of planting pattern and AMF inoculation (*: 0.01≤p<0.05, **: 0.001≤p<0.01, ***: p<0.001, two-way anova; n=3-4).

[0048] Figure 1 Agronomic traits of flue-cured tobacco (K) and corn (Y) in different treatment groups;

[0049] Figure 2 SPAD content and photosynthetic parameters of flue-cured tobacco (K) and corn (Y) leaves in different treatment groups;

[0050] Figure 3 The biomass of flue-cured tobacco (K) and corn (Y) in different treatment groups;

[0051] Figure 4 The nutrient contents of aboveground and underground parts of flue-cured tobacco (K) and corn (Y) in different treatment groups;

[0052] Figure 5 The physicochemical properties and rhizosphere soil respiration rate of flue-cured tobacco (K) and corn (Y) in different treatment groups;

[0053] Figure 6 Rhizosphere soil enzyme activities of flue-cured tobacco (K) and corn (Y) in different treatment groups;

[0054] Figure 7 The AMF (F. mosseae) structure in the root cells of potted flue-cured tobacco (K) (left) and corn (Y) (right);

[0055] Figure 8 This is a comparison of potted plants with different treatments 40 days after transplanting. M+ indicates inoculation with AMF, and M- indicates no inoculation with AMF. DETAILED DESCRIPTION

[0056] The present invention is further described below in conjunction with specific embodiments and drawings, but the protection scope of the present invention is not limited thereto:

[0057] The AMF (F. mosseae) of the present invention is from the Institute of Plant Nutrition and Resources of Beijing Agricultural and Forestry College, and the deposit number is BGC YN05,1511C0001BGCAM0013.

[0058] The formula of 1 / 2 Hoagland nutrient solution is: calcium nitrate 94.5g / L, ammonium nitrate 8g / L, potassium nitrate 50.6g / L, potassium dihydrogen phosphate 13.6g / L, magnesium sulfate heptahydrate 100.9g / L, Na2EDTA·2H2O 7.46g / L, FeSO4·7H2O 5.56g, MnSO4·4H2O 4.46g / L, H3BO3 1.24g / L, ZnSO4·7H2O 1.72g / L, CuSO4·5H2O 0.005g / L, CoCl2·6H2O 0.005g, KI 0.166g, Na2MoO4·2H2O 0.05g / L.

[0059] The tobacco floating seedling substrate can be purchased commercially, for example, from Yunnan Province Mile City Tobacco Materials Co., Ltd.

[0060] K326 coated seeds are provided by Yuxi China Tobacco Seed Co., Ltd.

[0061] Example 1: A method for jointly regulating the growth of corn and tobacco and rhizosphere soil nutrients by AMF and different planting patterns, the specific steps are as follows:

[0062] 1. Preparation of inoculant

[0063] (1.1) Preparation of cultivation medium:

[0064] The farmland soil was sieved through a 10-mesh sieve to remove large pieces of soil. The sieved soil was mixed evenly with vermiculite (horticultural vermiculite) in a volume ratio of 2:1, 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 soil was cooled to room temperature to obtain a sterile cultivation medium for inoculant propagation.

[0065] The horticultural vermiculite was sterilized in a high pressure steam autoclave at 121°C for 2 hours, and the sterilization was repeated every 24 hours for a total of 3 times. The vermiculite was then cooled to room temperature to obtain sterile vermiculite.

[0066] (1.2) Sterilize the surface of corn seeds and then culture until the corn sprouts to obtain sterile corn seedlings:

[0067] Select the Huidan No. 4 corn seeds with basically the same size, shape and fullness, disinfect them with 8% sodium hypochlorite for 8 minutes, rinse them with sterile water for 3 times, place them in a culture dish with double-layer sterile filter paper, and culture them in the dark until the corn sprouts (temperature is about 28°C); use sterile vermiculite to grow seedlings until they have three leaves and one heart, and obtain sterile corn seedlings. The seedling raising process is carried out in a greenhouse.

[0068] (1.3) Thoroughly scrub the flower pot (350×250mm) with 75% alcohol as a sterile flower pot (sterilized flower pot), dry it, and write a label. Lay the sterile cultivation substrate prepared in step (1.1) at the bottom of the sterilized flower pot for 3 to 5 cm as the bottom layer; weigh about 700g of rhizosphere soil containing AMF (F. mosseae) hyphae and spores purchased from the Institute of Plant Nutrition and Resources of Beijing Agricultural and Forestry College per pot, and spread it flat on the sterile cultivation substrate as the bottom layer in the sterile flower pot. Transplant the sterile corn seedlings obtained in step (1.2) into the rhizosphere soil of the sterile flower pot, and then cover it with 3 to 5cm of sterile cultivation substrate as the top layer, thus forming a "sandwich" method for propagation. AMF (F.mosseae); 1 / 2 Hoagland nutrient solution was used to irrigate the planting soil, and routine management was performed during the growth period to ensure the normal growth of the plants. 1 / 2 Hoagland nutrient solution was poured in (irrigated) every 20 days until the nutrient solution seeped out from the bottom. After culturing for about 30 days, the roots of corn were randomly inspected, and the colonization of AMF (F.mosseae) in the roots of corn was observed and counted using the cross-cross method under an optical microscope. Successful colonization was considered when typical structures of AMF, such as hyphae, arbuscules, vesicles, and hyphae circles, were observed.

[0069] After about 60 days of cultivation, when the colonization rate of AMF (F. mosseae) in the corn roots is not less than 90%, the above-ground part of the corn is cut off and the top 1-2 cm of the sterile cultivation substrate is removed (i.e., 1-2 cm of the topsoil is removed), and then the obtained materials (including the root system and the culture substrate, etc.) in the sterile container are collected to obtain the bacterial agent containing AMF.

[0070] Funneliformis mosseae colonization rate (%) = (total number of visual fields - number of blank visual fields) / total number of visual fields × 100%.

[0071] 2. Cultivation of flue-cured tobacco seedlings and corn seedlings:

[0072] (2.1) First, sterilize the tobacco floating seedling medium with high pressure steam (sterilization conditions are 121°C, 2h; repeat the sterilization once every 24h, for a total of 3 times, to obtain a sterile medium;

[0073] The sterile matrix is ​​mixed with appropriate sterile water (the moisture content is controlled to be 50% to 60%), that is, when the matrix can be kneaded into a ball in the hand and falls apart when touched, the matrix is ​​moistened;

[0074] After placing the moistened substrate into the holes of a sterile seedling tray (the holes of a seedling tray sterilized by wiping with 75% ethanol), the seedling tray is evenly shaken and filled with the moistened substrate (i.e., the seedling tray is raised 20-30 cm from the ground, and then thrown flat, repeated 2-3 times, and the substrate of the seedling tray is filled after shaking);

[0075] Press a 0.5cm deep seed hole on each hole, then put in 1-2 K326 coated seeds (provided by Yuxi Zhongyan Seed Co., Ltd.), and cover lightly with 0.5cm moistened substrate. Weigh 25g of tobacco seedling fertilizer (N:P2O4:K2O=18:12:13; Honghe Henglin Chemical Co., Ltd.) into the seedling pool, add water (about 25L of water) until the floating plate is flush with the surface of the seedling pool. Cultivate in the conventional way for about 45 days to obtain flue-cured tobacco seedlings of a certain seedling type.

[0076] (2.2) Aseptic germination of corn seedlings: Select Yunrui 319 corn seeds of basically uniform size, shape and fullness, sterilize them with 8% sodium hypochlorite for 8 minutes, rinse them with sterile water for 3 times, place them in a culture dish with sterile double-layer filter paper, and culture them in the dark until the corn sprouts (temperature is about 28°C); then use sterile vermiculite to grow the seedlings until they have three leaves and one heart, and then transplant them.

[0077] Note: Generally, corn (Yunrui 319) should be germinated aseptically 15 days before transplanting.

[0078] 3. Planting:

[0079] The farmland soil was sieved through a 10-mesh sieve to remove large pieces of soil. The sieved soil was mixed with horticultural vermiculite in a volume ratio of 3:1, placed in a high-pressure steam sterilizer, and sterilized at a temperature of 121°C for 2 hours. The sterilization was repeated once every 24 hours for a total of 3 times, and then naturally air-dried to obtain a sterile cultivation medium 2.

[0080] The inoculant containing AMF obtained in step (1.3) was divided into two equal parts, one of which was used as the inoculation group; the other was sterilized by high-pressure steam (121°C, sterilized for 2 hours, and repeated sterilization 3 times with an interval of 24 hours), and naturally air-dried to obtain inactivated AMF as the non-inoculated control group.

[0081] The planting patterns are monoculture and intercropping, namely: flue-cured tobacco K326 monoculture, corn Yunrui 319 monoculture and flue-cured tobacco intercropped with corn. Use 75% alcohol to thoroughly scrub the flower pots (50cm×20cm×18cm) as sterile containers, dry them, mark them with numbers, and lay a layer of sterile cultivation substrate 2 (at 2 / 3 of the height of the flower pot) on the bottom. The inoculation group adds an inoculant containing AMF on top of the sterile cultivation substrate 2 (200g / plant, added twice in equal amounts, added before and after transplanting), and the control group adds inactivated AMF on top of the sterile cultivation substrate 2 (200g / plant, added twice in equal amounts, added before and after transplanting). Transplant the flue-cured tobacco seedlings obtained in step (2.1) and the corn seedlings obtained in step (2.2). Plant 2 flue-cured tobacco plants in each pot in the flue-cured tobacco monoculture group, 2 corn plants in each pot in the corn monoculture group, and one flue-cured tobacco plant and one corn plant in the flue-cured tobacco and corn intercropping group, with a plant spacing of 30cm. The sterile cultivation substrate was spread evenly from the surface of the soil layer inoculated with the bacterial agent to the top of the cultivation container, forming a "sandwich" method to inoculate F. mosseae to the roots of flue-cured tobacco and corn.

[0082] Table 1. Experimental design and treatment groups

[0083]

[0084]

[0085] After the flue-cured tobacco seedlings and corn seedlings were cultivated in different planting modes in the cultivation containers for 45 days, the agronomic traits, photosynthetic parameters, chlorophyll and other indicators of the plants were measured. When the samples were harvested, the flue-cured tobacco and corn samples were collected separately, and 8 groups of samples were obtained:

[0086] KDM+ indicates that flue-cured tobacco monoculture is inoculated with AMF; KDM- indicates that flue-cured tobacco monoculture is not inoculated with AMF;

[0087] KJM+ indicates flue-cured tobacco intercropped with corn inoculated with AMF; KJM- indicates flue-cured tobacco intercropped with corn without AMF inoculation;

[0088] YDM+ means that maize monoculture is inoculated with AMF; YDM- means that maize monoculture is not inoculated with AMF;

[0089] YJM+ indicates corn intercropped with flue-cured tobacco and inoculated with AMF; YJM- indicates corn intercropped with flue-cured tobacco and not inoculated with AMF.

[0090] 4. Harvesting methods for flue-cured tobacco and corn plants:

[0091] Samples of flue-cured tobacco and corn were collected after 45 days of greenhouse cultivation. Before sampling, agronomic traits of crops were measured, and plant height, stem girth, leaf length, leaf width, and leaf number of crops were measured. Li-6400 portable photosynthetic meter (Li-Cor Inc., USA) was used to measure the transpiration rate (Tr), stomatal conductance (Gs), net photosynthetic rate (Pn), intercellular CO2 concentration (Ci) and other photosynthetic parameters of the fourth fully expanded leaf of corn from top to bottom and the fourth functional leaf of flue-cured tobacco (from top to bottom). SPAD instrument was used to measure the chlorophyll content of flue-cured tobacco and corn leaves, and the soil respiration rate was measured using SRS2000 T portable soil respiration meter. When the samples were harvested, the plants were carefully taken out, the soil attached to the rhizosphere was shaken off, the roots of the plants were washed with clean water, the excess water was absorbed with filter paper, and the fresh weight of the aboveground and underground parts of the crops was weighed respectively. A small amount of fresh root samples were taken and the acid fuchsin staining method and the cross method were used for the detection and statistics of the colonization rate of F. mosseae (the uninoculated group was checked for contamination, and the inoculated group was checked for colonization rate). Detection and statistics of the colonization rate of F. mosseae: Collect the flue-cured tobacco and corn root segments respectively, wash them with tap water, and put the root segments into test tubes. The flue-cured tobacco root segments are darker in color. Before analysis, they are first decolorized with root sample decolorizing solution (3ml ammonia water + 10ml 30% hydrogen peroxide to 600ml). After fading, 10% KOH solution is added to completely immerse the root sample, and the root sample is heated and analyzed in a water bath at a temperature of 90°C until the root sample is translucent. The corn is analyzed for 2-3 minutes, and the flue-cured tobacco root segments are analyzed for about 15 minutes. Then rinse with tap water 2-3 times, add lactic acid to acidify, and soak for 10-20 minutes. Cut the root sample into root segments of about 1 cm, move them onto the glass slide, add 0.5% acid fuchsin dye solution, and dye the root segments of flue-cured tobacco and corn plants. During the dyeing process, you can use an alcohol lamp to burn the back of the glass slide to accelerate the dyeing until white smoke is emitted 2-3 times. Use lactic acid glycerol to decolorize the stained root segments, select an appropriate amount of root segments and press them on the glass slide, and observe the pressed glass slide under an ordinary optical microscope. Use the cross method to count the observed colonization rate of Mosseae. At least 300 fields of view are tested for each root sample, and the total colonization rate of Mosseae is calculated according to the following formula:

[0092] Funneliformis mosseae colonization rate (%) = (total number of visual fields - number of blank visual fields) / total number of visual fields × 100%

[0093] The remaining samples were placed in kraft envelopes, placed in an oven at 105°C for 30 minutes, dried at 75°C to constant weight, and the dry weight of the samples was weighed. The samples were crushed and mixed with a micro-crusher to measure the content of crop nutrient elements N, P, and K. According to the method in the "Agricultural Industry Standard of the People's Republic of China NY / T2017-2011", 3 replicate samples were set for each treatment group.

[0094] 5. Harvesting methods of flue-cured tobacco and corn root soil:

[0095] The root soil related indicators of flue-cured tobacco and corn in the intercropping mode were sampled and measured separately, and the rhizosphere soil samples of the two plants in the potted monocropping mode were mixed evenly and then sampled and measured. The crops were pulled out of the pot with roots and soil, the surface soil was removed, the loose soil in the rhizosphere was gently shaken off, and the soil tightly bound to the root system was brushed off with a brush, and mixed well as the rhizosphere soil. The collected rhizosphere soil samples were transferred to a cool place and naturally air-dried to constant weight and passed through a 60-mesh sieve. Six replicates were selected for each treatment to determine the soil physical and chemical properties: pH, alkaline nitrogen, available phosphorus, available potassium, organic matter and nutrient-related soil enzyme activities: acid phosphatase, catalase, urease, phytase, sucrase, nitrate reductase. Soil pH was determined by water extraction method; soil alkaline nitrogen content was determined by alkaline diffusion method; soil available phosphorus content was determined by NaHCO3 extraction-molybdenum antimony anticolorimetry; soil available potassium content was determined by flame photometry; soil organic matter content was determined by potassium dichromate titration method, and the specific methods are referred to "Soil Agrochemical Analysis". Soil enzyme activity kits were purchased from Suzhou Grace Biotechnology Co., Ltd.

[0096] 6. Conclusion:

[0097] As shown in Table 2: The roots of flue-cured tobacco and corn inoculated with AMF were successfully colonized by various structures of AMF (F. mosseae): hyphae, arbuscules, vesicles, etc., with a colonization rate of 84.74%-88.27%, indicating that AMF can colonize and act on the roots of flue-cured tobacco and corn. Intercropping had no significant effect on the colonization rate of AMF, and no colonization was observed in the uninoculated group.

[0098] Table 2. AMF colonization rate of flue-cured tobacco and corn roots in different planting patterns

[0099]

[0100] The data in Table 2 are mean ± standard error.

[0101] like Figure 1 , 3The results showed that compared with non-inoculation, AMF inoculation significantly promoted the plant height, leaf length, leaf width and leaf number of flue-cured tobacco and corn, and significantly increased or increased the biomass of flue-cured tobacco and corn. Compared with monoculture, intercropping significantly reduced the plant height and leaf width of flue-cured tobacco and corn, but increased the stem girth of flue-cured tobacco. The root biomass of corn increased significantly after intercropping with flue-cured tobacco. The two-factor results showed that the planting pattern had a significant effect on the plant height, stem girth, leaf length and leaf width of flue-cured tobacco and corn, and the inoculation of AMF had a significant effect on the plant height, leaf length and leaf width of flue-cured tobacco and corn, and the interaction between the two had a significant effect on the plant height.

[0102] like Figure 2 The results show that: compared with the non-AMF inoculation treatment group, AMF inoculation significantly increased the chlorophyll content and net photosynthetic rate of flue-cured tobacco when planted alone. When AMF was inoculated, the intercropping of flue-cured tobacco and corn significantly increased the leaf transpiration rate and intercellular CO2 concentration compared with monoculture, and significantly reduced the chlorophyll content of flue-cured tobacco leaves. When AMF was not inoculated, intercropping of flue-cured tobacco significantly increased the leaf transpiration rate, stomatal conductance and net photosynthetic rate compared with monoculture. Bifactor analysis showed that the planting pattern had a significant effect on the SPAD value, intercellular CO2 concentration and stomatal conductance of flue-cured tobacco and corn, and AMF had a significant effect on the SPAD value and intercellular CO2 concentration of flue-cured tobacco and corn. The interaction between the two had a significant effect on the chlorophyll content and net photosynthetic rate of flue-cured tobacco and corn.

[0103] like Figure 4 The results showed that compared with the non-AMF inoculation group, AMF inoculation increased the total nitrogen, total phosphorus, and total potassium content of the aboveground and underground organs of flue-cured tobacco. Compared with monoculture, intercropping increased the total potassium content of flue-cured tobacco and corn roots. Bivariate analysis showed that planting pattern and AMF inoculation had a significant effect on the nitrogen, phosphorus, and potassium content of corn roots and aboveground and underground organs of flue-cured tobacco.

[0104] like Figure 5 The results show that inoculation with AMF significantly increased the content of alkaline nitrogen in the rhizosphere soil of flue-cured tobacco and the content of available phosphorus in the rhizosphere soil of corn under monoculture, and significantly increased the soil respiration rate of crop roots. Compared with monoculture, intercropping significantly increased the content of alkaline nitrogen, available phosphorus and available potassium in the rhizosphere soil of corn and the soil respiration rate of flue-cured tobacco roots. The two-factor analysis showed that the planting pattern had a significant effect on the pH and soil respiration rate of crop root soil, and inoculation with AMF had a significant effect on the alkaline nitrogen, available phosphorus and soil respiration rate of crop root soil.

[0105] like Figure 6The results show that: AMF inoculation significantly increased the activities of acid phosphatase, urease, phytase in the root soil of flue-cured tobacco and urease and sucrase in the root soil of corn compared with the non-inoculated treatment group. Compared with monocropping, intercropping significantly increased the activities of phytase in the root soil of flue-cured tobacco and urease and sucrase in the root soil of corn. Bivariate analysis showed that: planting pattern had a significant effect on catalase and phytase in the rhizosphere soil of flue-cured tobacco and corn, inoculation of AMF had a significant effect on urease, nitrate reductase, sucrase and phytase in the rhizosphere soil of flue-cured tobacco and corn, and the interaction between the two had a significant effect on urease and phytase activities.

[0106] According to the above test results, AMF inoculation and intercropping (KJM+, YJM+) jointly regulate the growth of corn and tobacco and rhizosphere soil nutrients. AMF inoculation promotes crop growth, and intercropping significantly increases the stem girth of flue-cured tobacco. Both AMF inoculation and intercropping increase the biomass of crop roots, promote crop absorption of nutrients, and improve soil nutrients and soil enzyme activity.

[0107] When tobacco and corn are intercropped, the alkaline nitrogen content in the rhizosphere soil of tobacco and corn is much higher than that in the monocropping mode. The nitrogen element has a great influence on the growth of tobacco and corn. After tobacco is intercropped with corn, the alkaline nitrogen content in the soil increases significantly compared with the monocropping mode. Therefore, the intercropping mode is better than the monocropping mode in improving the nutrients of the rhizosphere soil of tobacco and corn.

[0108] Comparative Example 1

[0109] The difference from Example 1 is that no exogenous inoculation of F. mosseae was performed.

[0110] That is, with respect to Example 1, the following changes are made:

[0111] Eliminate "Preparation of inoculant" in step 1;

[0112] Cancel the use of "inoculant containing AMF" in step (2.3), that is, specifically:

[0113] The planting patterns are flue-cured tobacco monoculture, corn monoculture and intercropping of flue-cured tobacco and corn. The flower pots (50cm×20cm×18cm) are thoroughly scrubbed with 75% alcohol, dried, and numbered respectively. The sterile cultivation substrate is loaded into the long flower pots, which is basically flush with the top of the flower pots.

[0114] The rest is the same as Example 1.

[0115] Tobacco and corn samples were collected at harvest time, and four groups of samples were obtained: KDM-: flue-cured tobacco monoculture without AMF inoculation; KJM-: flue-cured tobacco intercropped with corn without AMF inoculation; YDM-: corn monoculture without AMF inoculation; YJM-: corn intercropped with tobacco without AMF inoculation.

[0116] Conclusion: Comprehensively comparing Example 1 and Comparative Example 1, it was found that in both monocropping and intercropping modes, the agronomic traits, biomass, and photosynthetic indexes of flue-cured tobacco and corn not inoculated with F. mosseae were far inferior to those of the AMF-inoculated treatment group in Example 1; compared with the non-AMF-inoculated group, after AMF inoculation, the nutrient content of crop organs, the nutrient status of crop rhizosphere soil, and the activity of soil enzymes increased significantly, whether in monocropping or intercropping. The simultaneous action of AMF inoculation and intercropping mode on soil nutrient improvement was more obvious.

[0117] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A method for jointly regulating the growth of corn and tobacco and rhizosphere soil nutrients by AMF and planting pattern, characterized in that The steps include: 1) Using F. mosseae to prepare an AMF-containing bacterial agent; 2) Cultivation of flue-cured tobacco seedlings and corn seedlings; 3) Using the AMF-containing bacterial agent obtained in step 1), the flue-cured tobacco seedlings and corn seedlings obtained in step 2) are intercropped and planted together.

2. The method for regulating the growth of corn and tobacco and rhizosphere soil nutrients by AMF and planting pattern according to claim 1, characterized in that The step 1) is: The soil and vermiculite are mixed in a volume ratio of 2:1, and then sterilized to obtain a sterile cultivation medium 1; First, a sterile cultivation substrate is added to the bottom of the sterile container as a bottom layer, and then the surface of the bottom layer is covered with rhizosphere soil containing hyphae and spores of F. mosseae, and the surface of the rhizosphere soil is covered with a sterile cultivation substrate as a top layer to form a "sandwich"; Transplant the sterile corn seedlings into the rhizosphere soil of the above-mentioned sterile container, irrigate with 1 / 2 Hoagland nutrient solution, irrigate with 1 / 2 Hoagland nutrient solution once every 20±1 days, and co-cultivate until the colonization rate of F. mosseae at the roots of corn is not less than 90%, cut off the above-ground part of the corn and remove the top 1-2 cm sterile cultivation substrate, and then collect the obtained in the sterile container to obtain a bacterial agent containing AMF.

3. The method for regulating the growth of corn and tobacco and rhizosphere soil nutrients by AMF and planting pattern according to claim 2, characterized in that In the step 1), the soil is sieved through a 10-mesh sieve; and the corn is Huidan No.

4.

4. The method for regulating the growth of corn and tobacco and rhizosphere soil nutrients by AMF and planting pattern according to claim 2 or 3, characterized in that In the step 1), the co-cultivation time is 60±5 days.

5. The method for regulating the growth of corn and tobacco and rhizosphere soil nutrients by AMF and planting pattern according to claim 2 or 3, characterized in that The formula of the 1 / 2 Hoagland nutrient solution in the step 1) is: 94.5 g / L calcium nitrate, 8 g / L ammonium nitrate, 50.6 g / L potassium nitrate, 13.6 g / L potassium dihydrogen phosphate, 100.9 g / L magnesium sulfate heptahydrate, 7.46 g / L Na2EDTA·2H2O, 5.56 g FeSO4·7H2O, 4.46 g / L MnSO4·4H2O, 1.24 g / L H3BO3, 1.72 g / L ZnSO4·7H2O, 0.005 g / L CuSO4·5H2O, 0.005 g CoCl2·6H2O, 0.166 g KI, and 0.05 g / L Na2MoO4·2H2O.

6. The method for regulating the growth of corn and tobacco and rhizosphere soil nutrients by using AMF and planting patterns according to any one of claims 1 to 5, characterized in that The step 2) is: Flue-cured tobacco seeds are grown in a floating manner using a sterile substrate for 45±1 days to obtain flue-cured tobacco seedlings that can be transplanted; Corn seeds are grown with sterile vermiculite until they have three leaves and one heart, and then they are ready for transplanting.

7. The method for regulating the growth of corn and tobacco and rhizosphere soil nutrients using AMF and different planting patterns according to claim 6, characterized in that The step 2) is:

1. Preparation of flue-cured tobacco seedlings: First, the tobacco floating seedling medium is sterilized to obtain a sterile medium; sterile water is added to the sterile medium and mixed well to obtain a moistened medium; Place the moistened substrate into the holes of the sterile seedling tray, make a seed hole of 0.5±0.1cm deep in each hole, put 1-2 flue-cured tobacco seeds in, and then cover with the moistened substrate; Add tobacco seedling fertilizer to the seedling pool, and then add water until the floating plate is flush with the surface of the seedling pool; cultivate for 45±2 days to obtain flue-cured tobacco seedlings that can be transplanted; the tobacco seedling fertilizer: water = 1±0.1g / 1L; 2. Sterile corn seedlings: After the corn seeds are disinfected, they are first cultured on sterile double-layer filter paper in the dark until the corn sprouts, and then the seedlings are grown on sterile vermiculite until they have three leaves and one heart, and the corn seedlings are ready for transplanting.

8. The method for regulating the growth of corn and tobacco and rhizosphere soil nutrients using AMF and different planting patterns according to any one of claims 1 to 7, characterized in that The step 3) is: The soil and vermiculite were mixed in a volume ratio of 3:1, and then sterilized to obtain a sterile cultivation medium 2; A layer of sterile cultivation substrate 2 is spread on the bottom of the container, a fungus agent containing AMF is added, and then the flue-cured tobacco seedlings and corn seedlings obtained in step 2) are transplanted in an intercropping manner, and then the fungus agent containing AMF is added, and finally a layer of sterile cultivation substrate 2 is spread; The relationship between the total weight of the AMF-containing inoculant used twice and the total number of transplanted flue-cured tobacco seedlings and corn seedlings was 200±20 g / plant.

9. The method for regulating the growth of corn and tobacco and rhizosphere soil nutrients using AMF and different planting patterns according to claim 8, characterized in that In the step 3), the spacing between the flue-cured tobacco seedlings and the corn seedlings is 30±3 cm.

10. The method for regulating the growth of corn and tobacco and rhizosphere soil nutrients using AMF and different planting patterns according to any one of claims 1 to 9, characterized in that: The planting time of step 3) is 45±2 days.

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