Application of arbuscular mycorrhizal fungi in improving manganese stress resistance of tea seedlings

Abstract

The invention discloses application of arbuscular mycorrhizal fungi in improvement of manganese stress resistance of tea seedlings, relates to the field of ecological planting, and aims to solve the problem of poor manganese stress resistance effect of an existing tea seedling cultivation method. The method for improving the manganese stress resistance of the tea seedlings by using the arbuscular mycorrhizal fungi comprises the following steps: 1, selecting tea tree seeds, disinfecting, airing and accelerating germination; 2, soil and vermiculite are mixed and stirred evenly, sterilization treatment is conducted, and a culture medium is obtained; and 3, burying the tea tree seeds subjected to germination acceleration into a culture medium, inoculating the arbuscular mycorrhizal fungus inoculant below the tea tree seeds, and culturing. A symbiotic relationship is formed between the pyeosporium mosseae and root systems of the tea seedlings, so that biomass accumulation of underground parts of the tea seedlings under manganese stress is remarkably promoted, the content of osmotic regulation substances is increased, the membrane lipid peroxidation degree is reduced, an antioxidant enzyme system is regulated and controlled, the active oxygen scavenging capacity is enhanced, and the stress resistance of the tea seedlings is effectively improved. The method is used for improving the manganese stress resistance of the tea seedlings.

Description

Technical Field

[0001] This invention relates to the field of ecological planting, and in particular to the application of arbuscular mycorrhizal fungi in improving the manganese stress tolerance of tea seedlings. Background Technology

[0002] With the acceleration of industrialization and the irrational use of chemical fertilizers and pesticides in agricultural production, soil heavy metal pollution has become increasingly serious, among which manganese pollution has attracted much attention due to its wide distribution and severe harm. Manganese (Mn) is an essential trace element for the growth and development of plants and animals, and is the second most common trace element in the Earth's crust after iron (Fe). It is widely used in agriculture, medical and health industries, and other fields, bringing huge benefits to social and economic development. However, in recent decades, affected by large-scale mining of mineral resources and increased human activities, manganese pollution has become increasingly prominent, damaging the ecological environment and seriously threatening the sustainable development of human society. Its pollution is mainly manifested in excessively high manganese content in the soil, exceeding the soil's self-purification capacity. High manganese in the soil will damage the soil ecosystem, affect the structure and activity of microbial communities, and reduce soil fertility; it will also be absorbed and accumulated by plants, interfering with plant physiological and biochemical processes, leading to slow plant growth, reduced biomass, altered metabolism, and even death. More seriously, excessively high levels of manganese in the soil can enter the food chain through crop accumulation or contact with soil and dust, threatening human health.

[0003] As an important economic crop, tea trees are inevitably affected by manganese pollution in their growing environment. Tea seedlings are highly susceptible to manganese stress if grown in manganese-contaminated soil. High concentrations of manganese are absorbed and accumulated by the tea seedling roots, interfering with normal physiological and biochemical processes. This includes damaging root cell membranes, affecting water and nutrient absorption; inhibiting the activity of photosynthetic enzymes in leaves, reducing photosynthetic efficiency; and triggering oxidative stress, leading to cell damage. Ultimately, this is detrimental to the growth and development of tea seedlings and hinders the healthy development of the tea industry.

[0004] Arbuscular mycorrhizal fungi (AMF) are an important group of microorganisms widely present in soil. They can form symbiotic relationships with over 80% of terrestrial plants on Earth, constructing unique mycorrhizal structures. This symbiotic relationship greatly promotes the absorption of soil moisture and mineral nutrients by plants, and is crucial for the growth of most plants. In soil environments facing heavy metal pollution, the symbiotic relationship between AMF and plants exhibits remarkable regulatory functions: they can regulate the absorption, translocation, and accumulation of heavy metals in plants, significantly increasing the tolerance threshold of plants to heavy metal toxicity, thereby ensuring healthy plant growth and development. Furthermore, AMF also has the ability to fix heavy metal ions in the soil or plant roots, effectively preventing the migration of heavy metals into deeper soil layers and their infiltration into groundwater, building a barrier for environmental protection. In addition, as a highly efficient biofertilizer, AMF can not only significantly improve plant yield and nutrient utilization efficiency, but also play a positive role in optimizing plant quality, contributing valuable strength to the sustainable development of agriculture.

[0005] However, current methods for managing manganese stress in tea seedlings still have significant shortcomings: physical and chemical methods (such as soil replacement and application of chelating agents) are costly, easily disrupt the soil ecological balance, and may even cause secondary pollution; among bioremediation methods, research focusing on the synergistic resistance of "tea seedling-AMF" to manganese stress is scattered, lacking systematic and scalable application schemes, and the compatibility differences of different AMF strains with tea seedlings are not clear, and the response patterns of tea seedlings in symbiosis with AMF at different growth stages are unclear, making it difficult to accurately control the selection of strains and the timing of inoculation in practical applications. Summary of the Invention

[0006] The present invention aims to address the problem that existing tea seedling cultivation methods are not effective in resisting manganese stress, and provides the application of arbuscular mycorrhizal fungi in improving the manganese stress tolerance of tea seedlings.

[0007] This invention provides the application of arbuscular mycorrhizal fungi in improving the manganese stress tolerance of tea seedlings.

[0008] Furthermore, the arbuscular mycorrhizal fungus is *Funneliformis mosseae*.

[0009] Furthermore, the specific method for improving the manganese stress tolerance of tea seedlings using arbuscular mycorrhizal fungi includes the following steps:

[0010] 1. Select tea tree seeds, disinfect, dry, and germinate;

[0011] 2. Mix the soil with vermiculite, stir well, and sterilize to obtain the culture medium;

[0012] 3. Bury the tea seeds that have been germinated in step one into the culture medium, and inoculate the bottom of the tea seeds with arbuscular mycorrhizal fungi (AMF inoculant) for cultivation.

[0013] Furthermore, in step two, the volume ratio of soil to vermiculite is 1:(1-2).

[0014] Furthermore, the specific sterilization method in step two is: high-pressure steam sterilization at 121℃ for 2-4 hours.

[0015] Furthermore, the arbuscular mycorrhizal fungus mentioned in step three is *Funneliformismosseae*, and the number of active spores in the fungal agent is 176 per 10g.

[0016] Furthermore, in step three, the arbuscular mycorrhizal fungal inoculant is inoculated 1-2 cm below the tea seed, and the inoculation amount is 10 g per 550 g of culture medium.

[0017] Furthermore, the aforementioned tolerance to manganese stress refers to improving the overall growth of tea seedlings under manganese stress conditions.

[0018] Furthermore, the aforementioned tolerance to manganese stress refers to enhancing the overall osmotic regulation and antioxidant capacity of tea seedlings under manganese stress conditions.

[0019] This invention measures the biomass (fresh weight of aboveground and underground parts), osmotic regulators (content of proline, malondialdehyde, soluble sugar, and soluble protein in leaves and roots), antioxidant enzyme activities (activity of superoxide dismutase, peroxidase, catalase, and polyphenol oxidase in leaves and roots), and manganese content of tea seedlings under manganese stress after inoculation with AMF. The results show that AMF inoculation can significantly promote the accumulation of underground biomass in tea seedlings under manganese stress, increase the content of osmotic substances, reduce the degree of membrane lipid peroxidation, regulate the antioxidant enzyme system, enhance the ability to scavenge reactive oxygen species, and reduce the manganese content in roots. This indicates that AMF inoculation of tea seedlings under manganese stress can effectively improve stress resistance through enhanced growth, osmotic regulation, and antioxidant defense.

[0020] The beneficial effects of this invention are:

[0021] This invention addresses the problem of inhibited tea seedling growth caused by manganese pollution. It utilizes *Funneliformismosseae* to form a symbiotic relationship with the tea seedling roots, significantly promoting the accumulation of underground biomass under manganese stress, increasing the content of osmotic regulators, reducing membrane lipid peroxidation, regulating the antioxidant enzyme system, and enhancing reactive oxygen species scavenging capacity, thus effectively improving the stress resistance of tea seedlings. This method is low-cost, highly efficient, and easy to operate, without damaging the physical and chemical properties of the soil. It promotes plant growth and enhances stress resistance in tea seedlings under manganese stress conditions. It can replace physical / chemical remediation methods, achieving green and sustainable cultivation of stress-resistant tea seedlings, providing strong support for the cultivation of manganese-contaminated tea gardens and the sustainable development of the tea industry. It fills the gap in bioremediation technology for manganese stress in tea seedlings and provides a new path for the green development of the tea industry.

[0022] This invention is applicable to the cultivation of tea seedlings in manganese-contaminated tea gardens and can replace physical / chemical remediation methods to achieve green and sustainable stress-resistant cultivation of tea seedlings. Attached Figure Description

[0023] Figure 1 The figure shows the results of the biomass measurement of tea seedlings under manganese stress by arbuscular mycorrhizal fungi.

[0024] Figure 2 The figure shows the results of the determination of osmotic regulators in tea seedlings under manganese stress by arbuscular mycorrhizal fungi.

[0025] Figure 3 The figure shows the results of the determination of antioxidant enzyme activity in tea seedlings under manganese stress by arbuscular mycorrhizal fungi.

[0026] Figure 4 The figure shows the results of manganese content determination in tea seedlings under manganese stress by arbuscular mycorrhizal fungi. Detailed Implementation

[0027] The embodiments of the present invention will be described in detail below. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0028] Example 1:

[0029] This embodiment was conducted at Anhui Normal University in Wuhu City, Anhui Province, and the soil used was taken from the campus of Anhui Normal University.

[0030] This embodiment is the AMF+Mn group, in which the AMF agent of the present invention is inoculated at the roots of the tea seedlings during the cultivation process, and a manganese stress environment is created.

[0031] I. Experimental Material Processing:

[0032] Select healthy tea tree seeds that are plump, free from pests and diseases, undamaged, and uniform in size. Soak them in clean water for 24 hours to remove any floating, inferior seeds. Then, completely immerse the tea tree seeds in a 75% ethanol solution for disinfection. After 30 seconds, rinse several times with sterile distilled water. Next, soak them in a 2% sodium hypochlorite solution for 10 minutes, followed by rinsing the seed surface with sterile distilled water. Once clean, wipe off excess moisture with absorbent paper, air dry, and then store in sand for one month to promote germination.

[0033] II. Culture substrate:

[0034] Soil (taken from the campus of Anhui Normal University) and vermiculite were mixed at a volume ratio of 1:1, stirred thoroughly, and then autoclaved at 121℃ for 90 minutes to eliminate any remaining fungal spores in the soil, thus obtaining the culture medium. The culture medium was then dispensed into plastic flower pots, with 550g per pot.

[0035] III. Application of AMF bacterial agent:

[0036] Funneliformis mosseae (Fm) was selected as the AMF inoculum, with 176 active spores per 10g. It was purchased from the "Arbuscular Mycorrhizal Fungi Germplasm Resource Bank" of the Institute of Root Biology, Yangtze University, and can be used directly as an inoculum.

[0037] Select the seeds that have sprouted white leaves from the sand storage in step one and bury them in the culture medium for cultivation. Inoculate 10g of inoculant 1-2cm below the tea tree seeds.

[0038] IV. Manganese stress:

[0039] Thirty days after sowing, all tea seedlings emerged and sprouted. Once their growth stabilized, the seedlings were subjected to manganese stress treatment. The specific method was to dissolve MnSO4 in 200 mL of 1 / 4 Hoagland nutrient solution and then irrigate the seedlings. Dosage: Based on pure Mn, the concentration of pure manganese in the culture medium should reach 100 mg / kg.

[0040] Comparative Example 1:

[0041] This comparative example is the CK group (control group), in which the AMF inoculant of the present invention was not applied during the cultivation of tea seedlings, nor was a manganese environment created.

[0042] I. Experimental Material Processing:

[0043] Select healthy tea tree seeds that are plump, free from pests and diseases, undamaged, and uniform in size. Soak them in clean water for 24 hours to remove any floating, inferior seeds. Then, completely immerse the tea tree seeds in a 75% ethanol solution for disinfection. After 30 seconds, rinse several times with sterile distilled water. Next, soak them in a 2% sodium hypochlorite solution for 10 minutes, followed by rinsing the seed surface with sterile distilled water. Once clean, wipe off excess moisture with absorbent paper, air dry, and then store in sand for one month to promote germination.

[0044] II. Culture substrate:

[0045] Soil (taken from the campus of Anhui Normal University) and vermiculite were mixed at a volume ratio of 1:1, stirred thoroughly, and then autoclaved at 121℃ for 90 minutes to eliminate any remaining fungal spores in the soil, thus obtaining the culture medium. The culture medium was then dispensed into plastic flower pots, with 550g per pot.

[0046] Third, select the seeds that have sprouted white seeds from step one and bury them in the culture medium for cultivation.

[0047] Comparative Example 2:

[0048] This comparative example is the Mn group, in which the AMF inoculant of the present invention is not applied during the cultivation of tea seedlings, but a manganese environment is created.

[0049] I. Experimental Material Processing:

[0050] Select healthy tea tree seeds that are plump, free from pests and diseases, undamaged, and uniform in size. Soak them in clean water for 24 hours to remove any floating, inferior seeds. Then, completely immerse the tea tree seeds in a 75% ethanol solution for disinfection. After 30 seconds, rinse several times with sterile distilled water. Next, soak them in a 2% sodium hypochlorite solution for 10 minutes, followed by rinsing the seed surface with sterile distilled water. Once clean, wipe off excess moisture with absorbent paper, air dry, and then store in sand for one month to promote germination.

[0051] II. Culture substrate:

[0052] Soil (taken from the campus of Anhui Normal University) and vermiculite were mixed at a volume ratio of 1:1, stirred thoroughly, and then autoclaved at 121℃ for 90 minutes to eliminate any remaining fungal spores in the soil, thus obtaining the culture medium. The culture medium was then dispensed into plastic flower pots, with 550g per pot.

[0053] Third, select the seeds that have sprouted white seeds from step one and bury them in the culture medium for cultivation.

[0054] IV. Manganese stress:

[0055] Thirty days after sowing, all tea seedlings emerged and sprouted. Once their growth stabilized, the seedlings were subjected to manganese stress treatment. The specific method was to dissolve MnSO4 in 200 mL of 1 / 4 Hoagland nutrient solution and then irrigate the seedlings. Dosage: Based on pure Mn, the concentration of pure manganese in the culture medium should reach 100 mg / kg.

[0056] Comparative Example 3:

[0057] This comparative example is the AMF group, in which the AMF inoculant of the present invention was applied to the tea seedlings during cultivation, but without creating a manganese environment.

[0058] I. Experimental Material Processing:

[0059] Select healthy tea tree seeds that are plump, free from pests and diseases, undamaged, and uniform in size. Soak them in clean water for 24 hours to remove any floating, inferior seeds. Then, completely immerse the tea tree seeds in a 75% ethanol solution for disinfection. After 30 seconds, rinse several times with sterile distilled water. Next, soak them in a 2% sodium hypochlorite solution for 10 minutes, followed by rinsing the seed surface with sterile distilled water. Once clean, wipe off excess moisture with absorbent paper, air dry, and then store in sand for one month to promote germination.

[0060] II. Culture substrate:

[0061] Soil (taken from the campus of Anhui Normal University) and vermiculite were mixed at a volume ratio of 1:1, stirred thoroughly, and then autoclaved at 121℃ for 90 minutes to eliminate any remaining fungal spores in the soil, thus obtaining the culture medium. The culture medium was then dispensed into plastic flower pots, with 550g per pot.

[0062] III. Application of AMF bacterial agent:

[0063] Funneliformis mosseae (Fm) was selected as the AMF inoculum, with 176 active spores per 10g. It was purchased from the "Arbuscular Mycorrhizal Fungi Germplasm Resource Bank" of the Institute of Root Biology, Yangtze University, and can be used directly as an inoculum.

[0064] Select the seeds that have sprouted white leaves from the sand storage in step one and bury them in the culture medium for cultivation. Inoculate 10g of inoculant 1-2cm below the tea tree seeds.

[0065] Each treatment group was set up in 4 replicates, and all experimental groups were carried out under the same culture conditions and cultured for 60 days.

[0066] The tea seedlings in each group corresponding to the above examples were analyzed and measured using the following specific methods:

[0067] (1) Biomass: Two months after the tea seedlings were subjected to manganese stress, four complete tea seedlings were randomly selected and rinsed. The roots were washed with slow running water to remove surface mud and sand. After drying the surface of the plants, the fresh weight of the above-ground and underground parts was measured.

[0068] (2) Determination of osmotic conditioning substances: Proline (Pro) content was determined by acidic ninhydrin colorimetric method; malondialdehyde (MDA) content was determined by thiobarbituric acid method; soluble sugar content was determined by anthrone colorimetric method. Soluble protein content was determined by Coomassie brilliant blue staining method.

[0069] (3) Antioxidant enzyme activity assay: superoxide dismutase (SOD) was determined by nitroblue tetrazolium (NBT) method, peroxidase (POD) was determined by guaiacol method, catalase (CAT) was determined by ultraviolet spectrophotometry, and polyphenol oxidase (PPO) was determined by catechol method.

[0070] (4) Manganese content determination: The plant samples were digested by nitric acid and perchloric acid, and then the manganese content was determined by flame atomic absorption spectrophotometry.

[0071] Measurement results:

[0072] 1. Biomass determination:

[0073] The effects of arbuscular mycorrhizal fungi on the biomass of tea seedlings under manganese stress, such as Figure 1 As shown, -Mn represents no manganese stress, and +Mn represents manganese stress. From Figure 1 The experimental results show that after inoculation with AMF inoculant, the biomass of the underground parts of tea plants increased, while the biomass of the aboveground parts decreased. The underground biomass increased by 10% and 21.6% under no stress and manganese stress conditions, respectively. This indicates that AMF inoculant can significantly promote the growth of tea plant roots. This result fully demonstrates that the method of this invention can effectively promote the growth of the underground parts of tea seedlings, strengthen the root system's stress resistance structure, and help tea seedlings grow in manganese-contaminated soil.

[0074] 2. Osmotic conditioning measurement:

[0075] The effects of arbuscular mycorrhizal fungi on osmotic regulators in tea seedlings under manganese stress, such as Figure 2 As shown, -Mn represents no manganese stress, +Mn represents manganese stress, A, B, C, and D represent leaves, and A', B', C', and D' represent roots. (From...) Figure 2It can be seen that in leaves, AMF inoculation under manganese stress increased proline content by 65.28%, soluble protein content by 31.69%, decreased malondialdehyde content by 22.14%, and soluble sugar content by 2.82%, indicating that AMF can maintain leaf cell homeostasis by enhancing the accumulation of osmotic substances in tea seedlings and reducing membrane lipid peroxidation. In roots, AMF inoculation under stress increased proline content by 21.82%, decreased soluble sugar content by 4.3%, increased soluble protein content by 17.45%, and decreased malondialdehyde content by 22.58%, proving that AMF can protect root cell membrane structure and metabolic function, and work synergistically with leaves to form an osmotic regulation closed loop, effectively alleviating manganese toxicity and providing quantitative support for the stress resistance effect.

[0076] 3. Antioxidant enzyme assay:

[0077] Effects of arbuscular mycorrhizal fungi on the antioxidant enzyme activity of tea seedlings under manganese stress, such as Figure 3 As shown, -Mn represents no manganese stress, +Mn represents manganese stress, A, B, C, and D represent leaves, and A', B', C', and D' represent roots. (From...) Figure 3 As can be seen, in leaves, AMF inoculation under manganese stress significantly increased CAT activity by 222.9%, POD by 289.36%, SOD by 0.90%, and PPO by 66.92%, indicating that AMF can efficiently scavenge reactive oxygen species and avoid oxidative stress damage in tea seedlings by precisely regulating enzyme activity ratios. In roots, AMF inoculation under stress increased CAT activity by 48.06%, decreased POD by 12.17%, increased SOD by 211.28%, and significantly decreased PPO activity by 39.76%, demonstrating that this method optimizes the root antioxidant system by inoculating with AMF, forming a reactive oxygen species scavenging network in synergy with leaves, further validating the stress resistance mechanism of this method.

[0078] 4. Manganese content determination:

[0079] The effects of arbuscular mycorrhizal fungi on manganese content in tea seedlings under manganese stress, such as Figure 4 As shown, -Mn represents no manganese stress, and +Mn represents manganese stress. Figure 4 The results showed that AMF inoculation significantly increased the manganese content in tea seedling leaves by 44.2% and 27.38%, respectively; it reduced manganese accumulation in the underground parts, avoiding direct heavy metal toxicity to the roots by 23.07% and 27.56%, respectively; and the manganese content in the roots was lower than that in the leaves. These results demonstrate that AMF can regulate manganese tissue distribution, ensuring nutritional needs while reducing the risk of toxicity, providing a feasible basis for the application of this method in manganese-contaminated soils.

Claims

1. Application of arbuscular mycorrhizal fungi in improving the manganese stress tolerance of tea seedlings.

2. The application according to claim 1, characterized in that, The arbuscular mycorrhizal fungus is *Funneliformis mosseae*.

3. The application according to claim 1 or 2, characterized in that, The specific method for improving the manganese stress tolerance of tea seedlings using arbuscular mycorrhizal fungi includes the following steps:

1. Select tea tree seeds, disinfect, dry, and germinate; 2. Mix the soil with vermiculite, stir well, and sterilize to obtain the culture medium; 3. Bury the tea seeds that have been germinated in step one into the culture medium, inoculate the bottom of the tea seeds with arbuscular mycorrhizal fungi agent, and then cultivate them.

4. The application according to claim 3, characterized in that, In step two, the volume ratio of soil to vermiculite is 1:(1-2).

5. The application according to claim 3, characterized in that, The specific sterilization method in step two is: high-pressure steam sterilization at 121℃ for 2-4 hours.

6. The application according to claim 3, characterized in that, The number of active spores in the fungal agent described in step three is 176 per 10g.

7. The application according to claim 3, characterized in that, In step three, the arbuscular mycorrhizal fungi inoculant should be inoculated 1-2 cm below the tea seed, and the inoculation amount should be 10 g per 550 g of culture medium.

8. The application according to claim 1 or 2, characterized in that, The term "manganese stress tolerance" refers to improving the overall growth of tea seedlings under manganese stress conditions.

9. The application according to claim 1 or 2, characterized in that, The term "manganese stress tolerance" refers to enhancing the overall osmotic regulation and antioxidant capacity of tea seedlings under manganese stress conditions.

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