A composite fungus and its application in improving the cold resistance of gardenia

Abstract

The invention discloses a composite bacteria and an application thereof in improving the cold resistance of gardenia, the composite bacteria for improving the cold resistance of gardenia, the composite bacteria comprising heteromorphic rhizosporon and Glomus mosseae, the ratio of heteromorphic rhizosporon RI and Glomus mosseae GM being 1:1, and the composite bacteria for improving the cold resistance of gardenia. Experimental studies have shown that inoculation of GM and RI not only enhances the survival ability of gardenia under low temperature conditions, but also improves the overall cold resistance of the plant by regulating the physiological and molecular mechanisms inside the plant. This strategy of improving the cold resistance of plants by microbial inoculation provides an effective biotechnology means for improving the stress resistance of gardenia in agricultural production.

Description

Technical Field

[0001] The invention belongs to the technical field of improving plant cold resistance, and relates to a composite bacterium for regulating the cold resistance of gardenia, and application of the composite bacterium in improving the cold resistance of gardenia. Background Art

[0002] Gardenia ( Gardenia jasminoides Ellis ), a plant of the genus Gardenia in the Rubiaceae family, its dried mature fruits are widely used in traditional Chinese medicine. Gardenia is mainly distributed in the warmer climates of eastern, southwestern and southeastern China. As it is sensitive to temperature, extreme low temperatures pose a serious threat to the growth and yield of gardenia, which may lead to fewer flower buds, lower yields, and even frostbite and death of seedlings.

[0003] In order to improve the cold resistance of plants, people have tried a variety of methods, including the use of chemical regulators, improved planting techniques, breeding cold-resistant varieties, etc. However, these methods often have certain limitations, such as high costs, unstable effects, or negative impacts on the environment.

[0004] In recent years, the role of microorganisms in promoting plant growth and improving stress resistance has gradually received attention. In particular, mycorrhizal fungi (such as arbuscular mycorrhizal fungi) can form a symbiotic relationship with plant roots, improve plant nutrient absorption and regulate plant endogenous hormone levels, thereby enhancing plant resistance to abiotic stresses such as drought, salinity and low temperature.

[0005] Although studies have shown that mycorrhizal fungi can improve the cold resistance of some plants, there are relatively few studies on the specific species of Gardenia, and most of them focus on the application of a single species. In addition, issues such as the synergistic effect between different species and the optimal inoculation ratio have not been fully studied and applied.

[0006] In the process of implementing the present invention, the inventors found that the prior art has at least one of the following technical problems:

[0007] 1) There is a lack of methods to improve the cold resistance of Gardenia jasminoides;

[0008] 2) The use of a single strain cannot fully tap the potential of mycorrhizal fungi;

[0009] 3) There is a lack of research on the effects of different combinations of fungal strains and their proportions on the cold resistance of Gardenia. Summary of the invention

[0010] In view of this, the purpose of the present invention is to fill the gap in existing research, develop and provide a specific mycorrhizal fungus composite bacteria, and the application and method of using the composite bacteria to improve the cold resistance of gardenia. Through in-depth research on the interaction between gardenia and specific mycorrhizal fungi, the present invention aims to provide gardenia growers with an effective technical means to cope with the low temperature stress caused by climate change and ensure the stable production and supply of gardenia.

[0011] In order to solve the above technical problems, the technical solution provided by the present invention is to provide a composite bacteria for improving the cold resistance of gardenia, wherein the composite bacteria comprises:

[0012] a. Rhizosporium heteromorphum Rhizophagus irregularis (RI), and

[0013] b. Glomus mosseae Glomus mosseae (GM),

[0014] Among them, the ratio of Rhizospora heteromorpha RI and Glomus mosseae GM is 1:1.

[0015] Further, the Rhizosporium heteromorphum comprises mycelium, sporangia and spores;

[0016] The Glomus mosseae fungus includes mycelium, sporangia and spores.

[0017] The beneficial effects of the present invention are:

[0018] The composite bacteria of the present invention can significantly improve the cold resistance of gardenia plants. In addition, the composite bacteria can also promote the accumulation of plant endogenous hormones ABA, JA and JA-ME, which are key factors for improving plant cold resistance. Therefore, the composite bacteria of the present invention not only enhances the tolerance of gardenia to extreme low temperatures, but also may improve its growth quality and yield under cold conditions, providing a new biotechnology solution for the cultivation of gardenia, which has important agricultural and economic value.

[0019] The invention also provides an application of the composite bacteria for improving the cold resistance of gardenia.

[0020] Furthermore, the composite bacteria is inoculated onto gardenia seedlings to improve their survivability under extreme low temperature conditions.

[0021] The beneficial effects of the present invention are:

[0022] Experiments have shown that gardenia seedlings inoculated with the composite bacteria showed lower cell membrane damage indicators after experiencing low temperature stress, such as a significant decrease in relative conductivity and malondialdehyde content. At the same time, the activity of antioxidant enzymes such as POD, SOD and CAT was increased, indicating that the plant's ability to eliminate reactive oxygen was enhanced.

[0023] Further, the inoculation step comprises:

[0024] The heteromorphic rhizosporon is propagated with tobacco, the propagation substrate is yellow sand and quartz sand, after propagation, the tobacco plants are removed to obtain the first sandy bacterial agent;

[0025] The moses Glomus was propagated with tobacco respectively, and the propagation substrates were yellow sand and quartz sand. After propagation, the tobacco plants were removed to obtain a second sandy bacterial agent.

[0026] After the first sandy bacterial agent and the second sandy bacterial agent are mixed with nutrient soil, gardenia seedlings are planted.

[0027] The beneficial effects of this further technical solution are:

[0028] The present invention uses gardenia seedlings as inoculation objects, and can establish a symbiotic relationship with the composite bacteria in the early stage of plant growth, thereby providing continuous cold resistance protection for gardenia. This method not only helps to enhance the root development of the seedlings and improve their ability to absorb water and nutrients, but also can cultivate plants with stronger stress resistance from the beginning. In addition, the seedling inoculation operation is simple and easy to implement in large-scale cultivation, which helps to improve the survival rate and reduce subsequent maintenance costs, while promoting environmentally friendly and sustainable agricultural practices.

[0029] Further, the inoculated gardenia seedlings were cultured under alternating temperature conditions of 28°C and 24°C with a light / dark time of 14 / 10 hours.

[0030] Furthermore, the culture time is 1 to 3 months.

[0031] The beneficial effects of this further technical solution are:

[0032] The inoculated gardenia seedlings were cultured under alternating temperature conditions of 28°C and 24°C, with a photoperiod of 14 hours of light and 10 hours of darkness, to simulate the temperature changes and day and night changes in the natural environment, which helps the gardenia seedlings to adapt to the external environment more realistically and promote the natural rhythm of plant growth and development. This culture method can enhance the adaptability of gardenia seedlings to temperature changes, improve their physiological metabolic activities, and thus enhance their cold resistance. At the same time, the 1-3 month culture time can ensure that mycorrhizal fungi are fully integrated with the gardenia root system, establish a stable symbiotic relationship, provide long-term nutrition and protection for the gardenia seedlings, enable them to better resist the influence of adverse environments such as low temperature after transplanting, and improve the survival rate and growth quality.

[0033] Furthermore, the cold resistance of gardenia was evaluated by measuring the relative electrical conductivity, malondialdehyde content, POD, SOD and CAT enzyme activities, and ABA, JA and JA-ME contents of gardenia leaves.

[0034] The beneficial effects of this further technical solution are:

[0035] The cold resistance of gardenia can be comprehensively evaluated by measuring the relative conductivity, malondialdehyde content, POD, SOD and CAT enzyme activities, and the content of endogenous hormones ABA, JA and JA-ME in gardenia leaves. The beneficial effect of this evaluation method is that it can provide detailed information on the stability of gardenia cell membranes, antioxidant capacity, and the regulation status of plant hormones. Specifically, lower relative conductivity and malondialdehyde content indicate less cell membrane damage, while higher antioxidant enzyme activity shows that the plant has a stronger ability to scavenge reactive oxygen species, which are indicators of strong plant cold resistance. At the same time, hormones such as ABA, JA and JA-ME play a key regulatory role in plant response to low temperature stress, and the increase in their content helps to improve the cold resistance of plants. Therefore, this comprehensive evaluation method can accurately reflect the cold resistance of gardenia and provide a scientific basis for further optimizing the inoculation method and improving the cold resistance of gardenia.

[0036] The present invention also provides a method for improving the cold resistance of gardenia, using the composite bacteria, wherein Rhizosporon heteromorpha and Glomus mosseae are propagated with model plants respectively, after propagation, the plants used for propagation are removed, and the propagation matrix is ​​retained to obtain a first sandy bacterial agent and a second sandy bacterial agent respectively;

[0037] After the first sandy bacterial agent and the second sandy bacterial agent are mixed with nutrient soil, gardenia seedlings are planted for inoculation and cultivation.

[0038] Furthermore, the model plant is tobacco.

[0039] Furthermore, the propagation matrix is ​​a mixed matrix of yellow sand and quartz sand.

[0040] Preferably, the mass ratio of the yellow sand to the quartz sand is 2-4:1.

[0041] Preferably, the mass ratio of the yellow sand to the quartz sand is 3:1.

[0042] Furthermore, the mass ratio of the first sandy bacterial agent to the second sandy bacterial agent is 2:1~1:2.

[0043] Preferably, the mass ratio of the first sandy bacterial agent to the second sandy bacterial agent is 1:1.

[0044] Furthermore, the mass ratio of the total volume of the first sandy bacterial agent and the second sandy bacterial agent to the nutrient soil is 1:10~20.

[0045] Preferably, the mass ratio of the total volume of the first sandy bacterial agent and the second sandy bacterial agent to the nutrient soil is 1:15.

[0046] Furthermore, each 1 g of the first sandy bacterial agent contains 150 to 250 spores of Rhizospora heteromorpha.

[0047] Preferably, every 1 g of the first sandy bacterial agent contains about 200 spores of Rhizospora heteromorpha.

[0048] Furthermore, every 1 g of the second sandy bacterial agent contains 150 to 250 spores of Glomus mosseae.

[0049] Preferably, every 1 g of the second sandy bacterial agent contains about 200 spores of Glomus mosseae.

[0050] Furthermore, the first sandy bacterial agent and the second sandy bacterial agent also include naturally dried roots of model plants.

[0051] The beneficial effects of the present invention are:

[0052] Compared with the direct use of RI and GM composite bacteria mixed matrix for gardenia cultivation, the use of the expansion step has more technical advantages. The expanded matrix contains not only live RI and GM strains, but also rich in mycelium, sporocarps, spores, and dried plant root segments, which together constitute a complex microbial ecosystem. Mycelium can quickly form a network around the root system of gardenia, improving the efficiency of nutrient and water absorption; sporocarps and spores, as potential strain banks, can germinate under suitable conditions and enhance the symbiotic relationship between gardenia and mycorrhizal fungi; dried plant root segments provide additional organic matter, promote soil microbial diversity, and help maintain soil health and improve soil fertility. Therefore, the expansion step not only increases the possibility of establishing an effective symbiotic relationship between gardenia and mycorrhizal fungi, but also improves the biological activity and buffering capacity of the soil by enriching the soil microbial community, thereby improving the cold resistance of gardenia while also enhancing its resistance to other adversities, providing a healthier and more stable growth environment for gardenia.

[0053] The present invention also provides a substrate for gardenia cultivation, wherein the substrate contains the composite bacteria, wherein the first sandy bacterial agent and the second sandy bacterial agent containing mycelium, sporangium and spores are obtained after Rhizosporium heteromorphum and Glomus mosseae are propagated with tobacco respectively.

[0054] Furthermore, each 1g unit weight of the first sandy bacterial agent contains 150 to 250 spores of Rhizospora heteromorpha, and each 1g unit weight of the second sandy bacterial agent contains 150 to 250 spores of Glomus mosseae.

[0055] Furthermore, the first sandy bacterial agent and the second sandy bacterial agent also include naturally dried roots of model plants.

[0056] The experiment showed that the gardenia plants inoculated with GM and RI showed significant effects in cold resistance, which was mainly reflected in several key physiological indicators:

[0057] First, the inoculation treatment reduced the relative conductivity of Gardenia jasminoides under low temperature stress and reduced cell membrane damage, indicating that cell membrane stability was enhanced;

[0058] Secondly, the inoculation significantly increased the activities of antioxidant enzymes such as SOD and POD, strengthened the plant's ability to remove reactive oxygen species, and reduced oxidative damage;

[0059] In addition, inoculation with GM and RI also promoted the accumulation of cold-resistance-related plant hormones ABA, JA, and JA-ME, which play a central role in regulating plant cold-resistance responses.

[0060] Taken together, these results suggest that inoculation with GM and RI not only enhances the ability of gardenia to survive under low temperature conditions, but also improves the overall cold resistance of the plant by regulating the physiological and molecular mechanisms within the plant. This strategy of improving plant cold resistance through microbial inoculation provides an effective biotechnology means to improve the stress resistance of gardenia in agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0062] Figure 1 This is a microscopic observation view of GM mycorrhizal infection.

[0063] Figure 2 This is a microscopic observation view of RI mycorrhizal infection. DETAILED DESCRIPTION

[0064] The following describes the invention in conjunction with specific embodiments.

[0065] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention.

[0066] In this embodiment, the term "compound bacteria" may also be referred to as "compound bacterial agent".

[0067] Rhizospora heteromorpha Rhizophagus irregularis (RI), Glomus mosseae Glomus mosseae (GM) was purchased from the “Bank of Glomeromycota in China, BGC”, Institute of Plant Nutrition and Resources, Beijing Academy of Agricultural and Forestry Sciences.

[0068] RI and GM were propagated in pots respectively, and suitable host plants were selected, tobacco in this embodiment, and the potting medium was 3 parts by weight of yellow sand and 1 part by weight of quartz sand.

[0069] One of the reasons why composite bacteria use tobacco as the host plant for propagation is that tobacco has strong growth characteristics and wide adaptability, and can provide a suitable growth environment and sufficient nutrition sources for mycorrhizal fungi, thereby promoting the mass reproduction and active maintenance of the bacterial agent. The root system of tobacco plants is well developed, which can effectively establish a symbiotic relationship with composite bacteria. Through this symbiotic interaction, composite bacteria can obtain essential photosynthetic products from tobacco plants, while providing mineral nutrients and water to tobacco plants to enhance their growth. In addition, as a model plant, the biological characteristics and molecular mechanisms of tobacco are more thoroughly studied, which is convenient for monitoring and evaluating the effect of bacterial agent propagation and ensuring the quality and stability of the bacterial agent. Therefore, choosing tobacco as a host plant for the propagation of composite bacteria can improve production efficiency, ensure the activity and purity of the bacterial agent, and provide high-quality products for subsequent applications.

[0070] After multiplication, about 200 spores of heteromorphic root cysts are contained in every 1g of the first sandy bacterial agent, and about 200 spores of moses glomerulosa are contained in every 1g of the second sandy bacterial agent. In the present embodiment, the dosage mass ratio of the first sandy bacterial agent and the second sandy bacterial agent is 1:1, so the number ratio of heteromorphic root cysts and moses glomerulosa spores is also about 1:1. In the process of multiplication, environmental parameters, such as temperature, humidity and nutrient supply, need to ensure the best growth state of tobacco plants, and this process also makes the bacterial agent remain in a suitable growth state, thereby enhancing its stability and effect in practical applications. Multiplication also helps to screen out the most adaptable and energetic strains, reduce pollution, improve the purity and activity of the bacterial agent, so that it can more effectively establish a symbiotic relationship with the root system of gardenia, and enhance the cold resistance and other stress resistance of gardenia. Therefore, multiplication not only helps to improve the activity unit of the composite bacteria, but also helps to ensure its effectiveness in applications such as improving the cold resistance of gardenia.

[0071] Prepare the matrix for multiplication. The present embodiment selects a mixture of sterile yellow sand and quartz sand. The ratio of yellow sand to quartz sand is 3:1. Yellow sand and quartz sand provide ideal physical support and drainage for the growth of mycelium, ensure the air permeability of the matrix, and are conducive to the healthy multiplication of mycorrhizal fungi. The multiplication cycle is one or more cultivation cycles of tobacco plants. The present embodiment is a cultivation cycle of a tobacco plant. A cultivation cycle of a tobacco plant refers to the whole process from planting tobacco seedlings to the harvest of tobacco maturity. Throughout the growth cycle, mycorrhizal fungi will face selection pressures similar to the natural environment, which helps to screen out strains with strong adaptability and high growth vitality, and improve the quality and efficacy of the final product. A complete growth cycle allows spores and mycelium to fully mature, which is crucial for improving the activity and stability of the microbial agent. Using a life cycle of tobacco to multiply can make more effective use of time and resources, because a large amount of microbial agents can be harvested at the same time when the tobacco matures.

[0072] After propagation, the tobacco plants were removed to obtain the sandy inoculant. The sandy inoculant contains mycelium, sporangia, spores, yellow sand, quartz sand and dried plant root segments of arbuscular mycorrhizal fungi. During the propagation process, RI and GM grew in a mixed matrix of yellow sand and quartz sand, forming a dense mycelial network and producing a large number of sporangia and spores. The dried plant root segments were mainly the root segments of tobacco plants, and also the roots of weeds. The dried root segments are conducive to the preservation of fungal spores. In addition, as the normal growth cycle of tobacco proceeds, the number of spores in its root system during the dry stage is more than that of fresh root segments. These dried plant root segments provide rich organic matter and suitable growth matrix for RI and GM in the sandy inoculant. In the early stage of transplanting gardenia seedlings, since their root system has not yet fully developed, these dried root segments can create favorable conditions for the generation of RI and GM, including providing a microbial environment similar to that in natural soil, increasing the opportunity for microbial attachment and reproduction. In addition, the nutrients released by these root segments during the decomposition process can serve as a nutrient source for RI and GM, promoting their growth and reproduction, thereby providing more mycorrhizal fungi for the gardenia seedlings during their root development, helping the seedlings to absorb water and nutrients from the soil more effectively, and enhancing their early growth and resistance to adversity.

[0073] Using 3-month-old gardenia potted seedlings as test materials, the sandy inoculant containing mycorrhizal fungal spores was mixed with nutrient soil to obtain a cultivation matrix. The mass ratio of the first sandy inoculant, the second sandy inoculant and the nutrient soil was 1:1:30. In the present embodiment, the nutrient soil was purchased from the golden moss seedling matrix produced by Zhejiang Taisheng Agricultural Technology Co., Ltd. Watering keeps the cultivation matrix moist, with a humidity of about 50%, and proper drying is more conducive to mycorrhizal symbiosis. The gardenia seedlings were moved into the cultivation matrix, covered with a transparent moisturizing cover, and the temperature was alternating between 28°C and 24°C, with a temperature difference of 14 / 10 hours of light and dark time. After 2 months, the infection effect was checked, and the gardenia seedlings were subjected to a low temperature stress test, with a low temperature treatment of 8°C / 2°C (light and dark time 14 / 10 h), and the treatment was carried out for 5 days to measure the relevant indicators.

[0074] The results of impregnation are as follows Figure 1 and Figure 2 The measurement results are shown in Tables 1 to 8.

[0075] In order to illustrate the effect of inoculating GM and RI, control treatments were set up in the process of completing this embodiment, including blank treatment, GM inoculation treatment and RI inoculation treatment. In the simultaneous inoculation of GM and RI treatments, 50g of the first sandy bacterial agent and 50g of the second sandy bacterial agent were added to 1500g of nutrient soil at the same time; the inoculation of RI treatment was to add 100g of the first sandy bacterial agent to 1500g of nutrient soil, the inoculation of GM treatment was to add 100g of the second sandy bacterial agent to 1500g of nutrient soil, and the blank treatment was to add 50g of the first sandy bacterial agent and 50g of the second sandy bacterial agent with spores inactivated at 121°C to 1500g of nutrient soil. The remaining operating steps of all treatments are the same.

[0076] Table 1 Detection results of relative conductivity under different treatments

[0077] Whitespace processing GM inoculation Inoculation of RI Inoculation of GM and RI normal temperature 17.15±0.17Ab 22.04±0.22Bb 23.21±0.16Bb 28.05±0.42Cb Low temperature 57.52±0.13Aa 38.92±0.01Ba 35.34±0.12Ba 29.26±0.34Ca

[0078] Relative conductivity is an important indicator to measure the integrity and damage of cell membranes. When plants are subjected to adverse stress, such as low temperature stress, the integrity of the cell membrane will be damaged, resulting in an increase in relative conductivity. In the blank treatment group (the control group without any inoculation of bacterial agents), the relative conductivity under low temperature conditions was significantly higher than that under normal temperature conditions, indicating that low temperature stress caused damage to the cell membrane and increased the permeability of the cell membrane. In the treatment groups inoculated with GM, RI, and GM and RI at the same time, the relative conductivity under low temperature conditions was lower than that of the blank treatment group, indicating that inoculation with GM and RI can reduce the damage to the cell membrane caused by low temperature stress and maintain the integrity of the cell membrane. In the treatment groups inoculated with GM, RI, and GM and RI at the same time, the relative conductivity under low temperature conditions was lower than that of the blank treatment group, indicating that inoculation with GM and RI can reduce the damage to the cell membrane caused by low temperature stress and maintain the integrity of the cell membrane.

[0079] However, it is worth noting that the effects of GM and RI inoculation on the relative conductivity of gardenia showed a bidirectional regulatory effect, which is quite interesting and has potential scientific value in the field of plant physiology. Under normal temperature conditions, gardenia plants inoculated with GM and RI showed relatively high conductivity, which may mean that under non-stress conditions, the inoculated microbial agents promoted the metabolic activity of the root system and enhanced the root system's absorption of water and minerals, which may lead to an increase in cell membrane permeability. However, under low temperature stress conditions, plants inoculated with GM and RI showed lower relative conductivity, indicating that under adverse conditions, the inoculated microbial agents significantly improved the stability of the cell membrane, reduced cell membrane damage and increased permeability, and effectively protected the cells from low temperature damage. This opposite regulatory effect under different temperature conditions led to a significant reduction in the change in relative conductivity of plants inoculated with GM and RI under low temperature stress. This phenomenon may be related to the plant cold resistance mechanism induced by the microbial agents, such as enhancing the antioxidant system, regulating intracellular hormone levels, or promoting the expression of protective proteins. These mechanisms work together to improve the survival ability of plants under low temperature conditions. Although this phenomenon is beyond traditional cognition, it reveals the great potential of microbial-plant interactions in improving plant stress resistance and provides new directions for further research.

[0080] Table 2 Detection results of MDA under different treatments

[0081] Whitespace processing GM inoculation Inoculation of RI Inoculation of GM and RI normal temperature 2.36±0.16bAb 3.31±0.07 Bb 3.42±0.38Bb 3.17±0.03Cb Low temperature 4.06±0.17aAa 3.80±0.06Aa 3.72±0.24Ba 3.36±0.12Ba

[0082] Under normal temperature conditions, the malondialdehyde (MDA) content of gardenia plants inoculated with GM, RI, and GM and RI was higher than that of the blank treatment, indicating that the inoculation agent had a certain effect on the level of membrane lipid peroxidation of gardenia plants under normal growth conditions. As a product of membrane lipid peroxidation, the increase in MDA content is usually associated with cell membrane damage. However, this effect was different among different inoculation treatments, and the MDA content of plants inoculated with GM and RI was relatively low, indicating that these two inoculation agents reduced the degree of membrane lipid peroxidation to a certain extent.

[0083] Under low temperature conditions, plants inoculated with GM and RI showed lower malondialdehyde content compared with the blank treatment, indicating that the inoculation treatment enhanced the tolerance of Gardenia to low temperature stress. Since low temperature stress increases the production of reactive oxygen species and leads to peroxidation of the membrane system, the ability of inoculation with GM and RI to reduce malondialdehyde content means that they help reduce the damage of reactive oxygen species to the membrane system, thereby protecting the integrity of the cell membrane.

[0084] The data on the changes in malondialdehyde showed that the increase in malondialdehyde content in plants inoculated with GM and RI was small when they were transferred from normal temperature to low temperature conditions. In particular, the change in the plants inoculated with GM and RI was the smallest, only 0.19 nmol / L, which was much lower than the 1.7 nmol / L of the blank treatment. This indicates that inoculation with GM and RI, especially the combined use of the two, can significantly reduce the increase in membrane lipid peroxidation under low temperature stress, thereby improving the cold resistance of gardenia.

[0085] Overall, the test results and their changes showed that inoculation with GM and RI had a positive effect on the malondialdehyde content of gardenia plants under both normal and low temperature conditions. Especially under low temperature stress conditions, inoculation with GM and RI could significantly reduce the increase in malondialdehyde content and reduce membrane lipid peroxidation, thereby improving the cold resistance of gardenia. These results further demonstrated the potential application value of inoculation with GM and RI in improving plant stress resistance, and provided important clues for further studying the molecular mechanism of the interaction between microbial agents and plants.

[0086] Table 3 Detection results of SOD under different treatments

[0087] Whitespace processing GM inoculation Inoculation of RI Inoculation of GM and RI normal temperature 16.43±0.35Ab 17.26±0.30Ab 18.91±0.20Bb 24.68±0.66Bb Low temperature 20.00±0.22Aa 29.26±0.24Ba 35.26±1.08Ca 42.68±0.66Da

[0088] Under normal temperature conditions, the superoxide dismutase (SOD) activity of gardenia plants inoculated with GM, RI, and GM and RI was higher than that of the blank treatment, indicating that inoculation with these inoculants may enhance the antioxidant capacity of gardenia plants under normal growth conditions. SOD is a key antioxidant enzyme that can scavenge superoxide free radicals and protect cells from oxidative stress. The increase in SOD activity in plants inoculated with GM and RI at normal temperature may help improve the adaptability of plants to various environmental stresses.

[0089] Under low temperature conditions, the SOD activities of all inoculated treatments were significantly higher than those of the blank treatment, especially those of the plants inoculated with GM and RI, whose SOD activities increased most significantly. This indicates that inoculation with GM and RI can significantly improve the antioxidant capacity of Gardenia under low temperature stress, help to remove excessive reactive oxygen caused by low temperature, and protect cell membranes and organelles from oxidative damage.

[0090] The change in SOD showed that the increase in SOD activity was the largest when the plants inoculated with GM and RI were transferred from normal temperature to low temperature. In particular, the change in SOD activity of the plants inoculated with GM and RI at the same time was the highest, at 18 pg / mL, much higher than the 3.57 pg / mL of the blank treatment. This shows that inoculation with GM and RI, especially the combined use of the two, can significantly improve the adaptability of Gardenia to low temperature stress and enhance its survival ability under adverse conditions.

[0091] These results further confirmed the application value of inoculation of GM and RI in improving the cold resistance of Gardenia jasminoides.

[0092] Table 4 POD detection results under different treatments

[0093] Whitespace processing GM inoculation Inoculation of RI Inoculation of GM and RI normal temperature 43.94±0.93Aa 63.99±0.90Bb 58.82±0.88Ba 73.28±1.26Ca Low temperature 52.70±0.23Aa 74.76±0.97Ba 92.32±6.48Ca 105.84±5.68Da

[0094] Under normal temperature conditions, the peroxidase (POD) activity of gardenia plants inoculated with GM, RI, and GM and RI was higher than that of the blank treatment, indicating that the inoculation of these inoculants may enhance the antioxidant capacity of gardenia plants under normal growth conditions. Under low temperature conditions, the POD activity of all inoculation treatments increased further, especially the plants inoculated with RI and GM and RI at the same time, whose POD activity was significantly higher than that of the blank treatment and GM inoculated plants. This shows that under low temperature stress, inoculation with GM and RI can significantly improve the antioxidant capacity of gardenia, help to remove excessive reactive oxygen caused by low temperature, and protect cell membranes and organelles from oxidative damage.

[0095] Inoculation of GM and RI, especially their combined use, can significantly improve the adaptability of Gardenia to low temperature stress and enhance its survival ability under low temperature stress conditions.

[0096] Table 5 CAT detection results under different treatments

[0097] Whitespace processing GM inoculation Inoculation of RI Inoculation of GM and RI normal temperature 30.20±0.42Ab 43.43±0.57Bb 44.37±0.14Ba 54.82±2.16Bb Low temperature 44.37±0.14Aa 52.54±0.87Aa 69.20±0.42Bb 94.82±2.16Ca

[0098] The results of catalase (CAT) activity detection under different treatments showed that under normal temperature conditions, the CAT activity of gardenia plants inoculated with GM, RI, and GM and RI was higher than that of the blank treatment, among which the combination of GM and RI showed the highest activity. This indicates that inoculation with these bacterial agents may enhance the antioxidant capacity of gardenia plants under normal growth conditions, help to scavenge hydrogen peroxide, and protect cells from oxidative stress damage. Under low temperature conditions, the CAT activity of all inoculation treatments increased further, especially the plants inoculated with RI and GM and RI at the same time, whose CAT activity was significantly higher than that of the blank treatment and GM inoculated plants. This indicates that under low temperature stress, inoculation with GM and RI can significantly improve the antioxidant capacity of gardenia, help to scavenge excessive reactive oxygen caused by low temperature, and protect cell membranes and organelles from oxidative damage. The synergistic effect of inoculation with GM and RI is related to their complementary mechanism in improving the antioxidant capacity of plants. This synergistic effect may enhance the ability of gardenia to scavenge reactive oxygen, thereby improving the cold resistance of gardenia. These results further confirmed the potential application value of inoculating GM and RI in improving the cold resistance of gardenia, and provided a scientific basis for using these bacterial agents to improve plant cold resistance.

[0099] Table 6 ABA detection results under different treatments

[0100] Whitespace processing GM inoculation Inoculation of RI Inoculation of GM and RI normal temperature 99.59±3.45Ab 144.55±0.90Bb 102.55±2.98 Bb 168.59±4.76Cb Low temperature 141.3±1.81Aa 254.65±6.19Ba 181.3±1.81Ba 262.42±4.82Ca

[0101] As an important plant hormone, ABA plays a key role in plant response to stress, especially low temperature stress. It can enhance plant stress resistance through multiple pathways such as regulating stomatal opening and closing and promoting the expression of antioxidant enzymes. Therefore, inoculation of GM and RI improves the cold resistance of gardenia by promoting the accumulation of ABA. Simultaneous inoculation of GM and RI still expressed their synergistic effect in regulating the hormone levels of gardenia, and further enhanced the cold resistance of gardenia.

[0102] Table 7 Detection results of CJA under different treatments

[0103] Whitespace processing GM inoculation Inoculation of RI Inoculation of GM and RI normal temperature 735.74±12.82Ab 813.72±7.84Bb 905.74±9.48Ba 998.88±14.36C Low temperature 910.44±15.91Aa 964.78±16.66Ba 1022.26±18.32Ca 1226.56±20.48Ca

[0104] Coronary acid (CJA), as a plant hormone, is involved in regulating plant growth, development and stress response. Inoculation with GM and RI improves the cold resistance of gardenia by promoting the accumulation of CJA. The results of CJA detection under different treatments showed that under normal temperature conditions, the CJA content of gardenia plants inoculated with GM, RI, and GM and RI was higher than that of the blank treatment, among which the combination of GM and RI inoculation showed the highest CJA content. This indicates that inoculation with these inoculants enhances the hormone regulation ability of gardenia plants under normal growth conditions and helps to improve plant growth and development. Under low temperature conditions, the CJA content of all inoculated treatments was significantly higher than that of the blank treatment, especially the treatment inoculated with GM and RI at the same time, which had the highest CJA content, indicating that low temperature stress significantly induced the accumulation of CJA.

[0105] Table 8 Detection results of JA-ME under different treatments

[0106] Whitespace processing GM inoculation Inoculation of RI Inoculation of GM and RI normal temperature 76.4±3.51Ab 144.02±0.40Bb 176.24±3.51Bb 238.42±5.86Cb Low temperature 86.31±0.27Aa 187.20±1.51Ba 206.48±7.82Ca 258.72±16.26Da

[0107] Methyl jasmonate (JA-ME), as a plant hormone, is involved in regulating plant defense responses, growth and development, and stress responses. Inoculation with GM and RI improves the cold resistance of Gardenia jasminoides by promoting the accumulation of JA-ME.

[0108] The results of JA-ME detection under different treatments showed that under normal temperature conditions, the JA-ME content of gardenia plants inoculated with GM, RI, and GM and RI was higher than that of the blank treatment, especially the combination of GM and RI, which showed the highest JA-ME content. This shows that the inoculation of these fungi enhances the hormone regulation ability of gardenia plants under normal growth conditions, which helps to improve the defense response and growth and development of plants. Under low temperature conditions, the JA-ME content of all inoculation treatments was significantly higher than that of the blank treatment, especially the treatment inoculated with GM and RI at the same time, which had the highest JA-ME content, indicating that low temperature stress further significantly induced the accumulation of JA-ME. This shows that inoculation with GM and RI, especially the combination of the two, can significantly improve the response of gardenia to low temperature stress and enhance its survival ability under adverse conditions.

[0109] It is particularly noteworthy that the detection results of JA-ME inoculated with GM and RI under normal temperature conditions are higher than those inoculated with GM or RI under low temperature conditions. This phenomenon indicates that the synergistic effect of GM and RI inoculation can significantly increase the level of JA-ME in gardenia plants under non-stress conditions, thereby enhancing the basic defense mechanism of the plants. This early hormone response enables the plants to activate related defense pathways such as cold resistance more quickly when encountering adverse stress, so that they can respond more effectively when adversity really comes. In addition, this also reflects that plants inoculated with GM and RI have higher regulatory capabilities in hormone balance and signal transduction, and can maintain appropriate hormone levels under different environmental conditions to support the growth and defense needs of plants. This improvement in regulatory ability helps plants maintain a stable physiological state in the face of environmental changes and enhances their adaptability and resistance to adversity.

[0110] Based on the above indicators, simultaneous inoculation of GM and RI can alleviate the damage of gardenia leaves under low temperature stress to the greatest extent, thereby improving the cold resistance of gardenia under low temperature.

[0111] In the description of the present invention, it should be understood that “—” and “~” represent the range between two values, and the range includes the endpoints. For example: “A—B” represents a range greater than or equal to A and less than or equal to B. “A~B” represents a range greater than or equal to A and less than or equal to B.

[0112] In the description of the present invention, the term "and / or" herein is merely a description of the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent three situations: A exists alone, A and B exist at the same time, and B exists alone.

[0113] In the description of the invention, the numerical values ​​of time, temperature, ratio, mass, etc. involved may be based on actual measurements, standard parameters of equipment, simplified rounding results, or within an acceptable error range, ensuring the practicability and repeatability of the invention.

[0114] In the description of the present invention, the term "about" or "approximately" is used to express the approximate value of a numerical value or range, allowing a certain error to ensure the flexibility and practicality of the description while remaining within an acceptable error range, with the maximum error range not exceeding 10% of the corresponding numerical value or numerical range.

[0115] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A composite bacteria for improving the cold resistance of Gardenia jasminoides, characterized in that: The composite bacterial agent comprises: a. Rhizosporium heteromorphum Rhizophagus irregularis ),as well as b. Glomus mosseae Glomus mosseae ), The heteromorphic rhizosporon is propagated with tobacco, the propagation substrate is yellow sand and quartz sand, after propagation, the tobacco plants are removed to obtain the first sandy bacterial agent; The moses Glomus was propagated with tobacco respectively, and the propagation substrates were yellow sand and quartz sand. After propagation, the tobacco plants were removed to obtain a second sandy bacterial agent. The mass ratio of the first sandy bacterial agent to the second sandy bacterial agent is 1:1; Every 1g unit weight of the first sandy bacterial agent contains 150 to 250 spores of Rhizospora heteromorpha, and every 1g unit weight of the second sandy bacterial agent contains 150 to 250 spores of Glomus mosseae.

2. An application of the composite bacteria according to claim 1, characterized in that: Used to improve the cold resistance of gardenia; The composite bacteria is inoculated onto the gardenia seedlings to improve their survivability under extreme low temperature conditions; The inoculation steps include: The heteromorphic rhizosporon is propagated with tobacco, the propagation substrate is yellow sand and quartz sand, after propagation, the tobacco plants are removed to obtain the first sandy bacterial agent; The moses Glomus was propagated with tobacco respectively, and the propagation substrates were yellow sand and quartz sand. After propagation, the tobacco plants were removed to obtain a second sandy bacterial agent. After the first sandy bacterial agent and the second sandy bacterial agent are mixed with nutrient soil, gardenia seedlings are planted.

3. The use according to claim 2, characterized in that: The inoculated gardenia seedlings were cultured under alternating temperature conditions of 28°C and 24°C with a light / dark period of 14 / 10 hours.

4. The use according to claim 3, characterized in that: The cultivation time is 1 to 3 months.

5. The use according to claim 3, characterized in that: The cold resistance of Gardenia jasminoides was evaluated by measuring the relative electrical conductivity, malondialdehyde content, POD, SOD and CAT enzyme activities, and ABA, JA and JA-ME contents in its leaves.

6. A method for improving the cold resistance of Gardenia jasminoides, characterized in that: Using the Rhizosporon heteromorphum and Glomus mosseae described in claim 1, respectively, to propagate with tobacco, after propagation, removing the plants used for propagation, retaining the propagation matrix, to obtain a first sandy bacterial agent and a second sandy bacterial agent, respectively; After the first sandy bacterial agent and the second sandy bacterial agent are mixed with nutrient soil, gardenia seedlings are planted for inoculation and cultivation.

7. A substrate for cultivating gardenia, characterized in that: The matrix contains the first sandy bacterial agent and the second sandy bacterial agent containing mycelium, sporangia and spores obtained by respectively propagating the heteromorphic Rhizospora and Glomus mosseae as claimed in claim 1 with tobacco.

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