A composition comprising arbuscular mycorrhizal fungi and biochar and its use in increasing cadmium tolerance in tobacco and / or improving soil

Abstract

The application provides a composition containing arbuscular mycorrhizal fungi (AMF) and biochar and application thereof in improving tobacco cadmium tolerance and / or improving soil, and belongs to the technical field of bio-agriculture. The application can effectively promote tobacco growth, improve tobacco agronomic traits and biomass, effectively improve tobacco cadmium tolerance, effectively reduce cadmium accumulation in tobacco, change the subcellular distribution of cadmium in tobacco, convert high-toxicity chemical state cadmium into medium-toxicity or low-toxicity cadmium, and is also beneficial to improving soil environment and nutrient regulation antioxidant enzyme activity, and provides a better soil environment for tobacco growth.

Description

Technical Field

[0001] This invention belongs to the field of bio-agricultural technology, specifically relating to a composition comprising arbuscular mycorrhizal fungi (AMF) and biochar and its application in improving tobacco cadmium tolerance and / or refining soil. Background Technology

[0002] Cadmium is a highly toxic heavy metal, exhibiting a high degree of toxicity to plants. If cadmium enters the soil, its toxic effects will disrupt the balance of the soil ecosystem; furthermore, due to its high stability, its migration and transformation out of the soil is extremely difficult. Furthermore, when plants are grown in Cd-contaminated soil, Cd will be transferred to the plants along with nutrient absorption, severely impacting plant production and yield.

[0003] Tobacco is a plant species where cadmium (Cd) accumulation in branches can be up to four times higher than in roots. Reducing Cd accumulation in tobacco has long been a goal for tobacco growers. Various attempts have been made to reduce Cd accumulation in tobacco leaves. One method involves chelating Cd in root vesicles to reduce Cd accumulation in branches; another method uses genetic engineering to inactivate specific genes to reduce Cd accumulation in plant leaves. While these methods are effective, they are complex and require long-term experimental research to achieve. Patent publication CN104789229A discloses a method for reducing heavy metal content in tobacco, improving tobacco quality, and improving the soil environment using tobacco biochar combined with heavy metal passivating agents. However, this method only has an ameliorative effect on contaminated soil and has little effect on plants grown on contaminated soil. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a composition comprising arbuscular mycorrhizal fungi and biochar and its application in improving tobacco cadmium tolerance and / or improving soil. Based on the synergistic effect of arbuscular mycorrhizal fungi and biochar, the composition improves tobacco's tolerance to Cd, reduces the toxic effects of heavy metals on tobacco growth and development, thereby promoting the vegetative growth of tobacco, and at the same time achieves the purpose of improving soil cadmium pollution.

[0005] The present invention provides a composition comprising arbuscular mycorrhizal fungi and biochar, wherein the mass ratio of the number of spores of the arbuscular mycorrhizal fungi to the mass of the biochar is 7000-9000 spores: 75-85 grams.

[0006] Preferably, the mass ratio of the number of spores of the arbuscular mycorrhizal fungus to the biochar is 7500-8500 spores: 78-82 grams.

[0007] Preferably, the biochar includes at least one of the following: rice husks, tobacco stalks, bamboo charcoal, and wood charcoal.

[0008] The present invention provides a fertilizer comprising the aforementioned composition.

[0009] Preferably, the composition accounts for 2% to 10% of the fertilizer by mass.

[0010] The present invention provides the use of arbuscular mycorrhizal fungi, the composition, or the fertilizer in at least one of promoting tobacco growth, improving tobacco cadmium tolerance, and improving soil.

[0011] Preferably, the improvement of tobacco cadmium tolerance includes at least one of the following: reducing the damage of cadmium stress to plant cells, improving the antioxidant capacity of plants, reducing cadmium accumulation in tobacco plants, changing the distribution of cadmium at the subcellular level and between organs, and the chemical distribution of cadmium in tobacco.

[0012] Preferably, the improved soil includes cadmium-free soil and / or cadmium-stressed soil.

[0013] Preferably, the improved soil includes at least one of the following: improving soil fertility, increasing the activity of soil antioxidant enzymes, and reducing the availability of cadmium in the soil.

[0014] Preferably, improving soil fertility includes increasing the content of at least one nutrient among available nitrogen, available phosphorus, available potassium, and organic matter in the soil.

[0015] This invention provides a composition comprising arbuscular mycorrhizal fungi (AMF) and biochar, wherein the mass ratio of AMF spores to biochar is 7000-9000:75-85g. The application of AMF combined with biochar in this invention can effectively promote tobacco growth: In the embodiments of this invention, inoculation with AMF and addition of biochar can promote tobacco growth to varying degrees under Cd stress, increase photosynthetic parameters and photosynthetic capacity, and improve agronomic traits and biomass. The combined use of AMF and biochar can effectively improve tobacco's tolerance to the heavy metal Cd: Simultaneously, the combined use of AMF and biochar can significantly reduce cadmium accumulation in tobacco, alter the subcellular distribution of cadmium within the tobacco plant, isolate more heavy metal cadmium in the less toxic cell wall or cytoplasm, and convert highly toxic chemically active cadmium into moderately or less toxic cadmium, thereby improving tobacco's tolerance to the heavy metal cadmium. Inoculation with AMF and the addition of biochar are beneficial to improving the soil environment: The addition of biochar improves soil nutrients (significantly increasing the content of available nitrogen, available phosphorus, available potassium and organic matter), related enzyme activities, pH value, heavy metal availability and physicochemical properties through multiple mechanisms, providing a better soil environment for tobacco growth. Attached Figure Description

[0016] Figure 1 A comparison chart of the growth of tobacco under different treatments during the vegetative growth stage;

[0017] Figure 2The flowchart shows the separation process of Cd subcellular components, where F1 is the cell wall and unbroken residue, F2 is the cell nucleus and other components, F3 is the mitochondrial and chloroplast components, and F4 is the nucleoprotein components and soluble components (soluble components include cytoplasm, macromolecules in vacuoles, inorganic salt ions and ribosomes).

[0018] Figure 3 Flowchart for the extraction of Cd in different chemical states;

[0019] Figure 4 Typical structural diagram of AMF colonized on tobacco roots after 35 days of growth; where a represents tufts and hyphae observed at 50 μm; b represents vesicles observed at 50 μm; c represents hyphal rings observed at 50 μm; d represents hyphae observed at 200 μm; Note: Hy represents hyphae, Hc represents hyphal rings, Ve represents vesicles, and Ar represents tufts;

[0020] Figure 5 The results show the effects of AMF and biochar on the biomass of tobacco during the vegetative growth period; where a represents the effect of different treatments on the fresh weight of tobacco aboveground parts; b represents the effect of different treatments on the dry weight of tobacco aboveground parts; c represents the effect of different treatments on the fresh weight of tobacco roots; and d represents the effect of different treatments on the dry weight of tobacco roots.

[0021] Figure 6 The results show the effects of AMF and biochar on photosynthetic parameters and SPAD values ​​of tobacco during the vegetative growth stage under Cd stress.

[0022] Figure 7 The results show the effects of AMF and biochar on root activity during the vegetative growth stage of tobacco under Cd stress.

[0023] Figure 8 Effects of AMF and biochar on leaf damage during the vegetative growth stage of tobacco under Cd-free stress; Notes (9-13 same): CW: cell wall, Mt: mitochondria, Ch: chloroplast, Li: liposome, Os: osmium-rich granules, St: starch granules, Pl: plastid globules, Nu: nucleus, Er: endoplasmic reticulum, Ga: Golgi apparatus, Le: leucoplasmic bodies, Va: vacuoles, Ve: vesicles, ICS: intercellular spaces, Pe: peroxisomes, P: [Plant] starch nucleus, [Biochemical] protein nucleus, Au: autophagosome; Yellow arrow: membrane dissolution, disappearance, indistinct; Green arrow: endoplasmic reticulum swelling; Blue box: mitochondrial vacuolation or disintegration; Yellow box: thickening of vascular cell walls;

[0024] Figure 9 The results show the effects of AMF and biochar on the degree of leaf damage in tobacco during the vegetative growth stage under 20 mg / kg Cd stress.

[0025] Figure 10The results show the effects of AMF and biochar on the degree of leaf damage in tobacco during the vegetative growth stage under 40 mg / kg Cd stress.

[0026] Figure 11 The results show the effects of AMF and biochar on the degree of root damage during the vegetative growth period of tobacco under Cd-free stress.

[0027] Figure 12 The results show the effects of AMF and biochar on the degree of root damage in tobacco during the vegetative growth stage under 20 mg / kg Cd stress.

[0028] Figure 13 The results show the effects of AMF and biochar on the degree of root damage in tobacco during the vegetative growth stage under 40 mg / kg Cd stress.

[0029] Figure 14 The results show the effects of AMF and biochar on enzyme activity and stress-resistance substances in leaves and roots of tobacco during the vegetative growth period.

[0030] Figure 15 The results show the effects of AMF and biochar on Cd concentrations in the aboveground and underground parts of tobacco during the vegetative growth period; where a represents the Cd concentration in the aboveground parts and b represents the Cd concentration in the underground parts.

[0031] Figure 16 Results of the effects (percentage) of AMF and biochar on the subcellular distribution of Cd in the aboveground and underground parts of tobacco during the vegetative growth period;

[0032] Figure 17 The results of the effects (percentage) of AMF and biochar on the distribution of different chemical forms of Cd in leaves and roots during the vegetative growth period of tobacco.

[0033] Figure 18 The results show the effects of AMF and biochar on soil enzyme activity during the vegetative growth stage of tobacco under Cd stress; where a represents S-CAT (catalase) activity and b represents GSH-Px (glutathione peroxidase) activity.

[0034] Figure 19 The results show the effects of AMF and biochar on the total Cd content and available Cd content in soil during the vegetative growth stage of tobacco under Cd stress; where a represents the total Cd content in the soil and b represents the available Cd content in the soil. Detailed Implementation

[0035] The present invention provides a composition comprising arbuscular mycorrhizal fungi and biochar, wherein the mass ratio of the number of spores of the arbuscular mycorrhizal fungi to the mass of the biochar is 7000-9000 spores: 75-85 grams.

[0036] In this invention, the preferred spore count to biochar mass ratio of the arbuscular mycorrhizal fungi is 7500-8500 spores: 78-82 grams, or alternatively 8000 spores: 80 grams. Each 50 grams of the arbuscular mycorrhizal fungi contains approximately 2000 spores. The biochar preferably includes at least one of the following: rice husks, tobacco straw, bamboo charcoal, and wood charcoal. In this embodiment of the invention, the arbuscular mycorrhizal fungus is preferably *Funneliformis mosseae*, with accession number BGC YN05, 1511C0001BGCAM0013. This strain is described in the prior art (Zhan F, Li B, Jiang M, Yue X, He Y, Xia Y, Wang Y. Arbuscular mycorrhizal fungi enhance antioxidant defense in the leaves and the retention of heavy metals in the roots of maize[J]. Environmental Science and Pollution Research, 2018, 25(24): 24338-24347.).

[0037] In this invention, the preparation method of the composition preferably includes mixing the arbuscular mycorrhizal fungi (AMF) and biochar. The AMF exists in the form of an inoculum. The preferred method for preparing the AMF inoculum involves sterilizing crop seeds and culturing them until germination. The resulting sterile crop seedlings are then inoculated into a sterile substrate and cultured until roots develop. The roots and substrate are collected, and the roots are pulverized and mixed with the substrate to obtain an inoculum containing AMF. The sterile substrate has a "sandwich" structure, wherein the middle layer of the "sandwich" structure is rhizosphere soil containing AMF hyphae and spores, while the bottom and top layers of the "sandwich" structure are sterile cultivation substrates. The sterile cultivation substrate can be any common crop cultivation substrate, such as a mixture of bulk soil and horticultural vermiculite in a 3:1 volume ratio, sterilized. The concentration of hyphae or spores in the AMF-containing inoculum is preferably 40 spores / g.

[0038] The present invention provides a fertilizer comprising the aforementioned composition.

[0039] In this invention, the composition preferably accounts for 2% to 10% of the fertilizer by mass, and can be 2.62% to 5.2%. The fertilizer is preferably prepared by uniformly mixing the composition with the base fertilizer, and then applying it according to the usage instructions for the base fertilizer; alternatively, the composition and the base fertilizer can be packaged separately and applied separately. This invention does not impose any particular limitation on the preparation method of the base fertilizer; any type of base fertilizer well-known in the art can be used, such as tobacco seedling fertilizer (N:P2O4:K2O = 18:12:13).

[0040] The present invention provides the use of arbuscular mycorrhizal fungi, the composition thereof, or the fertilizer thereof in at least one of promoting tobacco growth, improving tobacco cadmium tolerance, and improving soil.

[0041] In this invention, promoting tobacco growth preferably includes increasing leaf length, leaf width, plant height, stem circumference, number of effective leaves, and biomass accumulation. In one embodiment of this invention, under different concentrations of cadmium stress, compared with the cadmium stress group, both the arbuscular mycorrhizal fungi group and the composition treatment group significantly increased leaf length, leaf width, plant height, stem circumference, number of effective leaves, and biomass of tobacco. Simultaneously, when arbuscular mycorrhizal fungi and the composition were applied to cadmium-free soil, the results showed that the arbuscular mycorrhizal fungi significantly improved leaf length, leaf width, and number of effective leaves compared to the control, increasing them by 17.40%, 14.86%, and 23.68%, respectively. This indicates that arbuscular mycorrhizal fungi can also promote tobacco growth under cadmium-free conditions.

[0042] In this invention, the improvement of cadmium tolerance in tobacco preferably includes at least one of the following: reducing cadmium stress damage to plant cells, improving the plant's antioxidant capacity, reducing cadmium accumulation in tobacco plants, altering the distribution of cadmium at the subcellular level and between organs, and the chemical distribution of cadmium in tobacco, as well as reducing the translocation of cadmium from the roots to the aboveground parts. Reducing cadmium stress damage to plant cells includes alleviating damage to leaves and root cortex. Improving the plant's antioxidant capacity includes reducing POD and SOD activity in leaves and roots, reducing MDA content, and increasing PCs and MT content. Reducing cadmium accumulation in tobacco plants preferably includes reducing cadmium content in the roots and aboveground parts of tobacco.

[0043] In one embodiment of the present invention, experiments showed that compared with the treatment without cadmium stress, cadmium stress caused damage to the leaf and root cortex, such as membrane dissolution and mitochondrial vacuolation; while inoculation with AMF or application of biochar could alleviate the cell damage to varying degrees, with AMF alone showing better mitigation effects. Regarding enzyme activity and antioxidants, compared with the treatment without cadmium stress, cadmium treatment significantly increased the POD and SOD activities in leaves and roots, and significantly increased the MDA content in leaves. At this point, biochar, AMF, and their combined effects had a significant impact on the MDA content in leaves and roots; it also significantly increased the PC content in leaves and roots, with different treatments all having a significant effect. Simultaneously, experiments showed that under higher cadmium pollution stress (40 mg / kg), the combined effect of AMF and biochar was more effective in promoting tobacco growth and biomass accumulation, while under lower cadmium pollution stress, AMF was more effective in promoting tobacco growth and biomass accumulation.

[0044] In one embodiment of the present invention, under cadmium stress, the addition of biochar and / or AMF significantly reduced the cadmium content in the roots and aboveground parts of tobacco. Experimental results showed that the cadmium content in the roots treated with biochar (BCd20) was reduced by 20.14% compared to the control group (Cd20); while the cadmium content in the roots treated with AMF alone (MCd20) and with a combination of biochar and AMF (MBCd20) was significantly reduced by 30.14% and 37.22%, respectively, indicating that the combined application of biochar and AMF has a significant effect in reducing cadmium accumulation in the roots. The cadmium content in the aboveground parts of AMF alone (MCd20) was significantly lower than that of the control group (Cd20), while the combined treatment (MBCd20) had the lowest cadmium content in the aboveground parts, indicating that the combined application of AMF and biochar has a synergistic effect in reducing cadmium accumulation in the aboveground parts.

[0045] In this invention, the alteration of cadmium distribution at the subcellular level and between organs preferably involves reducing the accumulation of cadmium in organelles and nucleoproteins in roots and leaves, which helps reduce cellular toxicity; it also reduces the proportion of cadmium in soluble components in roots and leaves. The alteration of the chemical distribution of cadmium in tobacco preferably includes reducing the proportion of highly toxic chemical cadmium in tobacco leaves, which helps mitigate the toxic effects of cadmium on leaves. The highly toxic chemical cadmium includes those extracted with sodium chloride and those extracted with deionized water.

[0046] In this invention, the improved soil preferably includes at least one of the following: improving soil fertility, increasing the activity of soil antioxidant enzymes, and reducing the availability of cadmium in the soil. The improved soil preferably includes cadmium-free soil and / or cadmium-stressed soil. Improving soil fertility preferably includes increasing the content of at least one nutrient among available nitrogen, available phosphorus, available potassium, and organic matter in the soil. Reducing the availability of cadmium in the soil preferably includes reducing the total cadmium content or the content of available cadmium, thereby reducing the toxic stress of cadmium on plants and the soil ecosystem. The soil antioxidant enzyme is preferably soil glutathione peroxidase (GSH-Px). In embodiments of this invention, the biochar combined with AMF can effectively increase the content of soil nutrients (available nitrogen, available phosphorus, and available potassium), enhance the activity of soil antioxidant enzymes (GSH-Px and S-CAT), and reduce the content of available cadmium. These changes help alleviate the oxidative damage of cadmium stress to soil microorganisms and plant roots, promote the growth and development of tobacco, and significantly reduce the absorption and accumulation of Cd by tobacco.

[0047] The following detailed description, in conjunction with embodiments, illustrates a composition comprising arbuscular mycorrhizal fungi and biochar provided by the present invention and its application in improving tobacco cadmium tolerance and / or refining soil, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0048] The AMF (F mosseae) of this invention is from the Institute of Plant Nutrition and Resources, Beijing Academy of Agriculture and Forestry, with accession number BGCYN05, 1511C0001BGCAM0013.

[0049] Explanation of technical abbreviations in the embodiments:

[0050] M: AMF; B: Biochar; +: Added; -: Not added;

[0051] M+B+ indicates AMF inoculation with added biochar; M+B- indicates AMF inoculation without added biochar; MB- indicates no AMF inoculation with no added biochar; M-B+ indicates no AMF inoculation with added biochar.

[0052] Cd: Cd addition; B: biochar; M: AMF; Cd×B: interaction between Cd and biochar; Cd×M: interaction between Cd and AMF; B×M: interaction between biochar and AMF; Cd×B×M: interaction between AMF, biochar and Cd.

[0053] ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001.

[0054] POD: Peroxidase; SOD: Superoxide dismutase; CAT: Catalase; MDA: Malondialdehyde; PCs: Plant chelate peptides; MT: Metallothionein. L: Leaf; R: Root. Note: S-CAT: Soil catalase, GSH-Px: Glutathione peroxidase.

[0055] The experimental setup in this embodiment of the invention is shown in Table 1.

[0056] Table 1. Experimental group settings for Examples 1-6 and Comparative Examples 1-3 of the present invention.

[0057] Group Handling method Cd0 Cd concentration 0 mg / kg, no biochar added, inoculated with inactivated AMF BCd0 Cd concentration 0 mg / kg, biochar added, inoculated with inactivated AMF MCd0 Cd concentration 0 mg / kg, no biochar added, inoculated with AMF MBCd0 Cd concentration 0 mg / kg, biochar added, AMF inoculated Cd20 Cd stress concentration 20 mg / kg, no biochar added, inoculated with inactivated AMF BCd20 Cd stress concentration 20 mg / kg, biochar added, inoculated with inactivated AMF MCd20 Cd stress concentration 20 mg / kg, no biochar added, inoculated with AMF MBCd20 Cd stress concentration 20 mg / kg, biochar added, AMF inoculated Cd40 Cd stress concentration 40 mg / kg, no biochar added, inoculated with inactivated AMF BCd40 Cd stress concentration 40 mg / kg, biochar added, inoculated with inactivated AMF MCd40 Cd stress concentration 40 mg / kg, no biochar added, inoculated with AMF MBCd40 Cd stress concentration 40 mg / kg, biochar added, AMF inoculated

[0058] Example 1

[0059] The effects of a combined application of AMF and biochar on the growth and cell damage of tobacco under 20 mg / kg cadmium stress were investigated. The specific steps are as follows:

[0060] 1. Preparation of inoculum

[0061] 1.1 Preparation of cultivation substrate

[0062] Large clumps of farmland soil were removed by passing it through a 10-mesh sieve. The mixture was then thoroughly mixed with horticultural vermiculite at a volume ratio of 3:1. The mixture was placed in a high-pressure steam sterilizer and sterilized at 121°C for 2 hours. The sterilization process was repeated three times after 24 hours. The mixture was then cooled to room temperature to obtain a sterile cultivation substrate for inoculum propagation.

[0063] 1.2 After disinfecting the surface of the corn seeds, they were cultured until the corn germinated to obtain sterile corn seedlings.

[0064] Select corn seeds of uniform size, shape and plumpness, disinfect them with 8% sodium hypochlorite 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 at 28°C in the dark until the corn sprouts.

[0065] 1.3 Thoroughly wipe the flowerpot (35cm×25cm) with 75% alcohol to sterilize it, let it dry, and write a label on it. The sterile cultivation substrate prepared in step 1.1 was laid at the bottom of the sterilized flowerpots. 700g of rhizosphere soil containing AMF (F. mosseae) mycelia and spores, purchased from the Institute of Plant Nutrition and Resources, Beijing Agricultural and Forestry College, was weighed and spread evenly on top of the cultivation substrate in the sterile flowerpots. The sterile corn seedlings obtained in step 1.2 were transplanted into the sterile flowerpot substrate, and then covered with 3-5cm of sterile cultivation substrate to form a "sandwich" method for propagating AMF (F. mosseae). 1 / 2 Hoagland nutrient solution (Hoagland nutrient solution formula: 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) was used. 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, Na2MoO4·2H2O 0.05 g / L) water thoroughly until nutrient solution seeps out from the bottom of the pot. During the growing season, manage the plants normally to ensure normal growth. Then, every 15 days, regularly water with 300 ml of 1 / 2 Hoagland nutrient solution. After about 35 days of cultivation, randomly check the corn roots and observe the colonization of AMF (F. mosseae) in the corn roots under an optical microscope using the cross-cross method. If typical structures such as mycelium, arbuscular branches, vesicles, and mycelial rings of AMF are observed, the colonization is considered successful.

[0066] After culturing for about 60 days until the colonization rate of AMF (F. mosseae) 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.

[0067] 2. Cultivation of flue-cured tobacco seedlings

[0068] 2.1 First, sterilize the tobacco floating seedling substrate (Yunnan Mile City Tobacco Supplies Co., Ltd.) with high-pressure steam (sterilization conditions: 121℃, 2h; sterilize again 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 formed into a ball in your hand but crumble easily 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 seeds (provided by Yuxi China Tobacco Seed Co., Ltd.), and gently cover with substrate. Weigh 25g of tobacco-specific seedling fertilizer (N:P2O4:K2O = 18:12:13; purchased from Honghe Henglin Chemical Co., Ltd.) and pour it into the seedling pool. Add pure water (approximately 25L) until the floating tray is level with the surface of the seedling pool. After about 45 days of cultivation, flue-cured tobacco seedlings with four leaves and two buds are obtained.

[0069] 2.2 Commercially available horticultural vermiculite and farmland soil were used as the basic cultivation substrate for flue-cured tobacco at a volume ratio of 1:3. The farmland soil was passed through a 10-mesh sieve to remove larger impurities. It was then mixed evenly with commercially available vermiculite and sterilized by high-pressure steam (sterilization conditions: 121℃, 2h; sterilization was repeated every 24h, for a total of 3 times) as the cultivation substrate.

[0070] 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 autoclaving (121℃, sterilization for 2 hours, repeated 3 times at 24-hour intervals) and air-dried to obtain inactivated AMF as the non-inoculation control group.

[0071] 3. Experiment Implementation

[0072] To investigate the effects of biochar and arbuscular mycorrhizal fungi on tobacco growth and cell damage under 20 mg / kg cadmium stress, four different treatment combinations were established. The specific implementation is as follows:

[0073] First, 20 mg / kg cadmium (Cd) was added to the soil as a basal treatment for Cd stress. Based on this, four different treatment conditions were further established. The first treatment (Cd20) was a control group, where soil with 20 mg / kg cadmium was added, but no biochar was added, and each pot (2.7 kg of sterilized soil) was inoculated with 200 g of inactivated AMF (2000 spores / 50 g). The second treatment (BCd20) was a control group, where soil with 20 mg / kg cadmium was added, 80 g of biochar was added to each pot, but each pot was also inoculated with 200 g of inactivated AMF to evaluate the effect of biochar alone. The third treatment (MCd20) was a control group, where soil with 20 mg / kg cadmium was added, but no biochar was added, but each pot was inoculated with 200 g of live AMF to study the alleviating effect of AMF alone on cadmium stress. The final treatment (MBCd20) involved adding 20 mg / kg cadmium to the soil, along with 80 g of biochar per pot and inoculating with 200 g of live AMF, to investigate the combined effect of biochar and AMF on alleviating cadmium stress.

[0074] All treatments were conducted under identical potting conditions, with each pot containing 2.7 kg of soil. The soil consisted of a mixture of red soil from the topsoil layer of farmland and vermiculite in a 3:1 volume ratio. Throughout the experiment, all treatments were operated according to uniform fertilization and management standards to ensure consistency of experimental conditions.

[0075] 4. Sample collection and determination

[0076] On the 35th day after transplanting, agronomic traits of tobacco plants were measured before collection, including plant height, stem circumference, leaf length, leaf width, and number of leaves. Before sampling, photosynthetic indices were measured at the same part of leaves of tobacco seedlings with uniform growth in different treatment groups. Chlorophyll content was measured using a portable SPAD-502 instrument, and photosynthetic parameters such as transpiration rate (Tr), stomatal conductance (Gs), net photosynthetic rate (Pn), and intercellular CO2 concentration (Ci) were measured at the same part of functional leaves on the fourth leaf (out of 12 leaves). Samples were taken from both aboveground and underground parts, placed in kraft paper envelopes, and dried in an oven at 105℃ for 30 minutes to kill the enzymes, then dried at 75℃ to constant weight. The dry weight was measured using a balance, and the fresh weight of the aboveground and underground parts, the dry weight of the aboveground and underground parts, leaf length, and leaf width were determined. The determination method refers to "YC / T 142-2010 Method for Survey and Measurement of Tobacco Agronomic Traits"; root activity was determined according to the reagent kit method of Suzhou Greens Biotechnology Co., Ltd., using modified nitrogen tetrazolium salt as hydrogen acceptor. The colored formazan substance generated is easily soluble in water, and its absorbance value was measured at 460 nm to obtain dehydrogenase activity.

[0077] Samples were collected from the roots to detect the colonization of arbuscular mycorrhizal fungi (AMF). 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. The colonization rate of *F. mosseae* was analyzed as follows: Flue-cured tobacco root segments were collected, washed thoroughly with tap water, and then placed in test tubes. If the root segments were dark in color, they were first decolorized with a root sample decolorizing solution (3 ml ammonia + 10 ml 30% hydrogen peroxide, diluted to 600 ml) before analysis. After decolorization, 10% KOH solution was added until the root samples were completely submerged. The samples were then heated in a 90°C water bath until translucent, 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 staining, the back of the slide can be ignited with an alcohol lamp to accelerate the staining process, continuing until white smoke is emitted 2-3 times. Decolorize the stained root segments with lactic acid glycerol. Select root segments of moderate thickness and approximately 1 cm in length 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 I:

[0078] Colony rate of Funneliformis mosseae (%) = (Total number of fields of view - Number of blank fields of view) / Total number of fields of view × 100% Formula I.

[0079] Physiological parameters were measured on tobacco leaf and root samples. Leaves and roots from the same part of tobacco seedlings with uniform growth in each treatment were taken, rinsed with tap water and then washed with distilled water. After drying the surface moisture, 2.0 g of each sample was weighed for the determination of various physiological parameters. Peroxidase (POD) activity, catalase (CAT) activity, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content were determined according to the kit method of Suzhou Keming Biotechnology Co., Ltd.; plant chelate peptide (PCs) content and metallothionein (MT) content were determined according to the kit method of Shanghai Huyu Biotechnology Co., Ltd.

[0080] 5. Conclusion

[0081] See AMF colonization status of tobacco roots Figure 4 And Table 2.

[0082] Table 2. AMF colonization rate in tobacco roots after 35 days of growth under different Cd concentrations.

[0083]

[0084] Note: Data represent mean ± standard error. Within-group comparisons: MCd0 vs MBCd0, MCd20 vs MBCd20, MCd40 vs MBCd40 (significance is indicated by A, B, C, etc.). Between-group comparisons: MCd0 vs MCd20 vs MCd40, MBCd0 vs MBCd20 vs MBCd40 (significance is indicated by A', B', C', etc.).

[0085] 5.1 Growth Performance

[0086] At a Cd concentration of 20 mG / kg, the tobacco agronomic traits of the AMF-inoculated group without biochar were the best, with significantly better leaf length, leaf width, plant height, stem circumference, and number of effective leaves compared to other treatment groups at the same concentration (see [link to treatment]). Figure 1 (See Tables 3 and 4). Compared with the control Cd20 treatment, this treatment group showed a 23.88% increase in leaf length, a 32.33% increase in leaf width, a 63.64% increase in plant height, a 25.16% increase in stem circumference, and a 13.16% increase in the number of effective leaves. In terms of biomass, AMF inoculation alone resulted in the highest biomass at this concentration (see Tables 3 and 4). Figure 5 (and Table 5).

[0087] Table 3 Effects of AMF and biochar on agronomic traits of tobacco during the vegetative growth stage

[0088]

[0089] Between-group comparisons: Cd0 vs Cd20 vs Cd40 (significance is indicated by a, b, c, etc.); Cd0 vs Cd20 vs Cd40 (significance is indicated by a', b', c', etc.); MCd0 vs MCd20 vs MCd40 (significance is indicated by A, B, C, etc.); MBCd0 vs MBCd20 vs MBCd40 (significance is indicated by A', B', C', etc.). The above are the results of one-way ANOVA.

[0090] Table 4. Results of three-way ANOVA on agronomic traits by biochar, AMF, and Cd addition treatments.

[0091]

[0092] Table 5. Results of three-way ANOVA on biomass after biochar, AMF, and Cd addition treatments.

[0093]

[0094] Under 20 mg / kg Cd stress, the combined treatment of AMF and biochar (MBCd20) significantly improved tobacco photosynthetic parameters and SPAD values. These treatments significantly alleviated the inhibitory effect of Cd stress on tobacco photosynthesis and promoted tobacco growth and development by increasing net photosynthetic rate, stomatal conductance, intercellular carbon dioxide concentration, transpiration rate, and SPAD value (see [link to treatment]). Figure 6 (and Table 6).

[0095] Under 20 mg / kg Cd stress, both AMF (MCd20) inoculation alone and biochar (BCd20) alone significantly reduced root activity, indicating that they alleviated the negative impact of Cd stress on root activity to some extent. The combined application of AMF and biochar (MBCd20) was even more effective, further reducing root activity. This may be because the combined effect works through multiple mechanisms, more effectively mitigating the damage of Cd stress to tobacco roots, thereby promoting the recovery and maintenance of root activity (see...). Figure 7 (and Table 6).

[0096] Table 6. Photosynthetic parameters and root activity of tobacco leaves in different treatment groups

[0097]

[0098]

[0099] Note: 1. Different superscript letters (a, b, c, d) indicate significant differences within treatment groups (P<0.05);

[0100] 2. M represents AMF; B represents biochar; Cd0 / Cd20 / Cd40 represents cadmium concentration (unit: mg·kg⁻¹). 1 );

[0101] 3. The data are the average value of each parameter ± standard error.

[0102] 5.2 Cell damage and changes in enzyme activity and antioxidants

[0103] Transmission electron microscopy analysis of the ultrastructure of tobacco roots and leaves in different treatment groups revealed that, without Cd stress, tobacco leaf and root cells exhibited normal morphology and intact organelle structures. However, the addition of biochar caused slight damage to some membrane structures. Figure 8 (e.g., f). At Cd concentrations of 20 and 40 mg / kg, damage was observed in tobacco leaves and root cortex, such as mitochondrial vacuolation. Figure 8 h, Figure 10 e, Figure 12 j, Figure 12 Middle l, Figure 13 (j), membrane dissolution ( Figure 8 f, Figure 9 f, Figure 9 g, Figure 10 e, Figure 10 f, Figure 11 f, Figure 11 Phenomena such as [e.g., [unclear]] indicate that the degree of cell damage increases with increasing Cd concentration. AMF or biochar can alleviate cell damage to varying degrees, with the combined effect of AMF and biochar showing the best repair effect at a Cd concentration of 40 mg / kg. Overall, AMF inoculation alone at Cd concentrations of 0 and 20 mg / kg is more effective in alleviating crop cell damage.

[0104] Compared to the treatment without Cd addition, 20 mg / kg Cd stress caused damage to the leaf and root cortex, such as membrane dissolution and mitochondrial vacuolation. Inoculation with AMF or application of biochar could alleviate cell damage to varying degrees, with AMF inoculation alone showing better mitigation effects. Figure 12 Regarding enzyme activity and antioxidants, compared with no Cd stress, the 20 mg / kg Cd treatment significantly increased POD and SOD activities in leaves and roots; significantly increased MDA content in leaves, at which point biochar, AMF, and their combined effects had a significant impact on MDA content in leaves and roots; and significantly increased PC content in leaves and roots, with different treatments having a significant effect on it. Figure 14 ).

[0105] Under 20 mg / kg Cd stress, AMF inoculation had a good effect on promoting tobacco growth and development and biomass accumulation. It could effectively alleviate cell damage, increase the activity of some antioxidant enzymes in tobacco, regulate the content of antioxidant substances, and enhance the tolerance of tobacco to Cd stress. Moreover, AMF inoculation alone was more effective than biochar treatment in promoting growth and alleviating stress.

[0106] Example 2

[0107] The effects of a combined application of AMF and biochar on the growth and cell damage of tobacco under 40 mg / kg cadmium stress were investigated. The specific steps are as follows:

[0108] 1. Preparation of inoculum

[0109] The same method as the preparation of AMF inoculum in Example 1.

[0110] 2. Cultivation of flue-cured tobacco seedlings

[0111] The same method of cultivating tobacco seedlings as in Example 1.

[0112] 3. Experiment Implementation

[0113] To investigate the effects of biochar and arbuscular mycorrhizal fungi on tobacco growth and cell damage under 40 mg / kg cadmium stress, four different treatment combinations were established. The specific implementation is as follows:

[0114] First, 40 mg / kg cadmium (Cd) was added to the soil as a basal treatment for Cd stress. Based on this, four different treatment conditions were further established. The first treatment (Cd40) was soil with 40 mg / kg cadmium added, without biochar, and inoculated with inactivated AMF, serving as a control group. The second treatment (BCd40) was soil with 40 mg / kg cadmium added, with biochar added, but also inoculated with inactivated AMF, to evaluate the effect of biochar alone. The third treatment (MCd40) was soil with 40 mg / kg cadmium added, without biochar, but inoculated with live AMF, to study the alleviating effect of AMF alone on cadmium stress. The last treatment (MBCd40) was soil with 40 mg / kg cadmium added, with both biochar and live AMF inoculated, to explore the alleviating effect of combined application of biochar and AMF on cadmium stress.

[0115] All treatments were conducted under identical potting conditions, with each pot containing 2.7 kg of soil. The soil consisted of a mixture of red soil from the topsoil layer of farmland and vermiculite in a 3:1 volume ratio. Throughout the experiment, all treatments were operated according to uniform fertilization and management standards to ensure consistency of experimental conditions.

[0116] 4. Sample collection and determination

[0117] The same method for collecting and measuring tobacco samples as in Example 1.

[0118] 5. Conclusion

[0119] 5.1 Growth Performance

[0120] At a Cd concentration of 40 mg / kg, the combined treatment of AMF and biochar resulted in the best agronomic traits in tobacco, followed by the AMF-only treatment group (see [link to article]). Figure 1 Compared to Cd40, MCd40 treatment increased leaf length by 39.70%, leaf width by 29.14%, plant height by 88.24%, stem circumference by 51.02%, and the number of effective leaves by 32.35%. Compared to Cd40 treatment, MBCd40 treatment increased leaf length by 33.70%, leaf width by 27.84%, plant height by 73.65%, stem circumference by 54.29%, and the number of effective leaves by 20.59%. In terms of biomass, both AMF inoculation alone and biochar combined with AMF treatment significantly increased tobacco biomass (see...). Figure 5 ).

[0121] 5.2 Cell damage and changes in enzyme activity and antioxidants

[0122] See results Figure 14 Under 40 mg / kg Cd stress, the degree of damage to leaves and roots increased with increasing Cd concentration. Inoculation with AMF or application of biochar could alleviate the damage, and the combined effect had a certain effect on alleviating cell damage under high concentration Cd stress. Regarding enzyme activity and antioxidants, compared with no Cd stress, 40 mg / kg Cd stress significantly reduced CAT activity in leaves and roots; significantly increased MDA content in leaves and roots; and significantly increased MT content in roots. Overall, 20 mg / kg and 40 mg / kg Cd treatments significantly increased or increased leaf MT content, and different treatments had a significant effect on it.

[0123] Under 40 mg / kg Cd stress, the combined action of AMF and biochar significantly promoted tobacco growth and development and biomass accumulation, to a certain extent reduced cell damage, regulated enzyme activity and antioxidant content in tobacco, and alleviated the toxic effects of heavy metals on tobacco.

[0124] Comparative Example 1

[0125] The effects of a combination of AMF and biochar on tobacco growth and cell damage under cadmium-free stress.

[0126] The specific steps are as follows:

[0127] 1. Preparation of inoculum

[0128] The same method as the preparation of AMF inoculum in Example 1.

[0129] 2. Cultivation of flue-cured tobacco seedlings

[0130] The same method of cultivating tobacco seedlings as in Example 1.

[0131] 3. Experiment Implementation

[0132] To investigate the effects of AMF and biochar on tobacco growth and cell damage under cadmium-free stress (0 mg / kg Cd), four different treatment groups were established: The first, Cd0: cadmium-free stress (0 mg / kg Cd), no biochar added, inoculated with inactivated AMF, served as the control group. The second, BCd0: cadmium-free stress (0 mg / kg Cd), biochar added, inoculated with inactivated AMF, to evaluate the effect of biochar alone. The third, MCd0: cadmium-free stress (0 mg / kg Cd), no biochar added, inoculated with live AMF, to study the promoting effect of AMF alone on tobacco growth. The fourth, MBCd0: cadmium-free stress (0 mg / kg Cd), biochar added and live AMF inoculated simultaneously, to explore the effect of combined application of biochar and AMF.

[0133] All treatments were conducted under identical potting conditions, with each pot containing 2.7 kg of soil. The soil consisted of a mixture of red soil from the topsoil layer of farmland and vermiculite in a 3:1 volume ratio. Throughout the experiment, all treatments were operated according to uniform fertilization and management standards to ensure consistency of experimental conditions.

[0134] 4. Sample collection and determination

[0135] The same method for collecting and measuring tobacco samples as in Example 1.

[0136] 5. Conclusion

[0137] 5.1 Growth Performance

[0138] When the Cd concentration stress was 0 mg / kg, the MCd0 treatment showed significant differences from the control in leaf length, leaf width, and number of effective leaves (see [reference]). Figure 1 The biomass of the treatment group increased by 17.40%, 14.86%, and 23.68% respectively, and the biomass of this treatment group also showed good performance at this time (see...). Figure 5 ).

[0139] 5.2 Cell damage and changes in enzyme activity and antioxidants

[0140] See results Figure 14 Without Cd stress, tobacco leaf and root cell morphology remained largely normal, and organelle structures were intact; however, the addition of biochar caused slight damage to some membrane structures. Regarding enzyme activity and antioxidants, without Cd stress, biochar significantly increased soil catalase (S-CAT) activity; AMF inoculation significantly increased GSH-Px activity.

[0141] Under Cd-free conditions, AMF inoculation can promote tobacco growth. Biochar and AMF have a certain regulatory effect on soil enzyme activity, but the addition of biochar will cause slight damage to cells.

[0142] Example 3

[0143] The effects of a combined application of AMF and biochar on cadmium content and distribution in tobacco under 20 mg / kg cadmium stress were investigated. The specific steps are as follows:

[0144] 1. Preparation of inoculum

[0145] The same method as the preparation of AMF inoculum in Example 1.

[0146] 2. Cultivation of flue-cured tobacco seedlings

[0147] The same method of cultivating tobacco seedlings as in Example 1.

[0148] 3. Experiment Implementation

[0149] To investigate the effects of biochar and arbuscular mycorrhizal fungi on the cadmium content and distribution in tobacco under 20 mg / kg cadmium stress, four different treatment combinations were established. The specific implementation was the same as in Example 1.

[0150] 4. Sample collection and determination

[0151] Samples were collected from flue-cured tobacco plants after 35 days of cultivation in a greenhouse. Before sampling, photosynthetic indices were measured at the same part of the leaves of tobacco seedlings with uniform growth in different treatment groups. Chlorophyll content was measured using a portable SPAD-502, and photosynthetic parameters such as net photosynthetic rate (A), transpiration rate (E), stomatal conductance (GH2O), and intercellular CO2 concentration (Ci) were measured using a Li-6400 portable photosynthesis meter. Then, tobacco agronomic traits were measured according to the method in "YC / T 142-2010 Method for Survey and Measurement of Tobacco Agronomic Traits".

[0152] At the time of sampling, tobacco seedlings of uniform growth from each treatment were collected, and leaves and roots from the same part were taken. These samples were first rinsed with tap water and then washed with distilled water. After drying the surface moisture, 2.0g of each sample was weighed for the determination of various physiological indicators. The activities of peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) content in tobacco leaves and roots were determined according to the reagent kit methods of Suzhou Keming Biotechnology Co., Ltd.; the content of plant chelate peptides (PCs) and metallothionein (MT) was determined according to the reagent kit methods of Shanghai Huyu Biotechnology Co., Ltd.

[0153] Roots were removed from the rhizosphere soil, and root activity was measured according to the reagent kit method of Suzhou Keming Biotechnology Co., Ltd.

[0154] On day 35 of cultivation, aboveground and belowground samples of tobacco were collected from each treatment group. Aboveground samples included leaves and stems, while belowground samples included roots. These samples were used for subsequent determination of cadmium content and subcellular distribution of cadmium.

[0155] Upon harvesting, the plants were carefully removed, the soil adhering 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 the colonization rate of *F. mosseae* was detected and statistically analyzed using the acid fuchsin staining method and the cross-swapping method. The colonization rate of *F. mosseae* was detected in the same manner as in Example 1.

[0156] Samples from both aboveground and underground parts of the tobacco plant were collected for heavy metal (Cd) concentration determination. The heavy metal concentration in different organs of the tobacco plant was measured using the microwave digestion method as described in the national standard GB 5009.268-2016. The Cd content in the digested samples was then determined using ICP-MS (NEXION 1000G, Perkin Elmer). The subcellular components of Cd in different organs of the tobacco plant were separated using differential centrifugation (see [link to ICP-MS]). Figure 2 and Figure 3 F1 consists of cell walls and unbroken residues; F2 consists mainly of the cell nucleus; F3 consists of mitochondria and chloroplasts; and F4 consists of nucleoproteins and soluble components (cytoplasm, macromolecules in vacuoles, inorganic salt ions, and ribosomes). The separation of different chemical forms of Cd from different aboveground and underground organs of tobacco was carried out using chemical extraction methods: Ethanol-extractable forms (E1) consist of soluble salts such as inorganic salts and amino acid salts, exhibiting the highest biotoxicity and mobility; deionized water-extractable forms (E2) consist of water-soluble organic acid salts, etc., with relatively high biotoxicity and mobility; sodium chloride-extractable forms (E3) consist of protein-bound or adsorbed forms and pectinates, etc., with moderate biotoxicity and mobility; acetic acid-extractable forms (E4) refer to water-insoluble phosphates, with low biotoxicity and mobility; hydrochloric acid-extractable forms (E5) refer to oxalates, etc., with biotoxicity and mobility similar to acetic acid-extractable forms, both relatively low; and residue forms (E6) are difficult to migrate and transform, and are hard to be absorbed and utilized by plants, exhibiting the lowest biotoxicity and mobility. Cd concentrations were determined after digestion using the wet digestion method according to the national standard GB5009.15-2014. Different chemical states and subcellular forms of Cd were extracted from different organs of tobacco plants, then digested and brought to a constant volume. The Cd content was determined using a flame atomic absorption spectrophotometer.

[0157] Tobacco Cd enrichment coefficient = Cd content in various plant organs / Total Cd content in soil (Formula II)

[0158] Tobacco Cd transfer coefficient = Cd content in aboveground organs / Cd content in roots (Formula III)

[0159] 5. Conclusion

[0160] 5.1 Changes in cadmium content in tobacco products

[0161] See results Figure 15Root cadmium content: Under cadmium stress of 20 mg / kg, the addition of biochar and AMF significantly reduced the cadmium content in tobacco roots. Specifically, the cadmium content in roots treated with biochar (BCd20) was reduced by 20.14% compared to the control group (Cd20); while the cadmium content in roots treated with AMF alone (MCd20) and with combined biochar and AMF treatment (MBCd20) was significantly reduced by 30.14% and 37.22%, respectively, indicating that the combined application of biochar and AMF has a significant effect on reducing cadmium accumulation in roots.

[0162] Cadmium content in aboveground parts: Biochar, AMF, and their combined treatments all significantly reduced the cadmium content in the aboveground parts of tobacco. The cadmium content in the aboveground parts of AMF alone (MCd20) was significantly lower than that of the control group (Cd20), while the cadmium content in the aboveground parts of the combined treatment (MBCd20) was the lowest, indicating that the combined application of AMF and biochar has a synergistic effect in reducing cadmium accumulation in the aboveground parts.

[0163] 5.2 Distribution changes of cadmium in tobacco:

[0164] See results Figure 16 Subcellular distribution of cadmium (Cd): Under 20 mg / kg cadmium stress, treatments with biochar and AMF significantly altered the subcellular distribution of cadmium in tobacco roots and leaves. In roots, cadmium was mainly concentrated in cell walls and their residual components, while in leaves, cadmium was predominantly distributed in nucleoproteins and soluble components. Treatments with biochar and AMF reduced the accumulation of cadmium in organelles and nucleoproteins in roots and leaves, thus decreasing the toxic effects of cadmium on cells. Furthermore, the combined treatment (MBCd20) further reduced the proportion of cadmium in soluble components in roots and leaves, indicating its significant advantage in promoting cadmium compartmentalization.

[0165] See results Figure 17 Cd chemical distribution: Under 20 mg / kg cadmium stress, in the untreated control group (Cd20), cadmium in the roots was mainly in the highly toxic ethanol-extractable (E1) and deionized water-extractable (E2) forms. These two forms of cadmium have high biotoxicity and mobility, are easily absorbed by plants, and cause serious damage to cells. Biochar treatment (BCd20) and AMF treatment (MCd20) significantly reduced the proportion of highly toxic cadmium in the roots and increased the proportion of acetic acid-extractable (E4) and hydrochloric acid-extractable (E5) forms. These forms of cadmium have relatively low toxicity and are difficult for plants to absorb, thus reducing the toxic effects of cadmium on root cells. The combined treatment (MBCd20) further optimized the chemical distribution of cadmium, significantly increasing the proportion of low-toxicity cadmium in the roots, especially the proportion of hydrochloric acid-extractable (E5) form, indicating that the combined treatment has a significant advantage in reducing cadmium toxicity.

[0166] The chemical distribution of cadmium in leaves was as follows: In the control group (Cd20), cadmium was mainly in sodium chloride extractable form (E3) and deionized water extractable form (E2). These two chemical forms of cadmium have high biotoxicity and mobility, and have a strong toxic effect on leaf cells. The treatment with biochar and AMF significantly reduced the proportion of highly toxic chemical forms of cadmium in leaves and increased the proportion of acetic acid extractable form (E4) and hydrochloric acid extractable form (E5). These chemical forms of cadmium have relatively low toxicity and are difficult to be absorbed by cells, thereby reducing the toxic effect of cadmium on leaf cells. The combined treatment (MBCd20) further reduced the proportion of highly toxic chemical forms of cadmium in leaves, so that cadmium existed more in low-toxic chemical forms, reducing the toxic effect of cadmium on leaf cells.

[0167] Under 20 mg / kg cadmium stress, treatment with biochar and AMF significantly reduced the accumulation of cadmium in organelles and nucleoproteins by altering the subcellular and chemical distribution of cadmium, thus reducing the toxic effects of cadmium on cells. The combined treatment (MBCd20) showed significant advantages in promoting cadmium compartmentalization and reducing cadmium toxicity, resulting in greater distribution of cadmium in the cell wall and its residual components, and its presence in a low-toxicity chemical state. This effectively mitigated the toxic effects of cadmium on tobacco root and leaf cells, enhancing tobacco's tolerance to cadmium stress.

[0168] Cd enrichment and translocation: Results are shown in Table 7. Biochar and AMF treatments significantly reduced the cadmium enrichment factor (BCF) and translocation factor (TF) in the aboveground parts of tobacco, indicating their important role in reducing cadmium translocation from roots to aboveground parts. AMF inoculation alone (MCd20) significantly reduced cadmium content in the aboveground parts, while the combined treatment (MBCd20) further reduced the cadmium translocation factor, demonstrating a synergistic effect of the combined application of biochar and AMF in regulating cadmium distribution within tobacco plants.

[0169] Table 7. Effects of AMF and biochar on Cd enrichment and transfer coefficients in tobacco.

[0170] deal with Cd enrichment coefficient in aboveground parts Cd enrichment coefficient in underground part Cd transfer coefficient Cd0 0.31±0.05g 0.25±0.02h 1.21±0.12ab BCd0 0.15±0.02g 0.11±0.01h 1.33±0.16ab MCd0 0.31±0.10g 0.19±0.09h 2.31±1.06a MBCd0 0.22±0.10g 0.34±0.05h 0.63±0.22b Cd20 3.76±0.12ab 2.25±0.07c 1.68±0.06ab BCd20 3.31±0.44bc 1.96±0.12d 1.67±0.14ab MCd20 4.13±0.15a 3.05±0.10b 1.36±0.09ab MBCd20 2.31±0.07e 1.67±0.12e 1.41±0.12ab Cd40 3.11±0.15cd 2.89±0.09b 1.08±0.07b BCd40 2.57±0.13de 1.11±0.06f 2.32±0.14a MCd40 2.75±0.17cde 3.46±0.07a 0.80±0.06b MBCd40 1.54±0.10f 0.68±0.06g 2.28±0.15a

[0171] In summary, the individual application of biochar and AMF, as well as their combined treatment, demonstrated significant effects in reducing cadmium content in tobacco plants and regulating cadmium distribution. The combined treatment (biochar + AMF) showed even stronger effects in reducing cadmium content in roots and shoots, promoting cadmium compartmentalization, reducing cadmium toxicity, and regulating cadmium distribution. The combined application of biochar and AMF was most effective in alleviating cadmium stress and enhancing tobacco tolerance, exhibiting a significant synergistic effect. These measures significantly enhanced tobacco's tolerance to cadmium stress by reducing cadmium accumulation in tobacco roots and shoots, and altering cadmium distribution at the subcellular level and across organs, providing a scientific basis for the safe production of tobacco in cadmium-contaminated soils.

[0172] Example 4

[0173] The effects of a combined application of AMF and biochar on cadmium content and distribution in tobacco under 40 mg / kg cadmium stress were investigated. The specific steps are as follows:

[0174] 1. Preparation of inoculum

[0175] The same method as the preparation of AMF inoculum in Example 1.

[0176] 2. Cultivation of flue-cured tobacco seedlings

[0177] The same method of cultivating tobacco seedlings as in Example 1.

[0178] 3. Experiment Implementation

[0179] The same test implementation method as in Example 2.

[0180] 4. Sample collection and determination

[0181] The same method for sample collection and measurement as in Example 3.

[0182] 5. Conclusion

[0183] 5.1 Changes in cadmium content in tobacco products

[0184] See results Figure 15 Root cadmium content: Under cadmium stress of 40 mg / kg, both AMF inoculation alone (MCd40) and the combined treatment of AMF and biochar (MBCd40) significantly reduced the cadmium content in tobacco roots. The combined treatment showed the lowest root cadmium content, a reduction of 231.58% compared to the control group (Cd40). This indicates that the combined application of AMF and biochar has a significant advantage in reducing cadmium accumulation in roots.

[0185] Aboveground cadmium content: The application of AMF and biochar also significantly reduced the cadmium content in the aboveground parts of tobacco. Under cadmium stress of 40 mg / kg, AMF inoculation significantly reduced the aboveground cadmium content, while the combined treatment of biochar and AMF further reduced the aboveground cadmium concentration, demonstrating stronger cadmium fixation and barrier capabilities.

[0186] 5.2 Distribution changes of cadmium in tobacco

[0187] See results Figure 16Subcellular distribution of cadmium (Cd): Under 40 mg / kg cadmium stress, the application of AMF and biochar significantly altered the subcellular distribution of cadmium in tobacco leaves and roots. In leaves, cadmium was mainly concentrated in the cell walls and their residual components, while in roots, cadmium was more distributed in nucleoproteins and soluble components. The combined treatment (MBCd40) led to a greater transfer of cadmium from leaves to the cell walls and their residual components, reducing the accumulation of cadmium in organelles and nucleoproteins, thereby mitigating the toxic effects of cadmium on cells.

[0188] See results Figure 17 Cd chemical form distribution: Under Cd stress conditions without the addition of biochar and AMF (Cd40), cadmium in tobacco leaves mainly exists in the highly toxic E1 and E2 forms. These two chemical forms of cadmium have high biotoxicity and mobility, posing a significant potential threat to plant cells. The combined treatment of AMF and biochar (MBCd40) significantly reduced the proportion of highly toxic chemical forms of cadmium in leaves, while increasing the proportion of moderately toxic chemical forms (such as sodium chloride-extractable E3 and acetic acid-extractable E4). Specifically, the MBCd40 treatment significantly increased the proportion of E3 chemical form cadmium, indicating that cadmium exists more in moderately or lowly toxic forms, thereby reducing the toxicity of cadmium to leaf cells.

[0189] Under Cd stress conditions without the addition of biochar and AMF (Cd40), cadmium in tobacco roots is mainly in the highly toxic forms E1 and E2. These chemical forms of cadmium are more easily absorbed by the plant and translocated to the aboveground parts, causing significant harm to plant growth and development. The combined treatment with AMF and biochar (MBCd40) significantly altered the chemical distribution of cadmium in the roots. Compared to the Cd40 treatment, the combined treatment reduced the proportion of highly toxic chemical forms of cadmium while increasing the proportion of moderately toxic chemical forms (such as sodium chloride-extractable E3) and low-toxicity chemical forms (such as hydrochloric acid-extractable E5). This change indicates that the combined treatment promoted the conversion of cadmium to low-toxicity or less mobile chemical forms, reduced the bioavailability of cadmium in the roots, and thus mitigated the toxic effects of cadmium on root cells.

[0190] Cd enrichment and translocation: Results are shown in Table 7. The application of AMF and biochar reduced the cadmium translocation coefficient from roots to shoots. Under cadmium stress of 40 mg / kg, the combined treatment of biochar and AMF (MBCd40) showed the best effect in reducing the cadmium translocation coefficient from roots to shoots. The combined treatment not only significantly reduced the cadmium translocation coefficient but also further enhanced the cadmium fixation and barrier capacity, demonstrating a significant synergistic effect. Therefore, the combined treatment of AMF and biochar can more effectively reduce cadmium translocation within plants and reduce the toxic effects of cadmium on the shoots of plants.

[0191] In summary, the application of AMF and biochar significantly reduced the cadmium content in the roots and aboveground parts of tobacco under 40 mg / kg cadmium stress, and reduced the toxic effects of cadmium on tobacco cells by altering the subcellular distribution, chemical state distribution, and distribution among aboveground and underground organs. In particular, the combined treatment of AMF and biochar showed a stronger effect in reducing cadmium accumulation and translocation. These results indicate that the combined application of AMF and biochar is an effective cadmium pollution remediation strategy that can significantly enhance tobacco's tolerance to cadmium stress, providing a theoretical basis for the remediation of cadmium-contaminated soils and safe tobacco production.

[0192] Comparative Example 2

[0193] The effects of a combined application of AMF and biochar on cadmium content and distribution in tobacco under 0 mg / kg cadmium stress were investigated. The specific steps are as follows:

[0194] 1. Preparation of inoculum

[0195] The same method as the preparation of AMF inoculum in Example 1.

[0196] 2. Cultivation of flue-cured tobacco seedlings

[0197] The same method of cultivating tobacco seedlings as in Example 1.

[0198] 3. Experiment Implementation

[0199] The specific implementation is the same as the comparison ratio 1.

[0200] 4. Sample collection and determination

[0201] The same method for sample collection and measurement as in Example 3.

[0202] 5. Conclusion

[0203] 5.1 Changes in cadmium content in tobacco products:

[0204] Root cadmium content: Under 0 mg / kg cadmium stress, the total Cd content in the soil was <0.5 mg / kg, meeting the national standard for heavy metal content in arable land soil in 2023. The combined application of AMF and biochar significantly reduced the cadmium content in tobacco roots. This indicates that AMF and biochar, through their adsorption and fixation effects, reduced cadmium accumulation in roots, thereby mitigating the potential toxic effects of cadmium on roots. The effect of AMF and biochar addition on root cadmium content is complex: adding biochar alone or inoculating with AMF can significantly reduce root cadmium content, but the combined application of both has a more significant effect. This suggests that AMF and biochar can reduce cadmium accumulation in roots through adsorption, fixation, or alteration of the chemical form of cadmium, thereby mitigating the toxic effects of cadmium on tobacco roots.

[0205] Aboveground cadmium content: Similar to the roots, the aboveground cadmium content in tobacco was low under 0 mg / kg Cd treatment, but increased significantly under cadmium stress. The addition of AMF and biochar significantly reduced the aboveground cadmium content, especially when applied in combination. This may be because AMF and biochar regulate cadmium transport within the plant, reducing the transfer of cadmium from the roots to the aboveground parts, thereby decreasing cadmium accumulation in the aboveground parts. Furthermore, AMF and biochar may indirectly mitigate the toxic effects of cadmium on the aboveground parts by enhancing the plant's antioxidant capacity and nutrient uptake.

[0206] 5.2 Distribution changes of cadmium in tobacco:

[0207] See results Figure 16 Subcellular distribution of cadmium (Cd): The subcellular distribution of cadmium in tobacco cells was significantly affected by AMF and biochar. Under cadmium-free stress conditions, cadmium was mainly distributed in the cell wall and its residual components, indicating that the cell wall is the primary site of cadmium fixation. Under cadmium stress conditions, the addition of AMF and biochar promoted the translocation of cadmium to the cell wall and cytoplasm, reducing the accumulation of cadmium in organelles and nucleoproteins. This change in distribution helps to mitigate the toxic effects of cadmium on organelles and protect normal cellular physiological functions.

[0208] See results Figure 17 Distribution of Cd chemical states: The combined application of AMF and biochar promotes the conversion of cadmium from a highly toxic chemical state to a less toxic chemical state. This change in chemical state further reduces the bioavailability of cadmium and decreases its toxic effects on tobacco.

[0209] Cd Distribution Across Organs: Under different cadmium treatment concentrations, the addition of AMF and biochar significantly altered the distribution of cadmium between the roots and shoots of tobacco. Under low cadmium stress, AMF and biochar primarily reduced the cadmium content in the shoots by decreasing cadmium translocation from the roots to the shoots. However, under high cadmium stress, the combined application of AMF and biochar not only reduced the cadmium content in the shoots but also further reduced the overall cadmium toxicity within the plant by increasing cadmium fixation in the roots. This change in organ distribution suggests that AMF and biochar can optimize cadmium distribution within the plant by regulating cadmium uptake and translocation, thereby improving tobacco's tolerance to cadmium stress.

[0210] In summary, the combined application of AMF and biochar significantly enhances tobacco's tolerance to cadmium by reducing cadmium content in tobacco, altering the subcellular distribution of cadmium, promoting the conversion of cadmium's chemical state, and regulating the distribution of cadmium in different organs.

[0211] Example 5

[0212] A method for regulating soil nutrients and antioxidant enzyme activity under 20 mg / kg cadmium pollution conditions by combining AMF and biochar.

[0213] 1. Preparation of inoculum

[0214] The same method as the preparation of AMF inoculum in Example 1.

[0215] 2. Cultivation of flue-cured tobacco seedlings

[0216] The same method of cultivating tobacco seedlings as in Example 1.

[0217] 3. Experiment Implementation

[0218] The specific implementation is the same as in Example 1.

[0219] 4. Sample collection and testing

[0220] Thirty-five days after transplanting, rhizosphere soil samples were collected from the tobacco plants. The flue-cured tobacco plants were carefully removed from their pots, roots and soil intact. The topsoil was removed, and the loose soil attached to the roots was gently shaken off. Soil tightly bound to the roots was brushed off with a small brush, mixed thoroughly, and used as rhizosphere soil. This was then transferred to a cool, shaded area to air dry and passed through a 60-mesh sieve. Three replicates were selected for each treatment to determine soil physicochemical properties: pH, organic matter, available nitrogen, available phosphorus, available potassium, total Cd concentration, and available Cd concentration. Simultaneously, the activities of antioxidant enzymes in the soil were measured: glutathione peroxidase (GSH-Px) activity and soil catalase (S-CAT) activity. Soil pH was determined by water extraction; soil organic matter (SOM) content was determined by potassium dichromate titration; available nitrogen was determined by potassium chloride extraction-indophenol blue colorimetric method; available phosphorus was determined by sodium bicarbonate-molybdenum antimony colorimetric method; available potassium was determined by ammonium acetate-flame photometry, with specific methods referring to "Soil Agrochemical Analysis"; total Cd concentration in tobacco rhizosphere soil was measured according to the method in national standard GB-T 17141-1997; available Cd concentration in tobacco rhizosphere soil was measured according to the diethylenetriaminepentaacetic acid (DTPA) method in national standard HJ804-2016, using DTPA extraction solution (GB / T23739-2009). Glutathione peroxidase (GSH-Px) activity and soil catalase (S-CAT) activity in tobacco rhizosphere soil were determined according to the kit methods of Suzhou Keming Biotechnology Co., Ltd.

[0221] 5. Conclusion

[0222] 5.1 Changes in Soil Nutrients

[0223] In the Cd20 treatment, the addition of biochar and inoculation with AMF (MBCd20) significantly increased the contents of available nitrogen, available phosphorus, and available potassium in the soil. Compared with the Cd20 treatment without biochar or AMF inoculation, the soil available nitrogen content in the MBCd20 treatment increased by 33.88%, available phosphorus content by 63.64%, and available potassium content by 51.02%. This indicates that the combined application of biochar and AMF can effectively improve the nutrient status of Cd-contaminated soil and provide better nutritional support for plant growth.

[0224] 5.2 Changes in soil antioxidant enzyme activity

[0225] See results Figure 18 Compared with the Cd20 treatment, the MBCd20 treatment significantly increased the activity of soil glutathione peroxidase (GSH-Px), with an increase of 41.03%. Furthermore, the activity of soil catalase (S-CAT) was also increased, indicating that the combined application of biochar and AMF can enhance the soil's antioxidant capacity and alleviate the oxidative damage to soil microorganisms and plant roots caused by Cd stress.

[0226] 5.3 Changes in Cd content in soil

[0227] See results Figure 19 In the Cd20 treatment, the addition of biochar and inoculation with AMF (MBCd20) significantly affected the total Cd content and available Cd content in the soil. Compared with the Cd20 treatment without biochar or AMF, the MBCd20 treatment reduced the total Cd content by 15.23% and the available Cd content by 20.45%. This indicates that the combined application of biochar and AMF not only improves soil nutrient status but also reduces the toxic stress of Cd on plants and soil ecosystems by decreasing Cd availability.

[0228] In Cd20 treatment, the combined application of biochar and AMF significantly increased the content of available nitrogen, available phosphorus, and available potassium in the soil, and enhanced the activity of soil antioxidant enzymes (GSH-Px and S-CAT) (see Table 8). These changes helped alleviate the oxidative damage of Cd stress to soil microorganisms and plant roots, promoted tobacco growth and development, and significantly reduced the absorption and accumulation of Cd by tobacco. Therefore, the combined application of AMF and biochar can serve as a green and efficient soil improvement and phytosanitary strategy for the remediation of Cd-contaminated soils and the sustainable production of economic crops such as tobacco.

[0229] Table 8 Effects of AMF and biochar on soil agrochemical properties

[0230] deal with pH Available nitrogen (mg / kg) Available phosphorus (mg / kg) Available potassium (mg / kg) Organic matter (mg / kg) Cd0 6.15±0.01c 62.70±2.12b 14.10±0.12a 129.33±2.40c 19.55±0.32a BCd0 6.15±0.00b’ *72.18±1.99c’ ***8.58±0.09c’ <![CDATA[ ** *271.33±0.88c']]> ***51.10±1.34a’ MCd0 ***6.38±0.00 65.28±3.98B ***9.46±0.18A <![CDATA[ ** *254.33±0.88C]]> **15.88±0.37B MBCd0 ***6.44±0.01 ***44.03±2.34C *13.51±0.23A’ ***511.00±9.02C ***31.78±0.21A’ Cd20 6.53±0.01a 67.73±3.08b 8.32±0.05b 404.33±29.48b 15.18±0.40c BCd20 6.49±0.00a’ **80.61±0.53b’ *9.57±0.19b’ ***618.33±11.84 ***32.83±0.48c’ MCd20 ***6.11±0.04 ***97.24±1.97A **6.81±0.40B ***793.00±15.28 ***23.08±0.89A MBCd20 ***6.09±0.02 ***90.98±2.33A **9.77±0.33B’ ***982.00±12.34 ***30.28±0.34B’ Cd40 6.18±0.01b 97.98±2.33a 6.48±0.32c 559.67±12.12a 16.78±0.38b BCd40 ***5.85±0.00 ***116.14±3.28 ***10.90±0.06a’ ***1033.33±2.85 ***43.52±0.24b’ MCd40 <![CDATA[ ** 6.34±0.02A]]> ***66.81±1.80B ***8.89±0.07A ***703.67±37.83 16.95±0.03B MBCd40 ***6.58±0.05 **81.14±2.63B’ ***9.28±0.16B’ ***1023.67±12.1 16.23±0.48C’

[0231] Note: Data represent mean ± standard error. Within-group comparisons: Cd0 vs BCd0 vs MCd0 vs MBCd0; Cd20 vs BCd20 vs MCd20 vs MBCd20; Cd40 vs BCd40 vs MCd40 vs MBCd40. (ns: P>0.05; *: P<0.05; **: P<0.01; ***: P<0.001) Intergroup comparisons: Cd0 vs Cd20 vs Cd40 (significance is indicated by a, b, c, etc.); Cd0 vs Cd20 vs Cd40 (significance is indicated by a', b', c', etc.); MCd0 vs MCd20 vs MCd40 (significance is indicated by A, B, C, etc.); MBCd0 vs MBCd20 vs MBCd40 (significance is indicated by A', B', C', etc.). The above are the results of one-way variance analysis.

[0232] Example 6

[0233] A method for regulating soil nutrients and antioxidant enzyme activity under 40 mg / kg cadmium pollution conditions by combining AMF and biochar.

[0234] 1. Preparation of inoculum

[0235] The same method as the preparation of AMF inoculum in Example 1.

[0236] 2. Cultivation of flue-cured tobacco seedlings

[0237] The same method of cultivating tobacco seedlings as in Example 1.

[0238] 3. Experiment Implementation

[0239] The specific implementation is the same as in Example 2.

[0240] 4. Sample collection and determination

[0241] The same method for collecting and measuring soil samples as in Example 5.

[0242] 5. Conclusion

[0243] 5.1 Changes in Soil Nutrients

[0244] At the 40 mg / kg Cd treatment, AMF alone significantly reduced soil available nitrogen content (by 31.8%). However, the combined application of biochar and AMF in MBCd40 mitigated this trend, reducing the decrease in available nitrogen to 17.2%, while significantly increasing available phosphorus, available potassium, and organic matter content. Available phosphorus content reached 8.89 mg / kg, approximately 37.2% higher than the Cd40 treatment without biochar and AMF (6.48 mg / kg). Available potassium content reached 703.67 mg / kg, approximately 25.7% higher than the Cd40 treatment without biochar and AMF (559.67 mg / kg). Organic matter content reached 16.95 mg / kg, slightly higher than the Cd40 treatment without biochar and AMF (16.78 mg / kg).

[0245] 5.2 Changes in antioxidant enzyme activity

[0246] See results Figure 18 At a Cd treatment level of 40 mg / kg, the combined application of AMF and biochar significantly increased the activity of soil catalase (S-CAT), indicating an enhanced ability to alleviate soil oxidative stress under Cd pollution conditions. The combined application of AMF and biochar also significantly increased the activity of glutathione peroxidase (GSH-Px), which helps to enhance soil antioxidant capacity and reduce oxidative damage to soil microorganisms and plants caused by Cd stress.

[0247] 5.3 Changes in Cd concentration in soil

[0248] See results Figure 19 When treated with 40 mg / kg Cd, the combined application of AMF and biochar significantly increased the concentration of total Cd in the soil at tobacco harvest, but significantly reduced the concentration of available Cd. This indicates that Cd was fixed or converted into a less toxic form, reducing its bioavailability, thus decreasing its absorption and accumulation by plants and reducing its toxicity to plants and microorganisms.

[0249] The combined application of AMF and biochar significantly improved soil nutrient status under 40 mg / kg Cd pollution conditions, increasing the content of available nitrogen, available phosphorus, and available potassium, while enhancing the soil's antioxidant capacity and reducing the bioavailability of Cd. This combined application, by improving soil nutrients and antioxidant enzyme activity, reduces the toxicity of Cd to plants and microorganisms, providing an effective strategy for the remediation of Cd-contaminated soils.

[0250] Comparative Example 3

[0251] A method for regulating soil nutrients and antioxidant enzyme activity under 0 mg / kg cadmium pollution conditions by combining AMF and biochar. The specific steps are as follows:

[0252] 1. Preparation of inoculum

[0253] The same method as the preparation of AMF inoculum in Example 1.

[0254] 2. Cultivation of flue-cured tobacco seedlings

[0255] The same method of cultivating tobacco seedlings as in Example 1.

[0256] 3. Experiment Implementation

[0257] The specific implementation is the same as the comparison ratio 1.

[0258] 4. Sample collection and determination

[0259] The same method for collecting and measuring soil samples as in Example 5.

[0260] 5. Conclusion

[0261] 5.1 Changes in Soil Nutrients

[0262] In the Cd0 treatment, the combined application of biochar and AMF inoculation significantly affected soil nutrients. The results showed that, compared with the control group without biochar or AMF inoculation, the combined application significantly increased the contents of available nitrogen, available phosphorus, and available potassium in the soil. Specifically, compared with the control group, available nitrogen increased by 15.12%, available phosphorus decreased by 39.15%, available potassium increased by 109.80%, and soil organic matter increased by 161.38%. These results indicate that the combined application of biochar and AMF can effectively improve soil fertility and provide better nutritional conditions for plant growth.

[0263] 5.2 Changes in antioxidant enzyme activity

[0264] See results Figure 18 Under Cd0 treatment, the combined application of biochar and AMF significantly regulated the activity of soil antioxidant enzymes. Specifically, soil catalase (S-CAT) activity was significantly increased. Simultaneously, glutathione peroxidase (GSH-Px) activity was also significantly enhanced. This increased activity of antioxidant enzymes indicates that the combined application of biochar and AMF can effectively alleviate the oxidative stress caused by Cd pollution in soil, enhance the soil's antioxidant capacity, and thus protect soil microorganisms and plant roots from oxidative damage.

[0265] 5.3 Changes in Cd concentration in soil

[0266] See results Figure 19In the Cd0 treatment, although there was no external Cd pollution in the soil, the impact of the combined application on the background Cd level could be assessed by detecting the concentrations of total Cd and available Cd in the soil. Table 7 shows that the combined application of biochar and AMF did not significantly change the total Cd content in the soil, but it effectively reduced the concentration of available Cd. This indicates that the combined application may reduce the potential toxicity of Cd to plants and soil ecosystems by altering the chemical form of Cd, transforming it from a bioavailable form that can be absorbed by plants into a more stable form.

[0267] This comparative study demonstrates that, even without Cd pollution, the combined application of AMF and biochar is an effective method for regulating soil nutrients and antioxidant enzyme activity. By increasing the content of available nitrogen, available phosphorus, available potassium, and organic matter in the soil, the combined application provides richer nutrient resources for plant growth. Therefore, the combined application of AMF and biochar not only improves soil fertility but also enhances the soil's antioxidant capacity and heavy metal tolerance.

[0268] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a composition of arbuscular mycorrhizal fungi and biochar in reducing the accumulation of cadmium in organelles and nucleoproteins in tobacco roots and leaves under cadmium stress, wherein the mass ratio of the number of spores of the arbuscular mycorrhizal fungi to the mass of biochar is 7000-9000 spores: 75-85 grams; The arbuscular mycorrhizal fungus is *M. mosieuri* (…). Funneliformis mosseae ).

2. The application according to claim 1, characterized in that, The ratio of the number of spores of the arbuscular mycorrhizal fungi to the mass of biochar is 7500-8500 spores: 78-82 grams.

3. The application according to claim 1, characterized in that, The types of biochar include at least one of the following: rice husks, tobacco stalks, bamboo charcoal, and wood charcoal.

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