Arbuscular mycorrhizal fungus inoculant, compound agent, method for realizing low cadmium and high quality of rice grains and application of arbuscular mycorrhizal fungus inoculant

Abstract

The invention belongs to the technical field of agriculture, and particularly relates to an arbuscular mycorrhizal fungus inoculant, a compound agent, a method for achieving low-cadmium and high-quality rice grains and application of the arbuscular mycorrhizal fungus inoculant and the compound agent. According to the arbuscular mycorrhizal fungi fungicide, arbuscular mycorrhizal fungi relieve the toxic effect of heavy metal on plants and improve the heavy metal toxicity resistance of the plants. The invention further provides a compound agent, the arbuscular mycorrhizal fungi fungicide and exogenous calcium are jointly applied, and the exogenous nano-calcium synergistically reduces cadmium absorption and cadmium accumulation of arbuscular mycorrhizal fungi symbiotic plants. The invention further provides a method for reducing cadmium absorption of the rice, the arbuscular mycorrhizal fungus inoculant is applied to the rice, the cadmium concentration of the roots and the overground part of the rice can be reduced, meanwhile, the content of Ca and Zn in rice grains is obviously increased, and therefore the quality of the rice is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of agriculture, and in particular relates to an arbuscular mycorrhizal fungal agent, a compound agent, and an application thereof for achieving low-cadmium and high-quality rice grains. Background Art

[0002] Cadmium (Cd) is extremely toxic, highly soluble in water, and easily absorbed and transported by plants. It is one of the most toxic pollutants in the environment. Since Cd accumulated in the edible parts of crops will eventually be transferred to the human body through the food chain, it will cause harm to human health. Therefore, how to effectively prevent and control the absorption and accumulation of heavy metal Cd by plants is an urgent problem to be solved in current green agricultural production. At present, technologies to reduce the accumulation of Cd in plants are emerging in an endless stream, among which exogenous addition of microorganisms is considered to be an economical, environmentally friendly and friendly strategy.

[0003] Arbuscular mycorrhizal fungi (AM fungi) are a type of beneficial fungi belonging to the phylum Glomeromycetes in the soil that form a mutualistic symbiosis with plant roots. As a microorganism that lives in symbiosis with plants, AM fungi can significantly improve soil structure, promote nutrient absorption, and reduce the emission of greenhouse gases such as nitrous oxide by regulating the nitrogen conversion process.

[0004] Prior art CN114885777A discloses a method for reducing cadmium absorption by rice, wherein the method for reducing cadmium absorption by rice includes applying alkaline passivation material as a base before rice planting and applying zinc fertilizer after rice planting; Prior art CN116171816A discloses an application of strengthening arbuscular mycorrhizal fungi to reduce cadmium absorption by rice in paddy fields, wherein arbuscular mycorrhizal fungi are added to rice, but the infection rate of arbuscular mycorrhizal fungi on rice is low, and the application effect of arbuscular mycorrhizal fungi under low cadmium concentration conditions of 0.23 mg / kg or 1.08 mg / kg is only involved. Summary of the invention

[0005] The invention aims to provide an arbuscular mycorrhizal fungal agent, a compound agent, a method for achieving low-cadmium and high-quality rice grains and applications. The arbuscular mycorrhizal fungal agent has a high infection rate on rice and has a good application effect under high-concentration cadmium conditions.

[0006] In order to solve the above technical problems, the following technical solutions are proposed:

[0007] The invention provides an arbuscular mycorrhizal fungal agent, comprising leek roots infected by arbuscular mycorrhizal fungi and a matrix. The arbuscular mycorrhizal fungi are Rhizophagus irregularis and / or Rhizophagus intraradices.

[0008] Preferably, the substrate comprises river sand; and the arbuscular mycorrhizal fungi in the microbial agent exist in the form of spores and / or hyphae.

[0009] The invention provides a method for preparing the arbuscular mycorrhizal fungi agent described in the technical scheme, comprising: culturing leeks in a matrix containing arbuscular mycorrhizal fungi, collecting the root system and the matrix of the leek after culturing, and obtaining the arbuscular mycorrhizal fungi agent; the addition amount of the arbuscular mycorrhizal fungi is 2% of the mass of the matrix.

[0010] Preferably, the cultivation time is 5 to 6 months, the light cycle of the cultivation is 12 h / d of light and 12 h / d of darkness, the daytime temperature of the cultivation is 25 to 35°C, and the nighttime temperature is 17 to 22°C; water and low-phosphorus nutrient solution are applied during the cultivation process.

[0011] The present invention provides the use of the arbuscular mycorrhizal fungal agent described in the above technical solution or the arbuscular mycorrhizal fungal agent prepared by the preparation method described in the above technical solution in the following 1) and / or 2),

[0012] 1) Reduce the absorption of cadmium by rice;

[0013] 2) Increase the infection rate of arbuscular mycorrhizal fungi on rice.

[0014] The invention provides a compound agent, comprising packaged arbuscular mycorrhizal fungi agent and calcium fertilizer; the arbuscular mycorrhizal fungi agent is the arbuscular mycorrhizal fungi agent described in the above technical scheme or the arbuscular mycorrhizal fungi agent prepared by the preparation method described in the above technical scheme.

[0015] Preferably, the calcium fertilizer comprises nano calcium carbonate.

[0016] The present invention provides the use of the compound described in the above technical solution in any one of the following 1) to 3):

[0017] 1) Increase the infection rate of arbuscular mycorrhizal fungi on rice;

[0018] 2) Reduce the absorption of cadmium by rice;

[0019] 3) Increase the content of macroelements and trace elements in rice.

[0020] The invention provides a method for reducing cadmium absorption by rice, comprising: using an arbuscular mycorrhizal fungal agent for rice cultivation, wherein the arbuscular mycorrhizal fungal agent is the arbuscular mycorrhizal fungal agent described in the above technical solution or the arbuscular mycorrhizal fungal agent prepared by the preparation method described in the above technical solution.

[0021] The present invention provides a method for achieving low-cadmium and high-quality rice grains, comprising: applying the compound agent described in the above technical solution during rice cultivation.

[0022] Beneficial effects of the present invention: The present invention provides an arbuscular mycorrhizal fungal agent, which can affect the absorption, accumulation and detoxification process of heavy metals by plants through multiple pathways, such as reducing the effectiveness of heavy metals in the soil by producing secretions such as glycoproteins and globulins, enhancing the plant antioxidant enzyme system (superoxide dismutase, catalase and peroxidase), promoting phosphorus absorption and plant growth, etc., alleviating the toxic effects of heavy metals on plants, and improving the ability of plants to resist heavy metal poisoning. In addition, leek roots and substrates are used to cultivate arbuscular mycorrhizal fungi, which increases the number of hyphae and spores of arbuscular mycorrhizal fungi, further improving the ability of arbuscular mycorrhizal fungi to resist heavy metal poisoning.

[0023] The present invention also provides a compound agent, which combines the packaged arbuscular mycorrhizal fungi agent with exogenous calcium, and introduces exogenous nano-calcium to enhance the symbiotic effect between plants and arbuscular mycorrhizal fungi, and synergistically reduce the cadmium absorption and cadmium accumulation of arbuscular mycorrhizal fungi symbiotic plants. The results of the embodiment show that applying the compound agent to rice can reduce the cadmium absorption of rice roots and the cadmium accumulation of rice aboveground parts and grains.

[0024] The invention also provides a method for reducing the absorption of cadmium by rice. The cadmium concentration in the root and aboveground part of the rice can be reduced by applying the arbuscular mycorrhizal fungi agent to the rice.

[0025] The present invention also provides a method for achieving low cadmium and high quality of rice grains. The compound is applied to rice, which can increase the infection rate of arbuscular mycorrhizal fungi on rice, reduce the absorption of cadmium by rice, and increase the content of macroelements and trace elements in rice. The results of the embodiment show that exogenous calcium combined with inoculated arbuscular mycorrhizal fungi can not only effectively control the absorption of Cd by rice roots and the accumulation of Cd in grains under high cadmium concentration conditions, but also significantly increase the content of Ca and Zn in rice grains, thereby improving the quality of rice. Today, when food safety and nutritional health issues are becoming increasingly prominent, the research results of the present invention not only conform to the sustainable development concept of modern agriculture, but also provide new ideas and methods for improving people's nutritional levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a diagram showing the effect of exogenous calcium addition on the symbiotic relationship between arbuscular mycorrhizal fungi and rice under Cd stress;

[0027] Figure 2 This is the effect of exogenous calcium-binding bacterial agent 2 on the Cd content in rice roots;

[0028] Figure 3 This is the result of detecting and analyzing the Cd content in the aboveground part and grains of rice under the treatment of exogenous calcium-binding bacterial agent 2. DETAILED DESCRIPTION

[0029] The invention provides an arbuscular mycorrhizal fungal agent, comprising leek roots infected by arbuscular mycorrhizal fungi and a matrix. The arbuscular mycorrhizal fungi are Rhizophagus irregularis and / or Rhizophagus intraradices.

[0030] The Rhizophagus irregularis and Rhizophagus intraradices of the present invention improve the ability of plants to resist heavy metal poisoning. The present invention does not specifically limit the sources of the Rhizophagus irregularis and Rhizophagus intraradices, and conventional products can be used. As an optional embodiment, the Rhizophagus irregularis of the present invention is a Rhizophagus irregularis strain numbered JX04B for effect verification; the Rhizophagus intraradices of the present invention is a Rhizophagus intraradices strain numbered BJ09 for effect verification. The application effect of Rhizophagus intraradices BJ09 is better than that of Rhizophagus irregularis JX04B.

[0031] As an optional embodiment, the substrate of the present invention includes river sand, which does not contain organic matter and is not easy to breed bacteria, thereby reducing the risk of bacterial contamination during the propagation process. River sand is a cheap and readily available material. Using river sand as a propagation medium can significantly reduce production costs, has good air permeability, and has a certain water retention capacity, which is convenient for the collection and use of fungal agents in the later stage. The substrate of the present invention is sterilized at high temperature before use.

[0032] The arbuscular mycorrhizal fungal agent of the present invention comprises leek roots infected by arbuscular mycorrhizal fungi. The reason why leek is selected as a culture matrix is ​​that the root system of leek is relatively developed, the spores and hyphae produced by propagation are many, and the propagation effect is good. Before the arbuscular mycorrhizal fungal agent is collected, the root system of leek is collected in advance for microscopic examination, and a large number of hyphae are seen in the root system, indicating that the propagation effect is good, and the fungal agent can be harvested at this time. Leek has a dense fibrous root system and a large root surface area, which provides sufficient space for the infection and colonization of AMF. The growth rate is fast, and the cycle from sowing to harvesting is short. The root segments of leek are attached with a large number of spores. After harvesting, the root segments of leek are collected and mixed evenly with the matrix to obtain the arbuscular mycorrhizal fungal agent, thereby improving the infection rate of the agent. The present invention cuts the leek roots infected by arbuscular mycorrhizal fungi to root segments with a length of about 0.5 cm, and then mixes them with the matrix used for cultivating leek.

[0033] As an optional embodiment, the arbuscular mycorrhizal fungi in the microbial agent of the present invention exist in the form of spores and / or hyphae, the number of spores in the arbuscular mycorrhizal fungi microbial agent is 12 / g, and the number of spores and hyphae of the arbuscular mycorrhizal fungi microbial agent is high.

[0034] The arbuscular mycorrhizal fungal agent of the invention has high quantities of spores and hyphae, can reduce the absorption of cadmium by plants, and improve the infection rate of arbuscular mycorrhizal fungi to plants.

[0035] The present invention provides a method for preparing the arbuscular mycorrhizal fungi agent described in the above technical solution, comprising: culturing leek in a matrix containing arbuscular mycorrhizal fungi, collecting the root system and matrix of the leek after culturing, and obtaining the arbuscular mycorrhizal fungi agent; the addition amount of the arbuscular mycorrhizal fungi is 2% of the mass of the matrix. As an optional embodiment, the method for preparing the matrix containing arbuscular mycorrhizal fungi of the present invention comprises: adding arbuscular mycorrhizal fungi to the matrix, and the method of adding arbuscular mycorrhizal fungi to the matrix includes a cave method, and the cave method includes: poking a cave in the matrix, adding the arbuscular mycorrhizal fungi to the cave, and there is no special limitation on the depth and size of the cave. As an optional embodiment, after adding arbuscular mycorrhizal fungi to the matrix, a mixture is obtained; and leek seeds are cultured in the mixture. Before culturing, the present invention first disinfects the leek seeds to prevent diseases and improve the germination rate of the seeds. As an optional embodiment, the reagent used for disinfection of the present invention comprises a sodium hypochlorite solution with an effective chlorine content of 30%. As an optional embodiment, the culture time of the present invention is at least 5 months, or 5 to 6 months, more preferably 6 months. The longer the culture time, the more plant roots, and the more spores harvested; the light cycle of the culture is 12h / d of light and 12h / d of darkness, and the daytime temperature of the culture is 25 to 35°C, or 28 to 31°C; the nighttime temperature is 17 to 22°C, or 18 to 21°C. As an optional embodiment, water and low-phosphorus nutrient solution are applied during the culture. The present invention does not specifically limit the amount and method of applying the water, and conventional methods can be used. In a specific embodiment of the present invention, the water and low-phosphorus nutrient solution are applied to the planting pot to drip water or nutrient solution. The present invention starts to add the low-phosphorus nutrient solution when the leek grows two fully expanded leaves. As an optional embodiment, the low-phosphorus nutrient solution of the present invention includes 1 / 8 low-phosphorus nutrient solution, 1 / 4 low-phosphorus nutrient solution, 1 / 2 low-phosphorus nutrient solution and low-phosphorus full nutrient solution. The composition of the first full nutrient solution of the present invention includes: 1mMNH 4 + , 4 mM NO 3 - , 2mM K + , 1 mM PO 4 3-, 0.75 mM Ca 2+ , 0.5 mM Mg 2+ , 0.25mMCl - , 0.5 mM SO 4 2- , 20 μM Fe 2+ , 9 μM Mn 2+ , 46 μM BO 3 3- , 8 μM Zn 2+ , 3 μM Cu 2+ and 0.03 μM MoO 4 2- . The components of the 1 / 8 low-phosphorus nutrient solution of the present invention are the same as those of the first complete nutrient solution. Except for phosphorus, the contents of other components are 1 / 8 of those of the first complete nutrient solution, and the phosphorus concentration in the 1 / 8 low-phosphorus nutrient solution is 25 μmol / L. The components of the 1 / 4 low-phosphorus nutrient solution are the same as those of the first complete nutrient solution. Except for phosphorus, the contents of other components are 1 / 4 of those of the first complete nutrient solution, and the phosphorus concentration in the 1 / 4 low-phosphorus nutrient solution is 25 μmol / L. The components of the 1 / 2 low-phosphorus nutrient solution are the same as those of the first complete nutrient solution. Except for phosphorus, the contents of other components are 1 / 2 of those of the first complete nutrient solution, and the phosphorus concentration in the 1 / 2 low-phosphorus nutrient solution is 25 μmol / L. The components of the low-phosphorus complete nutrient solution are the same as those of the first complete nutrient solution. Except for phosphorus, the contents of other components are the same as those of the first complete nutrient solution, and the phosphorus concentration in the low-phosphorus complete nutrient solution is 25 μmol / L. When the leek grows two fully expanded leaves during the leek cultivation process, 1 / 8 low-phosphorus nutrient solution is irrigated in the first week, 1 / 4 low-phosphorus nutrient solution is irrigated in the second week, 1 / 2 low-phosphorus nutrient solution is irrigated in the third week, and low-phosphorus full nutrient solution is irrigated in the fourth week, and then low-phosphorus full nutrient solution is irrigated once a week until the end of cultivation. The reason why 1 / 8 low-phosphorus nutrient solution, 1 / 4 low-phosphorus nutrient solution and 1 / 2 low-phosphorus nutrient solution are selected for irrigation in sequence is that in the early stage of cultivation, the seeds carry part of the nutrition and do not need too much nutrition. The concentration of the nutrient solution is from low to high, and the nutrient concentration in the culture environment is gradually increased, which can avoid the death of plants due to nutritional maladaptation. After 5 to 6 months of cultivation, watering and nutrition are stopped, the leek is allowed to die naturally, the aerial part of the leek is removed, the leek root is cut to a length of 0.5 cm, and after mixing, an arbuscular mycorrhizal fungus agent consisting of leek root segments, substrate river sand, spores and hyphae of arbuscular mycorrhizal fungi is finally obtained, and the interior of the leek root segment contains arbuscular mycorrhizal fungus hyphae. The arbuscular mycorrhizal fungus agent is applied to rice to reduce the cadmium concentration in the root and aerial part of the rice.

[0036] The present invention provides the use of the arbuscular mycorrhizal fungal agent described in the above technical solution or the arbuscular mycorrhizal fungal agent prepared by the preparation method described in the above technical solution in reducing the absorption of cadmium by rice. As an optional embodiment, the application period of the arbuscular mycorrhizal fungal agent includes the period when the rice grows two fully expanded leaves; the application method includes inoculating the arbuscular mycorrhizal fungal agent in the soil, and the inoculation method includes the cave burial method. In an embodiment of the present invention, the application amount is 200 to 220 spores per seedling. In the potted experiment of a specific embodiment of the present invention, the application amount of the arbuscular mycorrhizal fungal agent is 200 spores per seedling. As an optional embodiment, the reduction of cadmium absorption by rice in the present invention includes one or more of reducing the absorption of cadmium by rice roots, reducing the absorption of cadmium by rice aboveground parts, and the accumulation of cadmium in rice grains.

[0037] The present invention provides the use of the arbuscular mycorrhizal fungi agent described in the above technical solution or the arbuscular mycorrhizal fungi agent prepared by the preparation method described in the above technical solution in increasing the infection rate of arbuscular mycorrhizal fungi on rice. The application period, application method and application amount of the arbuscular mycorrhizal fungi agent have been discussed above and will not be repeated here.

[0038] The invention provides a compound agent, comprising a packaged arbuscular mycorrhizal fungal agent and a calcium fertilizer, wherein the arbuscular mycorrhizal fungal agent is the arbuscular mycorrhizal fungal agent described in the above technical solution or the arbuscular mycorrhizal fungal agent prepared by the preparation method described in the above technical solution. The packaged agent described in the invention is used separately at different times for the arbuscular mycorrhizal fungal agent and the calcium fertilizer. As an optional embodiment, the calcium fertilizer comprises nano calcium carbonate. The nano calcium carbonate described in the invention is prepared into a solution for application. The preparation method of the nano calcium carbonate solution of the invention comprises: mixing nano calcium carbonate, sodium polyacrylate, water and xanthan gum and then ultrasonicating. As an optional embodiment, the mass volume ratio of the nano calcium carbonate, sodium polyacrylate, water and xanthan gum is 0.5g, 1ml, 499ml: 0.5g; the sodium polyacrylate and xanthan gum are used to promote the dissolution of nano calcium carbonate in water. As an optional embodiment, the mixing method comprises stirring; and the ultrasonic time is 20min. A uniformly mixed nano calcium carbonate solution can be obtained by the mixing and ultrasonication. Calcium ions play an important role as a second messenger in plant cells, especially when responding to external stimuli such as heavy metal stress. Arbuscular mycorrhizal fungi can increase the level of calcium ions in plants, thereby prompting plants to activate antioxidant defense systems and reduce damage caused by oxidative stress, thereby enhancing the plant's defense mechanism against Cd toxicity.

[0039] Nano calcium carbonate has a very small particle size and can quickly enter plant cells, making it easily absorbed by plants. Whether the exogenous addition of nano calcium combined with arbuscular mycorrhizal fungi has a synergistic or antagonistic effect on rice Cd absorption, there are few reports on the relevant research mechanism. The present invention proves that the exogenous addition of nano calcium can promote the blocking effect of arbuscular mycorrhizal fungi on rice Cd absorption.

[0040] The present invention provides the use of the compound described in the above technical solution in improving the infection rate of arbuscular mycorrhizal fungi to rice. As an optional embodiment, the application period of the arbuscular mycorrhizal fungi agent in the compound described in the present invention includes the period when the rice grows two fully expanded leaves; the application method includes inoculating the arbuscular mycorrhizal fungi agent in the soil, and the inoculation method includes any one of the cave burial method, the layered addition method and the mixing and adding method; the application method includes the cave burial method; the layered addition method includes: dividing the substrate into two parts, first spreading the first part of the substrate, then spreading the agent on the upper part of the first part of the substrate, and then spreading the remaining substrate on the agent; the mixing and adding method includes mixing the substrate and the agent evenly.

[0041] The amount of spores applied in the present invention is 200 to 220 per seedling. In a potted plant experiment of a specific embodiment of the present invention, the amount of spores applied of the arbuscular mycorrhizal fungi agent is 200 per seedling. The arbuscular mycorrhizal fungi agent in the compound prepared by the present invention has a high infection rate on rice.

[0042] The application method of the nano calcium carbonate solution in the compound of the present invention includes foliar spraying, the period of foliar spraying includes the heading period and / or the grain filling period of rice, and the spraying amount is 15-16 mL per rice seedling.

[0043] The present invention provides an application of the compound described in the above technical solution in reducing the absorption of cadmium by rice. The application method has been discussed above and will not be repeated here. The reduction of cadmium absorption by rice in the present invention includes one or more of reducing the absorption of cadmium by rice roots, reducing the absorption of cadmium by rice aboveground parts, and the accumulation of cadmium in rice grains.

[0044] The present invention provides the use of the compound described in the above technical solution in increasing the content of macroelements and trace elements in rice. As an optional embodiment, the macroelements include calcium; the trace elements include zinc. The application method has been discussed above and will not be repeated here.

[0045] The present invention provides a method for reducing cadmium absorption by rice, comprising: using an arbuscular mycorrhizal fungal agent for rice cultivation, wherein the arbuscular mycorrhizal fungal agent is the arbuscular mycorrhizal fungal agent described in the above technical solution or the arbuscular mycorrhizal fungal agent prepared by the preparation method described in the above technical solution. The application method, application period and application amount of the present invention have been discussed above and will not be repeated here.

[0046] The present invention provides a method for achieving low cadmium and high quality rice grains, comprising: applying the compound described in the above technical solution during rice cultivation. The application method, application period and application amount of the present invention have been discussed above and will not be repeated here.

[0047] The present invention proposes a method and application of using exogenous calcium to strengthen arbuscular mycorrhizal fungi to achieve low Cd and high quality rice grains. The method aims to enhance the symbiotic effect between rice and mycorrhizal fungi by introducing exogenous nano-calcium, synergistically reduce the Cd absorption of mycorrhizal symbiotic rice roots and the accumulation of Cd to the aboveground parts and grains, while significantly enhancing the ability of rice grains to accumulate calcium and zinc, and ultimately improve the nutritional value of rice.

[0048] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0049] Example 1 Preparation of test bacterial agent

[0050] Rhizophagus irregularis JX04B and Rhizophagus intraradices BJ09 were purchased from the Institute of Plant Nutrition and Resources, Beijing Academy of Agriculture and Forestry Sciences.

[0051] For Rhizophagus irregularis JX04B and Rhizophagus intraradices BJ09, see “Wang Youshan, Zhang Junling. Collection, sharing services and research and utilization of arbuscular mycorrhizal fungi in China[J]. Journal of Mycology, 2019, 38(11): 1760-1807.”

[0052] The purchased Rhizophagus irregularis JX04B bacterial agent was recorded as initial bacterial agent 1, and the purchased Rhizophagus intraradices BJ09 bacterial agent was recorded as initial bacterial agent 2.

[0053] The leek seeds are disinfected by using a sodium hypochlorite solution with an effective chlorine content of 30% to obtain disinfected seeds.

[0054] The composition of the first complete nutrient solution is: 1mM NH 4 + , 4mM NO 3- , 2mM K + , 1mM PO 4 3- , 0.75 mM Ca 2+ , 0.5 mM Mg 2+ , 0.25 mM Cl - , 0.5 mM SO 4 2- 、20μMFe 2+ , 9μM Mn 2+ 、46μMBO 3 3- , 8μM Zn 2+ , 3μM Cu 2+ , 0.03μM MoO 4 2- , the phosphorus concentration of the first complete nutrient solution is 1mM.

[0055] 1 / 8 low-phosphorus nutrient solution: Its components are the same as the first complete nutrient solution. Except for phosphorus, the concentration of all other elements is 1 / 8 of that in the complete nutrient solution. The phosphorus concentration in the 1 / 8 low-phosphorus nutrient solution is 25μmol / L.

[0056] 1 / 4 low-phosphorus nutrient solution: Its components are the same as the first complete nutrient solution. Except for phosphorus, the concentration of all other elements is 1 / 4 of that of the complete nutrient solution. The phosphorus concentration in the 1 / 4 low-phosphorus nutrient solution is 25μmol / L.

[0057] 1 / 2 low-phosphorus nutrient solution: Its components are the same as the first complete nutrient solution. Except for phosphorus, the concentration of all other elements is 1 / 2 of that of the complete nutrient solution. The phosphorus concentration in the 1 / 2 low-phosphorus nutrient solution is 25μmol / L.

[0058] The components of the low-phosphorus complete nutrient solution are the same as those of the first complete nutrient solution. Except for phosphorus, the contents of other components are the same as those of the first complete nutrient solution. The phosphorus concentration in the low-phosphorus complete nutrient solution is 25 μmol / L.

[0059] Propagation of microbial agents:

[0060] The purchased river sand was subjected to high-temperature treatment at 120°C for 1h, then potted and irrigated with an appropriate amount of tap water overnight, and the initial bacterial agent 1 and the initial bacterial agent 2 were added to the matrix river sand at a ratio of 2% (W / W) using a cave method, and the disinfected leek seeds were sown into the matrix, and an appropriate amount of water was sprayed regularly. When the leek grew two fully expanded leaves, the nutrient solution for the first irrigation was 1 / 8 low-phosphorus nutrient solution, the nutrient solution for irrigation in the second week was 1 / 4 low-phosphorus nutrient solution, the nutrient solution for irrigation in the third week was 1 / 2 low-phosphorus nutrient solution, and the nutrient solution for irrigation in the fourth week was low-phosphorus full nutrient solution. After that, the low-phosphorus full nutrient solution was watered at a frequency of once a week until the cultivation was completed.

[0061] The culture conditions of leek are: 12h / d of light, 12h / d of darkness, daytime temperature: 25℃~35℃, nighttime temperature: 17℃~22℃. After 6 months of culture, stop watering and low-phosphorus nutrient solution, and let the leek die naturally. After death, remove the aboveground part of the leek, cut the leek roots to root segments with a length of 0.5cm, mix the root segments with the river sand used to culture the leek, and finally obtain the arbuscular mycorrhizal fungal inoculant, which is composed of leek root segments, river sand, spores and hyphae of arbuscular mycorrhizal fungi. The arbuscular mycorrhizal fungal inoculant prepared using the initial inoculant of Rhizophagus irregularis JX04B is recorded as inoculant 1, and the arbuscular mycorrhizal fungal inoculant prepared using the initial inoculant of Rhizophagus intraradices BJ09 is recorded as inoculant 2.

[0062] Example 2

[0063] 1. Rice seedling cultivation

[0064] The second complete nutrient solution consists of: 2.5 mM NH 4 + 、2.5mMNO 3- , 0.3 mM PO 4 3- , 2mM K + , 1mM SO 4 2- , 1mMCa 2+ , 2 mM Cl - 、1mM Mg 2+ , 1mM SO 4 2- , 0.5mMNa + , 1mM SiO 3 2- , 9μM Mn 2+ , 0.39μM MoO 4 2- , 20μM BO 33- , 0.77μM Zn 2+ , 0.32μM Cu 2+ and 20 μM Fe 2+ , the phosphorus concentration of the second complete nutrient solution is 0.3mM.

[0065] 1 / 3 rice nutrient solution, its components are the same as the second complete nutrient solution, and the concentration of each substance is 1 / 3 of that of the second complete nutrient solution.

[0066] The components of the second low-phosphorus complete nutrient solution are the same as those of the second complete nutrient solution. Except for phosphorus, the contents of other components are the same as those of the first complete nutrient solution. The phosphorus concentration in the low-phosphorus complete nutrient solution is 10 μmol / L.

[0067] Using 30% (v / v) H 2 O 2 The solution was used to disinfect rice seeds for 10 minutes, and then the seeds were repeatedly rinsed with deionized water and then soaked in water for 72 hours until the seeds turned white. The water was changed three times during the soaking period.

[0068] After soaking, select rice seeds with consistent germination potential, place them on a rice culture floating plate with fine holes, add 1 / 3 rice nutrient solution to further cultivate the seedlings, and replace 1 / 3 rice nutrient solution every 7 days. The culture conditions are: light 12h / d, dark 12h / d, daytime temperature 28℃, night temperature 20℃. When the plant grows two fully expanded leaves, the rice seedlings are obtained.

[0069] 2. Effects of inoculation with different arbuscular mycorrhizal fungi on Cd absorption by rice seedlings

[0070] (1) Experimental design

[0071] Three treatments were set up: control (CK): no AM fungus inoculation;

[0072] Treatment 1 (AM1): inoculation with bacterial agent 1 (JX04B);

[0073] Treatment 2 (AM2): Inoculation with bacterial agent 2 (BJ09), the grouping is shown in Table 1.

[0074] Table 1 Grouping

[0075]

[0076]

[0077] (2) Potted experiment with inoculation of different arbuscular mycorrhizal fungi

[0078] The purchased river sand was sterilized at 120℃ for 1h. After cooling naturally, the river sand was divided into pots, with 1.5kg of river sand in each pot. An appropriate amount of 1 / 3 rice nutrient solution containing Cd was added to the river sand so that the final Cd content in the test matrix river sand was 2mg / kg.

[0079] Randomly poke 5 holes on each pot substrate, each hole is 7-8cm deep, 15g of microbial agent 1 and 15g of microbial agent 2 are evenly added to the 5 holes, and then the holes are covered with soil. Select the healthy rice seedlings with uniform growth and size cultivated in step 1 and transplant them into pots. Plant one seed in each pot and divide them into three groups: control (CK), treatment 1 and treatment 2. Each group has 3 pots. Each group is treated according to Table 1 and irrigated with 300mL of the second low-phosphorus complete nutrient solution every week. Culture conditions: light 12h / d, dark 12h / d, daytime temperature: 28℃, night temperature: 20℃.

[0080] (3) Determination of Cd content in rice samples

[0081] After the rice was cultivated for 60 days, the root and aboveground samples of rice were collected from each treatment, rinsed and wiped clean with deionized water, transferred to marked kraft paper envelopes, placed in an oven, and after inactivation at 105°C for 1 hour, the temperature was adjusted to 70°C and maintained for 2 to 3 days before use.

[0082] Disinfection: First, use a grinder to fully grind the root and aerial parts of the rice after killing green, weigh 0.25g of the ground sample and transfer it to a disinfection tube, add 5mL of premium pure concentrated HNO 3 , overnight, digested by microwave digestion, transferred and fixed to volume flask. Cd content was determined by inductively coupled plasma mass spectrometry (ICP-MS).

[0083] (4) Test results and analysis

[0084] The Cd concentrations in the roots and aboveground parts of rice obtained from the control (CK), treatment 1 and treatment 2 were measured, and the results are shown in Table 2. Compared with the control, the Cd concentrations in the roots and aboveground parts of rice in treatment 1 were reduced by 15.6% and 34.4%, respectively; the Cd concentrations in the roots and aboveground parts of rice in treatment 2 were reduced by 25.4% and 38.0%, respectively. Therefore, both bacterial agents can significantly inhibit the absorption of Cd in the roots of rice and its transport to the aboveground parts, and the inoculation of bacterial agent 2 (BJ09) has a more significant effect on the control of Cd in rice.

[0085] Table 2 Cd concentrations in rice roots and shoots obtained from control (CK), treatment 1 and treatment 2

[0086] deal with Root Cd concentration (mg / kg) Aboveground Cd concentration (mg / kg) Control (CK) 2.07±0.2 0.78±0.1 Treatment 1 (AM1) 1.75±0.08 0.51±0.04 Process 2 (AM2) 1.54±0.17 0.48±0.04

[0087] 3. Effects of exogenous calcium-binding bacterial agent 2 on calcium, Cd and zinc accumulation in rice

[0088] (1) Preparation of exogenous nano-calcium carbonate solution

[0089] Compared with traditional calcium fertilizers, nano calcium carbonate has a very small particle size and can quickly enter plant cells, making it easily absorbed by plants. In addition, foliar spraying can allow nano calcium carbonate to quickly enter the plant body through the leaves, improving the plant's utilization of calcium.

[0090] Preparation method: First weigh 0.5g of nano-calcium carbonate, then add 1mL of sodium polyacrylate, 499mL of deionized water and 0.5g of xanthan gum in sequence, stir well, then place in an ultrasonic cleaning instrument, ultrasonicate for 20min to mix well, and obtain an exogenous nano-calcium carbonate solution for use.

[0091] (2) Experimental design

[0092] The results of step 2 showed that the inoculated bacterial agent 2 (i.e., the bacterial agent prepared by Rhizospora endocystis BJ09) had a more significant effect on the control of Cd in rice. Therefore, this experiment selected bacterial agent 2 for inoculation treatment. The specific experimental design and grouping treatment are shown in Table 3, where the control (CK): sprayed with a solution without exogenous calcium and not inoculated with bacterial agent 2;

[0093] Treatment 1 (T1): addition of exogenous calcium and no inoculation of inoculant 2;

[0094] Treatment 2 (T2): no addition of exogenous calcium and inoculum 2;

[0095] Treatment 3 (T3): Addition of exogenous calcium and inoculum 2.

[0096] Table 3 Experimental design

[0097] deal with Exogenous calcium Bacterial Agent 2 Control (CK) - - Treatment 1 (T1) + - Treatment 2 (T2) - + Treatment 3 (T3) + +

[0098] (3) Potted experiment of exogenous calcium-binding mycorrhizal fungi

[0099] The collected soil was sterilized at 120℃ for 1h. After cooling naturally, urea, KH 2 PO 4 and K 2 SO 4 As base fertilizer, N:P per 1kg of soil 2 O 5 :K 2 O mass ratio was 0.2:0.15:0.2, and then the soil was divided into pots, each pot contained 10 kg of soil, and the Cd content of the test soil was 13 mg / kg.

[0100] According to the grouping in Table 3, 5 holes were randomly poked in the test soil for treatments 2 and 3, with a depth of 7 to 8 cm, and an appropriate amount of microbial agent 2 was evenly added so that the final spore application amount for each seedling was 200, and each gram of microbial agent 2 contained 12 spores, and the holes were covered with soil; no microbial agent 2 was added to the control group and treatment 1. After that, the rice seedlings with uniform growth and size cultivated in step 1 were moved into pots, and 3 rice seedlings were moved into each pot. In treatments 1 and 3, nano calcium carbonate solution was sprayed on the leaves at the heading and filling stages of rice, respectively, with a spraying amount of 50 mL / pot. Culture conditions: light 12h / d, dark 12h / d, daytime temperature: 28℃, night temperature: 20℃.

[0101] (4) Determination of mycorrhizal fungal infection rate in rice roots

[0102] After rice is harvested, root segments of rice under different treatment conditions are collected, cleaned, cut into 1-2 cm long root segments, and completely immersed in 10% (w / v) KOH solution, treated in a 90°C water bath for 55 min, the solution is discarded, and a 5% (v / v) HCl solution is added, reacted for 10 min, rinsed with sterile water, placed in a 0.05% (w / v) trypan blue dye solution, and dyed in a boiling water bath for 45 min, the dye solution is discarded, rinsed with sterile water three times, and then an appropriate amount of sterile water is added. The roots are placed at 25°C for one week, samples are taken for observation under an ordinary microscope, and the total infection rate of the rice root system is statistically analyzed using the cross-hatch method.

[0103] (5) Determination of Cd, calcium and zinc contents in rice samples

[0104] After the rice is harvested, the roots, above-ground parts and rice grains are placed in marked kraft paper envelopes, transferred to an oven, and dried at 105°C. After 1 hour, the temperature is adjusted to 70°C and maintained for 2 to 3 days before use.

[0105] Dehulling: Hull the rice grains and grind the roots and aerial parts of the rice thoroughly with a grinder. Weigh 0.5 g of the dehulled grain sample and transfer it to a dehulling tube. Add 5 mL of premium pure concentrated HNO 3 , overnight, digested by microwave digestion, transferred and fixed to volume flask. Calcium, zinc and Cd contents were determined by ICP-MS.

[0106] Weigh 0.25 g of the root grind sample and transfer it to a sterilization tube. Add 5 mL of premium pure concentrated HNO 3 , overnight, digested by microwave digestion, transferred and fixed to volume flask. Calcium, zinc and Cd contents were determined by ICP-MS.

[0107] Weigh 0.25 g of the aboveground ground sample and transfer it to a sterilization tube. Add 5 mL of premium pure concentrated HNO 3 , overnight, digested by microwave digestion, transferred and fixed to volume flask. Calcium, zinc and Cd contents were determined by ICP-MS.

[0108] (6) Test results and analysis

[0109] 1) Effects of exogenous calcium addition on the symbiotic relationship between arbuscular mycorrhizal fungi and rice under Cd stress

[0110] The histochemical staining analysis of rice roots without and with calcium supplementation was performed, such as Figure 1 As shown in Figure 2, under the condition of AMF inoculation, regardless of whether exogenous calcium was added, arbuscules, hyphae and vesicles could be observed in rice roots ( Figure 1 Middle A and Figure 1 Middle B). Statistical data analysis found that ( Figure 1 C and Table 4), compared with no addition of exogenous calcium, the total infection rate of rice roots with exogenous calcium did not change significantly. These results show that exogenous calcium addition does not affect the infection of arbuscular mycorrhizal fungi to rice under Cd stress, and the arbuscular mycorrhizal fungi used in the present invention have a certain Cd tolerance.

[0111] Table 4 Effects of exogenous calcium addition on the symbiotic relationship between arbuscular mycorrhizal fungi and rice under Cd stress

[0112]

[0113] 2) Effects of exogenous calcium-binding AM fungi on Cd absorption by rice roots

[0114] Under Cd pollution, compared with the control, the Cd content in the rice roots of the treatments of adding exogenous calcium alone and inoculating with bacterial agent 2 alone decreased by 36% and 7%, respectively. The Cd content in the rice roots of the treatments of adding exogenous calcium and inoculating with bacterial agent 2 was significantly reduced by 39% (see Figure 2 These results indicate that exogenous calcium combined with inoculant 2 has a significant synergistic effect on controlling the uptake of Cd by rice.

[0115] Table 5 Results of determination of Cd content in rice roots under conditions of exogenous calcium combined with inoculation of bacterial agent 2 (unit: mg / kg)

[0116] average value Standard error of the mean Process 2 14.3217 1.05681 Process 3 13.5403 0.42379 Process 1 20.8128 1.96422 Comparison 22.294 2.19953

[0117] 3) Effects of exogenous addition of calcium-binding bacterial agent 2 on Cd accumulation in rice shoots and grains

[0118] The effect of exogenous calcium-binding inoculant 2 (i.e., T3) on Cd accumulation in the aboveground part and grain of rice was further analyzed, and the Cd content in the aboveground part and grain of rice under the treatment of exogenous calcium-binding inoculant 2 was detected and analyzed. The Cd content in the aboveground part and grain of rice in CK was regarded as 100%, and the percentage (%) was calculated by the formula: T3 treatment cadmium concentration / control CK cadmium concentration*100. Compared with the control, exogenous calcium-binding inoculant 2 could significantly reduce the concentration of Cd in the aboveground part and grain of rice, which was reduced by about 17% and 14%, respectively. Figure 3 And as shown in Table 6.

[0119] Table 6 The percentage results of the effect of exogenous addition of calcium-binding bacterial agent 2 on Cd accumulation in rice shoots and grains

[0120] CK(%) T3(％) Grains 100％ 87.0119±7.57857 above ground 100％ 82.8497±5.03011

[0121] 4) Effects of exogenous addition of calcium-binding bacterial agent 2 on the calcium and zinc contents in rice grains

[0122] As shown in Table 7, under Cd pollution conditions, compared with the control, the Ca and Zn contents of rice grains inoculated with AM fungi alone did not change significantly, and the Zn content of rice grains with exogenous calcium addition alone increased by 17%, while the Ca content did not change significantly. The Ca and Zn contents of rice grains inoculated with AMF increased significantly by 30% and 10% when exogenous calcium was added. Therefore, under Cd stress conditions, exogenous calcium addition can enhance the accumulation of calcium and zinc nutrients in rice grains by AM fungi.

[0123] Table 7 Effects of exogenous calcium addition combined with AM fungi on calcium and zinc contents in rice grains

[0124] deal with Ca(mg / kg) Zn(mg / kg) CK 1367.99±30.98 75.56±0.46 T1 1334.27±142.81 88.53±6.94 T2 1310.35±34.89 74.01±4.97 T3 1779.59±279.25 83.6±1.82

[0125] In summary, exogenous calcium combined with inoculation of AM fungi can not only effectively block the Cd absorption by rice roots and the accumulation of Cd in rice grains, but also significantly increase the content of Ca and Zn in rice grains, thereby improving the quality of rice.

[0126] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An arbuscular mycorrhizal fungal agent, characterized in that: The invention comprises leek roots infected by arbuscular mycorrhizal fungi and a substrate. The arbuscular mycorrhizal fungi are Rhizophagus irregularis and / or Rhizophagus intraradices.

2. The arbuscular mycorrhizal fungal agent according to claim 1, characterized in that The substrate includes river sand; the arbuscular mycorrhizal fungi in the microbial agent exist in the form of spores and / or hyphae.

3. The method for preparing the arbuscular mycorrhizal fungal agent according to claim 1 or 2, characterized in that: include: The leek is cultivated in a substrate containing arbuscular mycorrhizal fungi, and the root system and substrate of the cultivated leek are collected to obtain an arbuscular mycorrhizal fungi agent. The added amount of the arbuscular mycorrhizal fungi is 2% of the mass of the substrate.

4. The preparation method according to claim 3, characterized in that: The cultivation time is 5 to 6 months, the light cycle of the cultivation is 12 hours / day of light and 12 hours / day of darkness, the daytime temperature of the cultivation is 25 to 35°C, and the nighttime temperature is 17 to 22°C; water and low-phosphorus nutrient solution are applied during the cultivation process.

5. Use of the arbuscular mycorrhizal fungal agent according to claim 1 or 2 or the arbuscular mycorrhizal fungal agent prepared by the preparation method according to claim 3 or 4 in the following 1) and / or 2), 1) Reduce the absorption of cadmium by rice; 2) Increase the infection rate of arbuscular mycorrhizal fungi on rice.

6. A compound formulation, characterized in that: The invention comprises packaged arbuscular mycorrhizal fungi agent and calcium fertilizer; the arbuscular mycorrhizal fungi agent is the arbuscular mycorrhizal fungi agent according to claim 1 or 2 or the arbuscular mycorrhizal fungi agent prepared by the preparation method according to claim 3 or 4.

7. The compound according to claim 6, characterized in that: The calcium fertilizer comprises nano calcium carbonate.

8. Use of the compound according to claim 6 or 7 in any one of the following 1) to 3): 1) Increase the infection rate of arbuscular mycorrhizal fungi on rice; 2) Reduce the absorption of cadmium by rice; 3) Increase the content of macroelements and trace elements in rice.

9. A method for reducing cadmium absorption by rice, characterized in that: include: The arbuscular mycorrhizal fungal agent is used for rice cultivation, wherein the arbuscular mycorrhizal fungal agent is the arbuscular mycorrhizal fungal agent according to claim 1 or 2 or the arbuscular mycorrhizal fungal agent prepared by the preparation method according to claim 3 or 4.

10. A method for achieving low cadmium and high quality rice grains, characterized in that: include: The compound formulation according to claim 6 or 7 is applied in rice cultivation.

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