A stress-resistant agent for improving the cold resistance of crops, and its preparation method and application

Abstract

The present invention relates to the field of plant growth regulators, and discloses an anti-stress agent for improving the cold tolerance of crops, as well as its preparation method and application. The agent comprises Candida XHZG06-95A3 and / or its fermentation supernatant, and also comprises an antifreeze protection protein; the Candida XHZG06-95A3 belongs to Candida palmioleophila in taxonomy, and its deposit number is CGMCC No.29215; the amino acid sequence of the antifreeze protection protein is shown in SEQ ID NO:1. In the anti-stress agent of the present invention, Candida XHZG06-95A3 and / or its fermentation supernatant can be used to enhance the effect of the antifreeze protection protein, thereby improving the cold tolerance of crops to a greater extent.

Description

Technical Field

[0001] The present invention relates to the field of plant growth regulators, in particular to an anti-stress agent for improving the cold resistance of crops, and a preparation method and application thereof. Background Art

[0002] Low temperatures are a key adverse factor affecting plant growth, development, and yield quality. When plants are exposed to low-temperature stress, they experience physiological changes such as damage to cell membrane structures, reduced antioxidant enzyme activity, and decreased photosynthesis. Macroscopically, these changes manifest as slow germination and growth, wilting, yellowing leaves, tissue limpness, low fruit set, weak seedlings, and even dead seedlings.

[0003] Crop antifreeze agents are a class of exogenous plant growth regulators that stabilize cell membranes and induce a series of physiological and biochemical reactions in crops, thereby enhancing their cold tolerance. These agents typically work by soaking seeds or spraying them onto plants. Currently reported crop antifreeze agents include salicylic acid, abscisic acid, fulvic acid, ascorbic acid, potassium dihydrogen phosphate, paclobutrazol, chitosan, and locust bean gum. However, the effectiveness of a single antifreeze agent in improving crop cold tolerance is limited. Therefore, the current trend in crop antifreeze research and development is to design antifreeze compound solutions based on crop cold tolerance and the mechanism of action of crop antifreeze agents.

[0004] Patent CN112876545A discloses an antifreeze protein and its preparation method and application, which is obtained from Marinomonas arcticus ( Marinomonas arctica An antifreeze protein has been discovered in the BSI20414 strain. This protein inhibits ice crystal growth and recrystallization, thereby protecting cells in low-temperature environments and acting as a protective agent for cryopreservation. However, when the patented antifreeze protein is incorporated into crop antifreeze agents, while it improves the crops' cold tolerance to a certain extent, the effect is limited. Summary of the Invention

[0005] To address the technical problem of antifreeze protection proteins being ineffective in improving crop cold tolerance when used as antifreeze agents, the present invention provides an antifreeze agent for improving crop cold tolerance, as well as its preparation method and use. The antifreeze agent of the present invention utilizes Candida albicans XHZG06-95A3 and / or its fermentation supernatant to enhance the effectiveness of antifreeze protection proteins, thereby further improving crop cold tolerance.

[0006] The specific technical solutions of the present invention are:

[0007] In the first aspect, the present invention provides an anti-stress agent for improving the cold resistance of crops, characterized in that it comprises Candida sp. XHZG06-95A3 and / or its fermentation supernatant, and also comprises antifreeze protection protein; the Candida sp. XHZG06-95A3 belongs to Candida palmioleophila , the deposit number is CGMCC No.29215; the amino acid sequence of the antifreeze protection protein is shown in SEQ ID NO:1.

[0008] By adding an antifreeze protein with an amino acid sequence such as SEQ ID NO: 1 to an anti-stress agent, the present invention can prevent further growth of small ice crystals once they form in crops. Furthermore, it can also round the ice crystals formed in the crops, preventing them from puncturing cells. In this way, the antifreeze protein can improve the cold tolerance of crops.

[0009] Based on this, the present invention utilizes Candida albicans XHZG06-95A3 and / or its fermentation supernatant in combination with a specific antifreeze protein, creating a synergistic effect. This synergistic effect is strain-specific; not all Candida albicans strains combined with the antifreeze protein will achieve the same level of synergistic effects in improving crop cold tolerance as described in the present invention. The mechanism of this synergistic effect is hypothesized to be as follows: Candida albicans XHZG06-95A3 secretes specific glycoproteins that form stable complexes with antifreeze proteins of a specific structure. Furthermore, this strain may produce inhibitory metabolites (such as certain lipids) that weaken the active conformation of the antifreeze protein.

[0010] The deposit information of Candida sp. XHZG06-95A3 is as follows:

[0011] Depository: General Microbiology Center, China Culture Collection Administration;

[0012] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0013] Deposit number: CGMCC No.29215;

[0014] Deposit date: December 4, 2023;

[0015] Category naming: Candida palmioleophila ——This is Candida species ( Candida ) is a species in the family, for which there is currently no unified Chinese translation.

[0016] In previous studies by the present invention team, it was reported that Candida species XHZG06-95A3 has the function of improving acidified soil. However, the mechanism of improving acidified soil is different from that of improving crop cold tolerance. In previous studies, it was not reported that this strain has the ability to improve plant cold tolerance and enhance the effect of antifreeze protection protein. In other existing technologies, Candida species ( Candida ) contains strains that can enhance the effect of antifreeze protection proteins to improve the cold resistance of crops.

[0017] Preferably, the stress-resistant agent further comprises one or more of glycerol, trehalose and phosphate buffer.

[0018] Preferably, the mass ratio of the fermentation broth supernatant to the antifreeze protective protein is 1-2:1; the fermentation broth supernatant is formed by fermentation of Candida albicans XHZG06-95A3 in a yeast extract peptone dextrose (YPD) liquid medium supplemented with glycerol, and has a polysaccharide content of 0.10-0.15 g / mL.

[0019] Furthermore, the amount of glycerol added to the yeast extract peptone glucose liquid culture medium is 4-7 wt%.

[0020] In a second aspect, the present invention provides a method for preparing the stress-resistant agent, comprising the following steps: mixing Candida XHZG06-95A3 and / or its fermentation supernatant with antifreeze protection protein to prepare the stress-resistant agent.

[0021] Preferably, the preparation method comprises the following steps: 600 =0.5~1.5 of Candida XHZG06-95A3 bacterial suspension, antifreeze protection protein and dispersion medium are mixed to obtain an anti-stress agent; the volume ratio of the Candida XHZG06-95A3 bacterial suspension and the anti-stress agent is 1:1.5~2.5; the mass volume ratio of the antifreeze protection protein and Candida XHZG06-95A3 bacterial suspension is 0.08~0.15 g:50 mL.

[0022] Preferably, the preparation method comprises the following steps: mixing the fermentation supernatant of Candida XHZG06-95A3 and antifreeze protection protein, adding a dispersion medium until the solid content is 10-15 wt%, and then adding an emulsifier for homogenization and emulsification to obtain an anti-stress agent.

[0023] Furthermore, the dispersion medium is phosphate buffered saline (PBS) buffer.

[0024] Preferably, the step of preparing the fermentation broth supernatant comprises: inoculating Candida species XHZG06-95A3 into a yeast extract peptone glucose liquid medium supplemented with glycerol, fermenting and culturing until OD600=1.8-2.2, collecting the supernatant by centrifugation, and concentrating it to a polysaccharide content of 0.10-0.15 g / mL.

[0025] In a third aspect, the present invention provides the use of the stress-resistant agent in improving the cold resistance of crops.

[0026] Preferably, the improving cold tolerance of crops includes reducing intracellular electrolyte exudation, reducing the degree of cell membrane lipid peroxidation, increasing superoxide dismutase activity and improving crop survival rate under low temperature stress; the crops are tea or wheat.

[0027] In a fourth aspect, the present invention provides a use of Candida sp. XHZG06-95A3 and / or its fermentation supernatant in enhancing the effect of antifreeze protection protein, wherein the antifreeze protection protein improves the cold resistance of crops; the Candida sp. XHZG06-95A3 belongs to Candida palmioleophila , the deposit number is CGMCC No.29215; the amino acid sequence of the antifreeze protection protein is shown in SEQ ID NO:1.

[0028] Preferably, the crop is tea or wheat.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] The present invention uses Candida XHZG06-95A3 and / or its fermentation supernatant with a specific antifreeze protection protein. The two can cooperate with each other to produce a synergistic effect, thereby improving the cold resistance of crops to a greater extent. Candida ) have been found to have strains that can enhance the effect of antifreeze protection proteins to improve the cold resistance of crops. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to the embodiments.

[0032] First, the present invention relates to an anti-stress agent for improving the cold resistance of crops, comprising Candida sp. XHZG06-95A3 and / or its fermentation supernatant, and also comprising antifreeze protection protein; the Candida sp. XHZG06-95A3 belongs to Candida palmioleophila , the deposit number is CGMCC No.29215; the amino acid sequence of the antifreeze protection protein is shown in SEQ ID NO:1.

[0033] In some embodiments, the stress-resistant agent further comprises one or more of glycerol, trehalose and phosphate buffer.

[0034] In some specific embodiments, the mass ratio between the fermentation broth supernatant and the antifreeze protective protein is 1-2:1.

[0035] In some embodiments, the fermentation supernatant is produced by fermenting Candida species XHZG06-95A3 in a yeast extract peptone dextrose liquid medium supplemented with glycerol, and has a polysaccharide content of 0.10 to 0.15 g / mL. Optionally or preferably, the glycerol is added to the yeast extract peptone dextrose liquid medium in an amount of 4 to 7 wt%.

[0036] Secondly, the present invention relates to a method for preparing the stress-resistant agent, comprising the following steps: mixing Candida XHZG06-95A3 and / or its fermentation supernatant with antifreeze protection protein to prepare the stress-resistant agent.

[0037] In some embodiments, the preparation method comprises the following steps: 600 =0.5-1.5 of a suspension of Candida species XHZG06-95A3, an antifreeze protective protein, and a dispersion medium are mixed to obtain an anti-stress agent; the volume ratio of the Candida species XHZG06-95A3 suspension to the anti-stress agent is 1:1.5-2.5; and the mass-to-volume ratio of the antifreeze protective protein to the Candida species XHZG06-95A3 suspension is 0.08-0.15 g:50 mL. Optionally or preferably, the dispersion medium is a phosphate buffer.

[0038] In other specific embodiments, the preparation method comprises the following steps: mixing the fermentation supernatant of Candida species XHZG06-95A3 with an antifreeze protective protein, adding a dispersion medium until the solid content reaches 10-15 wt%, and then adding an emulsifier for homogenization and emulsification to obtain an anti-stress agent. Optionally or preferably, the dispersion medium is a phosphate buffer.

[0039] In some embodiments, the preparation step of the fermentation broth supernatant comprises: inoculating Candida species XHZG06-95A3 into a yeast extract peptone glucose liquid medium supplemented with glycerol, and fermenting and culturing the mixture until the OD 600 =1.8-2.2, centrifugation to collect the supernatant, and concentration to a polysaccharide content of 0.10-0.15 g / mL. Optionally or preferably, the amount of glycerol added to the yeast extract peptone glucose liquid medium is 4-7 wt%.

[0040] Third, the present invention relates to the use of the stress-resistant agent in improving the cold resistance of crops.

[0041] In some embodiments, the improving the cold tolerance of crops comprises reducing intracellular electrolyte extravasation, reducing the degree of cell membrane lipid peroxidation, increasing superoxide dismutase activity, and improving crop survival rate under low temperature stress. In some embodiments, the crops are tea or wheat.

[0042] Fourthly, the present invention relates to the use of Candida sp. XHZG06-95A3 and / or its fermentation supernatant in enhancing the effect of antifreeze protection protein, wherein the antifreeze protection protein effect is the effect of antifreeze protection protein in improving the cold resistance of crops; the Candida sp. XHZG06-95A3 belongs to Candida palmioleophila , the deposit number is CGMCC No.29215; the amino acid sequence of the antifreeze protection protein is shown in SEQ ID NO:1.

[0043] In some embodiments, the crop is tea or wheat.

[0044] The present invention is described below by way of specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit and scope of the inventive concept, any changes and advantages that can be imagined by those skilled in the art are included in the present invention, and the scope of protection of the present invention is defined by the appended claims and any equivalents thereof.

[0045] In the following examples and comparative examples, unless otherwise specified, the Candida species XHZG06-95A used are all strains deposited in the present invention with the number CGMCC No. 29215, and the antifreeze protective proteins used are all proteins with the amino acid sequence shown in SEQ ID NO: 1, the specific sequence of which is as follows:

[0046] DSATHHDYNYDQWHQFGSGNDQIVIDNDVEKWLDAGDGDNSIYVGDDVNRNNSAGIKTGSGDDDIFVKDNVDSTIQTGGGNDRVQIGYDLGNGYHSAHINLGDGDNNLIIKNDVNNYSTV HSGSGDDVVSIGDDVRYDADIQLGDGNDRLTIGDKIEKEVSINLGSGNDVLVVGGKVSDEAWVDGGSGSDSVWFESYSRSDYNSDKDGIKSRFANFENFKFSDGTVIGNASAFDGSSVSG.

[0047] Example 1: Effect of an anti-stress agent containing Candida XHZG06-95A on improving the cold resistance of crops

[0048] 1.1 Preparation of stress-resistant agents

[0049] 1.1.1 Raw material formula

[0050] (1) Candida albicans XHZG06-95A3 bacterial solution: OD 600 =1.0, dosage 50 mL.

[0051] (2) Antifreeze protein dispersion: concentration 0.5 mg / mL, dosage 20 mL.

[0052] (3) Excipients: glycerol, added at 5% v / v; trehalose, added at 0.05 g / mL; phosphate buffer (PBS), pH 7.0.

[0053] 1.1.2 Preparation steps of anti-stress agents

[0054] (1) Bacteria culture:

[0055] Candida species XHZG06-95A3 were inoculated into YPD liquid medium (1% yeast extract, 2% peptone, 2% glucose), cultured at 25°C and 180 rpm for 48 h, centrifuged at 5000 × g for 10 min, and the cells were collected and resuspended in PBS buffer to an OD of 600 =1.0, and obtain bacterial suspension.

[0056] (2) Mixing preparation:

[0057] The bacterial suspension (50 mL, OD 600 =1.0), antifreeze protective protein dispersion (20 mL, 0.5 mg / mL), glycerol (5 mL) and trehalose (5 g) were mixed, and the volume was adjusted to 100 mL with PBS buffer (pH = 7.0). The mixture was stirred at 200 rpm at 4°C for 30 min to obtain the anti-stress agent, which was then sterilely packaged.

[0058] 1.2 Testing the effect of improving crop cold tolerance

[0059] 1.2.1 Experimental Design

[0060] Three experimental groups and one control group were set up, and the treatment solutions used in each group were as follows:

[0061] (1) Experimental Group 1: The anti-adversity agent prepared in this example.

[0062] (2) Experimental group 2: Cryoprotectant dilution solution. The preparation method is as follows: Cryoprotectant dispersion (20 mL, 0.5 mg / mL) was diluted to 100 mL with PBS buffer (pH = 7.0) and stirred at 200 rpm for 30 min at 4 °C.

[0063] (3) Experimental group 3: Candida XHZG06-95A3 liquid, prepared as follows: Candida XHZG06-95A3 was inoculated into YPD liquid medium (1% yeast extract, 2% peptone, 2% glucose), cultured at 25°C and 180 rpm for 48 hours, centrifuged at 5000 × g for 10 minutes, and the cells were collected and resuspended in PBS buffer to an OD of 600 = 1.0, and obtain bacterial suspension. Then, the bacterial suspension (50 mL, OD 600 = 1.0) was diluted to 100 mL with PBS buffer (pH = 7.0) and stirred at 200 rpm for 30 min at 4°C.

[0064] (4) Control group: excipient solution, prepared as follows: glycerol (5 mL) and trehalose (5 g) were mixed, the volume was adjusted to 100 mL with PBS buffer (pH = 7.0), and stirred at 200 rpm for 30 min at 4 °C.

[0065] The treatment conditions were as follows: tea leaves were sprayed with each treatment solution until they were completely wetted, subjected to low-temperature stress at -20°C for 24 hours, and the following physiological indicators were measured after returning to normal temperature (25°C): relative conductivity, malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and plant survival rate.

[0066] 1.2.2 Experimental Results

[0067] The results of physiological index tests of each group are shown in Table 1. In Table 1, “▲” indicates that there are significant differences between Group 1 and Group 2 and Group 3 (p < 0.05). The data are the mean ± standard deviation of three repeated experiments.

[0068] Table 1 Test results of improving crop cold tolerance

[0069]

[0070] According to the experimental results in Table 1, we can see that:

[0071] (1) Compared with the control group, the crops in experimental groups 2 and 3 had lower relative conductivity and MDA content after being subjected to low temperature stress, but higher SOD activity and plant survival rate. This indicates that the antifreeze protection protein and Candida albicans XHZG06-95A3 in the present invention can improve the cold tolerance of crops to a certain extent.

[0072] (2) Compared with experimental groups 2 and 3, the crops in experimental group 1 had lower relative conductivity and MDA content after being subjected to low temperature stress, and higher SOD activity and plant survival rate. This indicates that the antifreeze protection protein in the invention and Candida albicans XHZG06-95A3 can produce a synergistic effect, and the combination of the two can further improve the cold tolerance of crops.

[0073] Example 2: Effect of an anti-stress agent containing the supernatant of Candida XHZG06-95A fermentation broth on improving the cold resistance of crops

[0074] 2.1 Preparation of stress-resistant agents

[0075] Candida species XHZG06-95A3 were inoculated into YPD liquid medium (pH = 6.0) supplemented with 5% glycerol and cultured at 30 °C and 180 rpm until OD 600 = 2.0, centrifuged at 8000 × g for 15 min, collected the supernatant, and concentrated to a polysaccharide content of 0.1 g / mL to obtain the fermentation supernatant. The fermentation supernatant and cryoprotectant were mixed at specific mass ratios (3:1, 2:1, 1:1, 1:2, and 1:3 for groups A–E, respectively) and diluted with PBS buffer to prepare a mixture with a solids concentration of 15 wt%. Tween-80 was added to the mixture at a concentration of 0.1 wt%, and homogenized and emulsified at 40°C and 5000 rpm for 20 min to obtain the stress-resistant agents for groups A–E.

[0076] Candida species XHZG06-95A3 were inoculated into YPD liquid medium (pH = 6.0) supplemented with 5% glycerol and cultured at 30 °C and 180 rpm until OD 600 = 2.0, centrifuged at 8000 × g for 15 min, collected the supernatant, and concentrated to a polysaccharide content of 0.1 g / mL to obtain the fermentation broth supernatant. The fermentation broth supernatant was diluted with PBS buffer to a solids concentration of 15 wt %, and then Tween-80 was added at a concentration of 0.1 wt %. Homogenization and emulsification were carried out at 40°C and 5000 rpm for 20 min to obtain the stress-resistant agent of Group F.

[0077] The antifreeze protective protein was mixed with PBS buffer to obtain an antifreeze protective protein dispersion with a solid concentration of 15 wt%, and then Tween-80 was added in an amount of 0.1 wt%. The mixture was homogenized and emulsified at 40°C and 5000 rpm for 20 min to obtain the anti-stress agent of group G.

[0078] 2.2 In vitro antifreeze activity assay

[0079] Each group of stress-resistant agents was diluted to a solids concentration of 1 wt% and placed in a -20°C freezer. The freezing point of the solution was measured using differential scanning calorimetry (DSC), and the freezing point depression (ΔTf) was calculated. The ΔTf values ​​measured for each group are shown in Table 2. The data in Table 2 are the means ± SD of three replicate experiments.

[0080] Table 2 In vitro antifreeze activity test results

[0081]

[0082] 2.3 Testing the Effect of Improving Crop Cold Tolerance: Winter wheat seedlings (three-leaf stage) were sprayed with each stress-resistant agent at a rate of 5 mL per plant. A control group was sprayed with PBS buffer at a rate of 5 mL per plant. After 24 hours of low-temperature stress at -8°C, the plants resumed growth for 7 days, and the following physiological indicators were measured: electrolyte permeability, MDA content, SOD activity, and plant survival rate. The test results are shown in Table 3. Data in Table 3 are the means ± SD of three replicate experiments.

[0083] Table 3 Test results of improving crop cold tolerance

[0084]

[0085] 2.4 Experimental Results Analysis

[0086] According to the experimental results in Table 2 and Table 3, it can be seen that:

[0087] (1) Compared with groups F and G, the freezing point of the solutions in groups A to E increased significantly. Moreover, after the crops were subjected to low temperature stress, the electrolyte permeability and MDA content were lower, while the SOD activity and plant survival rate were higher. This indicates that the fermentation supernatant of Candida sp. XHZG06-95A3 and the antifreeze protection protein can synergistically improve the cold tolerance of crops after the two are combined.

[0088] (2) Compared with Group A and Groups D to F, the freezing point of Groups B and C increased significantly. Moreover, after the crops were subjected to low temperature stress, the electrolyte permeability and MDA content were lower, while the SOD activity and plant survival rate were higher. This indicates that the ratio between the supernatant of the fermentation broth of Candida albicans XHZG06-95A3 and the antifreeze protection protein affects the synergistic effect between the two. When the mass ratio of the two is 1 to 2:1, the cold tolerance of crops can be improved to a greater extent, and the optimal mass ratio is 2:1.

[0089] Example 3: Storage stability of stress-resistant agent containing supernatant of Candida XHZG06-95A fermentation broth

[0090] 2.1 Preparation of stress-resistant agents

[0091] Candida species XHZG06-95A3 were inoculated into YPD liquid medium (pH = 6.0) supplemented with 5% glycerol and cultured at 30 °C and 180 rpm until the OD 600 = 2.0, centrifuged at 8000 × g for 15 minutes, collected the supernatant, and concentrated to a polysaccharide content of 0.1 g / mL to obtain the fermentation broth supernatant. The fermentation broth supernatant and cryoprotectant were mixed in a 2:1 mass ratio and diluted with PBS buffer to prepare a mixture with a solids concentration of 15 wt% in both solutions. Tween-80 was added to the mixture at a concentration of 0.1 wt%, and homogenized and emulsified at 40°C and 5000 rpm for 20 minutes to obtain the stress-resistant agent.

[0092] 2.2 Testing the effect of improving crop cold tolerance

[0093] The stress-resistant agent was divided into two groups, designated Group B1 and Group B2. Group B1 was immediately tested for its effect on improving crop cold tolerance, while Group B2 was stored at 4°C for 30 days and then tested again for its effect on improving crop cold tolerance. The testing method was as follows: winter wheat seedlings (three-leaf stage) were sprayed with each stress-resistant agent at a rate of 5 mL per plant; the control group was sprayed with PBS buffer at a rate of 5 mL per plant. After 24 hours of low-temperature stress at -8°C, growth was resumed for 7 days, and the following physiological indicators were measured: electrolyte permeability, MDA content, SOD activity, and plant survival rate. The test results are shown in Table 4. Data in Table 4 are the mean ± SD of three replicate experiments.

[0094] Table 4 Test results of the effect of improving crop cold resistance

[0095]

[0096] The experimental results in Table 4 show that after exposure to low temperature stress, the electrolyte permeability, MDA content, SOD activity, and plant survival rate of Group B2 were close to those of Group B1. This indicates that the stress-resistant agent of the present invention has good storage stability and can maintain its ability to improve crop cold tolerance even after long-term storage.

[0097] Example 4: Effect of the selection of Candida and antifreeze protection protein on the effect of stress resistance

[0098] 4.1 Preparation of stress-resistant agents

[0099] 4.1.1 Experimental Materials

[0100] In this example, Candida utilis ACCC 20060 was used as a comparison. This strain was purchased from the China Agricultural Microbiological Culture Collection Center (abbreviated as "ACCC 20060").

[0101] 4.1.2 Preparation steps of anti-stress agents

[0102] Candida species XHZG06-95A3 were inoculated into YPD liquid medium (pH = 6.0) supplemented with 5% glycerol and cultured at 30 °C and 180 rpm until the OD 600 = 2.0, centrifuged at 8000 × g for 15 minutes, collected the supernatant, and concentrated to a polysaccharide content of 0.1 g / mL to obtain the fermentation broth supernatant. The fermentation broth supernatant and antifreeze protectant were mixed in a 2:1 mass ratio and diluted with PBS buffer to prepare a mixture with a solids concentration of 15 wt%. Tween-80 was added to the mixture at a concentration of 0.1 wt%, and homogenized and emulsified at 40°C and 5000 rpm for 20 minutes to obtain the stress-resistant agent in Group B.

[0103] Candida species XHZG06-95A3 were inoculated into YPD liquid medium (pH = 6.0) supplemented with 5% glycerol and cultured at 30 °C and 180 rpm until the OD 600 = 2.0, centrifuged at 8000 × g for 15 min, collected the supernatant, and concentrated to a polysaccharide content of 0.1 g / mL to obtain the fermentation broth supernatant. The fermentation broth supernatant was diluted with PBS buffer to a solids concentration of 15 wt %, and then Tween-80 was added at a concentration of 0.1 wt %. Homogenization and emulsification were carried out at 40°C and 5000 rpm for 20 min to obtain the stress-resistant agent of Group F.

[0104] The antifreeze protective protein was mixed with PBS buffer to obtain an antifreeze protective protein dispersion with a solid concentration of 15 wt%, and then Tween-80 was added in an amount of 0.1 wt%. The mixture was homogenized and emulsified at 40°C and 5000 rpm for 20 min to obtain the anti-stress agent of group G.

[0105] Candida utilis ACCC 20060 was inoculated into YPD liquid medium (pH = 6.0) supplemented with 5% glycerol and fermented at 30°C and 180 rpm until OD 600 = 2.0, centrifuged at 8000 × g for 15 minutes, collected the supernatant, and concentrated to a polysaccharide content of 0.1 g / mL to obtain the fermentation broth supernatant. The fermentation broth supernatant and antifreeze protectant were mixed in a 2:1 mass ratio and diluted with PBS buffer to prepare a mixture with a solids concentration of 15 wt%. Tween-80 was added to the mixture at a concentration of 0.1 wt%, and homogenized and emulsified at 40°C and 5000 rpm for 20 minutes to obtain the stress-resistant agent of Group H.

[0106] Candida utilis ACCC 20060 was inoculated into YPD liquid medium (pH = 6.0) supplemented with 5% glycerol and fermented at 30°C and 180 rpm until OD 600 = 2.0, centrifuged at 8000 × g for 15 minutes, collected the supernatant, and concentrated to a polysaccharide content of 0.1 g / mL to obtain the fermentation supernatant. The fermentation supernatant was diluted with PBS buffer to a solids concentration of 15 wt%, and then Tween-80 was added at a concentration of 0.1 wt%. Homogenization and emulsification were carried out at 40°C and 5000 rpm for 20 minutes to obtain the stress-resistant agent of Group I.

[0107] 4.2 Testing the effect of improving crop cold tolerance

[0108] Winter wheat seedlings (three-leaf stage) were sprayed with each stress-resistant agent at a rate of 5 mL per plant. A control group was sprayed with PBS buffer at a rate of 5 mL per plant. After 24 hours of low-temperature stress at -8°C, the plants resumed growth for 7 days, and the following physiological indicators were measured: electrolyte permeability, MDA content, SOD activity, and plant survival rate. The test results are shown in Table 5. Data in Table 5 are means ± SD of three replicate experiments.

[0109] Table 5 Test results of the effect of improving crop cold resistance

[0110]

[0111] The experimental results in Table 5 show that after exposure to low-temperature stress, the electrolyte permeability, MDA content, SOD activity, and plant survival rate of Group I were similar to those of Group F. In contrast, the electrolyte permeability and MDA content of Group H were significantly higher than those of Group B, while the SOD activity and plant survival rate were significantly lower than those of Group B. This indicates that the synergistic effect between Candida and antifreeze protection proteins is strain-specific, and the selection of Candida strains can influence the realization of this synergistic effect. Not all Candida strains combined with antifreeze protection proteins can achieve the synergistic effect in improving crop cold tolerance achieved in the present invention. Based on this, it is speculated that the synergistic effect between Candida strain XHZG06-95A3 and antifreeze protection proteins in the present invention may be due to the secretion of specific glycoproteins by this strain, which can form stable complexes with antifreeze protection proteins of a specific structure. Furthermore, this strain may produce inhibitory metabolites (such as certain lipids) that weaken the active conformation of antifreeze proteins.

[0112] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this disclosure pertains. Unless otherwise specified, the raw materials and equipment used herein are conventional in the art and can be obtained from conventional commercial sources. The methods used herein are conventional in the art, unless otherwise specified.

[0113] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. Use of Candida species XHZG06-95A3 and / or its fermentation supernatant in enhancing the effect of antifreeze protective proteins, characterized in that: The antifreeze protection protein has the effect of improving the cold resistance of crops; the Candida species XHZG06-95A3 belongs to Candida palmioleophila , the deposit number is CGMCC No.29215; the amino acid sequence of the antifreeze protection protein is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that The Candida XHZG06-95A3 and / or its fermentation liquid supernatant and the antifreeze protection protein are prepared into an anti-stress agent.

3. The use according to claim 2, characterized in that The stress-resistant agent further comprises one or more of glycerol, trehalose and phosphate buffer.

4. The use according to claim 2, characterized in that The mass ratio between the fermentation broth supernatant and the antifreeze protective protein is 1-2:1; the fermentation broth supernatant is formed by fermenting Candida albicans XHZG06-95A3 in a yeast extract peptone glucose liquid medium supplemented with glycerol, and has a polysaccharide content of 0.10-0.15 g / mL.

5. The use according to claim 2, characterized in that The method for preparing the stress-resistant agent comprises the following steps: mixing Candida XHZG06-95A3 and / or its fermentation liquid supernatant with antifreeze protection protein to prepare the stress-resistant agent.

6. The use according to claim 5, characterized in that The preparation method of the anti-adverse agent comprises the following steps: 600 =0.5~1.5 of Candida XHZG06-95A3 bacterial suspension, antifreeze protection protein and dispersion medium are mixed to obtain an anti-stress agent; the volume ratio of the Candida XHZG06-95A3 bacterial suspension and the anti-stress agent is 1:1.5~2.5; the mass volume ratio of the antifreeze protection protein and Candida XHZG06-95A3 bacterial suspension is 0.08~0.15 g:50 mL.

7. The use according to claim 5, characterized in that The preparation method of the stress-resistant agent comprises the following steps: mixing the fermentation supernatant of Candida XHZG06-95A3 and antifreeze protection protein, adding a dispersion medium until the solid content is 10-15 wt%, and then adding an emulsifier for homogenization and emulsification to obtain the stress-resistant agent.

8. The use according to claim 5 or 7, characterized in that The preparation steps of the fermentation broth supernatant include: inoculating Candida species XHZG06-95A3 into a yeast extract peptone glucose liquid medium supplemented with glycerol, and fermenting and culturing the mixture until the OD 600 =1.8~2.2, collect the supernatant by centrifugation, and concentrate to a polysaccharide content of 0.10~0.15 g / mL.

9. The use according to claim 1, characterized in that The method for improving the cold tolerance of crops comprises reducing intracellular electrolyte exudation, reducing the degree of cell membrane lipid peroxidation, increasing superoxide dismutase activity and improving crop survival rate under low temperature stress; the crops are tea or wheat.

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