Cold-resistant resistance inducer for fruit trees as well as preparation method and application of cold-resistant resistance inducer

Abstract

The invention belongs to the technical field of fruit tree planting, and relates to a novel fruit tree cold-resistant resistance inducer as well as a preparation method and application thereof. According to the resistance inducer, a bacillus subtilis BS-Z15 fermentation extract (abbreviated as B-MES) is used as a main component and is matched with calcium, zinc, iron, boron, trehalose, abscisic acid and the like with different concentrations to form the special resistance inducer for different fruit trees or other plants. The resistance inducer provided by the invention can significantly improve the physiological function and stress resistance of fruit trees in a cold environment, reduce low-temperature damage, and effectively solve the problem of sudden cold damage possibly encountered by the fruit trees in early spring low temperature and late spring cold and other growth stages. The cold-resistant agent has an excellent cold-resistant effect in fruit trees such as apples, walnuts and apricots and crops such as cotton seedlings, can effectively improve the cold resistance of plants, is wide in application range and has important significance on cold resistance of the crops such as the fruit trees. Meanwhile, the cold-resistant resistance inducer is simple in preparation method, convenient to use and suitable for large-scale popularization.

Description

Technical Field

[0001] The present invention relates to the technical field of fruit tree cultivation, and particularly relates to a novel cold-resistant elicitor for fruit trees. The elicitor takes B-MES as the main component, and is formulated with calcium, zinc, iron, boron, trehalose, abscisic acid, etc. at different concentrations to form specific elicitors for different varieties of fruit trees or other plants, so as to improve their cold resistance, and also provides preparation and application methods of the elicitor. Background Art

[0002] Fruit trees are vulnerable to low-temperature stress in cold environments, especially during the early spring budding period and the period of cold snap in spring, which will not only lead to problems such as poor growth and reduced yield. Traditional cold-resistant measures mostly rely on physical warming such as smoking. It has also been found in research that chemical drugs such as trehalose and abscisic acid can enhance the cold resistance of plants. However, at present, no specific highly efficient cold damage elicitor for different fruit trees such as apples, walnuts, and apricots has been developed. Therefore, it is of great significance to develop specific, safe, and highly efficient cold-resistant elicitors for different fruit trees. Summary of the Invention

[0003] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide a cold-resistant elicitor. The elicitor takes B-MES as the main component, and is formulated with calcium, zinc, iron, boron, trehalose, abscisic acid, etc. at different concentrations to form specific elicitors for different varieties of fruit trees or other plants, significantly enhancing their physiological functions and stress resistance in cold environments, and reducing low-temperature damage (as Figure 1 shown).

[0004] The present invention also provides a preparation method of the above cold-resistant elicitor.

[0005] Another object of the present invention is to provide the application of the above cold-resistant elicitor in improving cold resistance in fruit trees or other crops.

[0006] In order to achieve the above objects, the present invention provides the following technical solutions:

[0007] (1) Main components:

[0008] B-MES: The concentration is 4.0 - 20.0 mg / L, specifically the fermentation product with cold-resistant activity produced by Bacillus subtilis under specific culture conditions. This substance can rapidly activate the plant's low-temperature and other stress defense signal pathways through root and leaf absorption, accumulate endogenous stress hormones such as ABA and JA, enhance the activities of multiple ethylene synthase enzymes, and can significantly increase the content of osmotic water-retaining substances such as betaine and proline, increase free radical scavenging enzymes such as SOD, POD, and CAT, reduce the damage of membrane lipid peroxidation, and synthesize cold-resistant substances such as lignin and callose, so as to induce and improve the ability of plants to resist cold stress. It has the advantages of fast regulation speed, remarkable effect, safety and non-toxicity

[0009] Calcium sources: such as CaCl2, CaCl2·2H2O, Ca(NO3)2 or calcium sugar alcohol chelate, etc., with a concentration of 0 - 6.0 g / L

[0010] Zinc sources: such as ZnSO4, ZnSO4·7H2O, zinc chelate, etc., with a concentration of 0 - 2.5 g / L

[0011] Iron sources: such as FeSO4, FeSO4·7H2O, iron chelate, etc., with a concentration of 0 - 1.5 g / L

[0012] Boron sources: such as Na2B4O7, Na2B4O7·10H2O (borax), H3BO3, etc., with a concentration of 0 - 6.0 g / L

[0013] Trehalose: with a concentration of 0 - 6.0 g / L

[0014] Abscisic acid (ABA): with a concentration of 0 - 1.0 mg / L

[0015] In the preferred example, for the type-specific cold-resistant elicitor for "Yan 3" apple trees and "Xinxin 2" walnut trees, the concentration ratio of each raw material is: B-MES 14.0 ± 0.5 mg / L, trehalose 4.0 ± 0.4 g / L, CaCl2·2H2O 5.0 ± 0.5 g / L, ZnSO4 2.0 ± 0.2 g / L, Na2B4O7·10H2O 5.0 ± 0.5 g / L, FeSO4·7H2O 1.0 ± 0.1 g / L, and the solvent is water (the above ratio is simply referred to as the E6 formula).

[0016] In the preferred example, for the type-specific cold-resistant elicitor for apricot trees, the concentration ratio of each raw material is: B-MES 14.0 ± 2.0 mg / L, and the solvent is water.

[0017] In the preferred example, the cold-resistant elicitor provided by the present invention can also be used for other crops besides fruit trees.

[0018] In the preferred example, taking cotton seedlings as an example, the concentration ratio of raw materials in its type-specific cold-resistant elicitor is: B-MES 7.0 ± 1.0 mg / L, and the solvent is water; for the type-specific cold-resistant compound for cotton seedlings, the concentration ratio of each raw material is: B-MES 8.0 ± 2.0 mg / L, ZnSO4 1.0 ± 0.1 mg / L, ABA 0.5 ± 0.1 mg / L, and the solvent is water.

[0019] In the preferred example, the other crops include but are not limited to crops such as cotton and wheat.

[0020] (2) Preparation method:

[0021] 1. Weigh B-MES, calcium source, zinc source, iron source, boron source, trehalose, and ABA according to the established ratio.

[0022] 2. Extraction method of B-MES, specifically including the following steps:

[0023] ① Loop pick a single colony of Bacillus subtilis and inoculate it into a conical flask containing 100 ml of beef extract peptone medium, and incubate it at a constant temperature of 37 °C and 180 rpm for 16 h to obtain a seed solution;

[0024] ② Take the seed solution obtained in step ①, inoculate it into a conical flask containing beef extract peptone medium at a volume ratio of 1:100, and incubate it at a constant temperature of 37 °C and 180 rpm for 24 h to obtain a fermentation broth. Subsequently, use high-speed centrifugation (10000 rpm, 15 min) to remove the bacteria, and collect the sterile fermentation broth;

[0025] ③ Use concentrated H2SO4 (or concentrated HCl) to adjust the pH of the sterile fermentation broth obtained in step ② to 1.0 - 2.0, and let it stand at 2 - 5 °C for 16 h. Subsequently, obtain the precipitate again by high-speed centrifugation (10000 rpm, 30 min), wash it out with as little sterile water as possible, and adjust the pH to 7 - 7.5 with NaOH to obtain a paste, which is B-MES;

[0026] ④ Freeze-dry the paste-like B-MES obtained in step ③ to obtain B-MES powder.

[0027] 3. Dissolve the above-mentioned B-MES, mineral elements (such as calcium, zinc, iron, boron, etc.), trehalose, abscisic acid, etc. weighed according to the established ratio in water and mix them evenly to obtain a cold-resistant inducing agent specific for fruit trees.

[0028] (3) Application method:

[0029] The application time of the inducing agent is 1 - 7 days before the sudden cold damage in the early spring cold snap and other growth stages (such as before autumn leaf fall, spring bud germination period, etc.). The application frequency is 1 - 3 times, and the application interval is 2 - 3 days. After preparing the inducing agent according to the established ratio, apply it to the fruit trees by foliar spraying or root irrigation. The degree of foliar spraying should be such that the leaf surface is evenly covered with mist-like water droplets, so that a layer of mist-like water droplets is evenly covered on the leaf surface; the irrigation should be preferably 40 - 45 L / acre of the medicament dissolved in the irrigation water to ensure that the medicament can be effectively absorbed by the roots.

[0030] The cold-resistant inducing agent provided by the present invention can not only be used to improve the cold resistance of fruit trees, but also has a significant inducing effect on crops such as cotton seedlings to resist cold damage.

[0031] The B-MES provided in the present invention is a fermentation product of Bacillus subtilis. This substance can rapidly activate the plant's stress defense signaling pathways against low temperature and other stresses through root and leaf absorption, accumulate endogenous stress hormones such as ABA and JA, enhance the activities of multiple ethylene synthase enzymes, and can significantly increase the contents of osmotic water-retaining substances such as betaine and proline, increase free radical scavenging enzymes such as SOD, POD, and CAT, reduce the damage of membrane lipid peroxidation, and synthesize cold-resistant substances such as lignin and callose, so as to induce and improve the plant's ability to resist cold stress. It has the advantages of fast regulation speed, remarkable effect, safety and non-toxicity. It can not only be used to improve the cold resistance of fruit trees, but also has a significant induction effect on crops such as cotton seedlings against cold damage. The present invention has formulated corresponding special cold-resistant inducer antagonists for different varieties of fruit trees and successfully developed special inducer antagonists suitable for fruit trees such as apples, walnuts, and apricots. At the same time, we also found that this invention has a good effect on cotton seedlings. Experiments show that the fruit tree cold-resistant inducer antagonist provided by the present invention and its preparation and application methods can effectively improve the cold resistance of plants, which is of great significance.

[0032] The beneficial effects of the present invention are as follows:

[0033] The fruit tree cold-resistant inducer antagonist provided by the present invention takes the fermentation extract B-MES of Bacillus subtilis BS-Z15 as the main component, and is formulated with calcium, zinc, iron, boron, trehalose, abscisic acid, etc. at different concentrations to form special inducer antagonists for different varieties of fruit trees or other plants. This inducer antagonist can significantly improve the physiological functions and stress resistance of fruit trees in cold environments, reduce low-temperature damage, and effectively solve the sudden cold damage problems that fruit trees may encounter during the late spring cold snap in early spring and other growth stages.

[0034] The inducer antagonist provided by the present invention has excellent cold resistance effects in fruit trees such as apples, walnuts, and apricots, and crops such as cotton seedlings, can effectively improve the cold resistance of plants, has a wide range of applications, and is of great significance for the cold resistance of fruit trees and crops.

[0035] The preparation method of the fruit tree cold-resistant inducer antagonist provided by the present invention is simple and easy to use, and is suitable for large-scale promotion. Description of the Drawings

[0036] Figure 1 It is a schematic diagram of the inducer antagonist formula, usage method and function;

[0037] Figure 2 For screening the special cold-resistant inducer antagonist of "Yan 3" apple trees with different ratios, Figure 2 Among them, A-B are the morphologies of each treatment group 3 days and 5 days after recovery from cold stress; C: the malondialdehyde content of each treatment group after cold stress (n = 3, * P<0.05, ** P<0.01,*** P < 0.001 vs the post - cold - stress treatment group 1); D: chilling injury index of each treatment group on the 3rd and 5th days after recovery from cold stress (n = 4, * P < 0.05, ** P < 0.01, *** P < 0.001 vs the treatment group 1 on the 3rd day after recovery from cold stress; n = 4, # P < 0.05, ## P < 0.01, ### P < 0.001 vs the treatment group 1 on the 5th day after recovery from cold stress);

[0038] Figure 3 For the optimization of the apple tree - specific cold - resistance inducing agent “Yan 3”, A - D are the morphologies of each treatment group before cold stress, just after the end of stress, 3 days after stress recovery, and 5 days after stress recovery respectively; E is the relative conductivity value measured after cold stress for each treatment group (n = 4, * P < 0.05, ** P < 0.01, *** P < 0.001 vs the post - cold - stress treatment group 1); F is: chilling injury index of each treatment group on the 3rd and 5th days after recovery from cold stress (n = 4, * P < 0.05, ** P < 0.01, *** P < 0.001 vs the treatment group 1 on the 3rd day after recovery from cold stress; n = 4, # P < 0.05, ## P < 0.01, ### P < 0.001 vs the treatment group 1 on the 5th day after recovery from cold stress);

[0039] Figure 4 For the field plant verification of the apple tree - specific cold - resistance inducing agent “Yan 3”, A is the morphology of each treatment group before cold stress and 3 days after stress recovery; B is the relative chlorophyll content of “Yan 3” apple leaves after cold stress; C is the relative conductivity of “Yan 3” apple leaves after cold stress (n = 4, * P < 0.05, ** P < 0.01, *** P < 0.001 vs the Control group before cold stress);

[0040] Figure 5 For the R & D of the walnut tree - specific inducing agent “Xinxin 2”, A is the morphology of each treatment group 1 day and 3 days after recovery from cold stress; B is the relative chlorophyll content of “Xinxin 2” walnut leaves after cold stress; C is the relative conductivity of “Xinxin 2” walnut leaves after cold stress (n = 5, * P < 0.05, ** P < 0.01, *** P < 0.001 vs the Control group);

[0041] Figure 6 For the research and development of a special type of elicitor for apricot trees, A - D are the morphologies of each treatment group before cold stress, just after the stress ends, 3 days after the stress recovery, and 5 days after the stress recovery, respectively; E is: the chilling injury index of each treatment group 3 days and 5 days after the cold stress recovery (n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001 vs the treatment group 1 3 days after the cold stress recovery; n = 3, # P < 0.05, ## P < 0.01, ### P < 0.001 vs the treatment group 1 5 days after the cold stress recovery);

[0042] Figure 7 For the role of B - MES elicitor in the cold resistance of cotton seedlings, A is the morphology of each treatment group 0 - 12 h after 48 h of cold stress recovery; B is the chilling injury index 1 day after the cold stress recovery; C is the plant height and main root length 3 days and 7 days after the cold stress recovery; D is the dry weight and fresh weight of the plants 3 days and 7 days after the cold stress recovery; E - G are the chlorophyll a, chlorophyll b, and total chlorophyll contents of each treatment group after the cold stress (different letters indicate significant differences between the means, P < 0.05);

[0043] Figure 8 For the research and development of a special type of elicitor for cotton seedlings, A is the spraying elicitor compound ratio of each treatment group; B is the morphology of each treatment group 0 - 12 after the cold stress recovery; C is the chilling injury index of each treatment group; D is the malondialdehyde content in the leaves of each treatment group after the cold stress ends. Specific embodiments

[0044] The technical solutions of the present invention will be further explained and illustrated below through specific examples.

[0045] The raw materials used in the present invention are all commercially available except for B - MES which is self - developed and extracted.

[0046] In some embodiments, the preparation method of B - MES is as follows:

[0047] (1) Inoculate Bacillus subtilis with the preservation number of CCTCC No: M2017309 (which has been disclosed in Chinese patent application CN201710658550.6) into the beef extract peptone medium, and perform constant - temperature shaking culture at 37°C and 180 rpm for 16 h to obtain the seed liquid;

[0048] (2) Inoculate the seed solution into a conical flask containing beef extract peptone medium at a volume ratio of 1:100, and perform constant temperature shaking culture at 37 °C and 180 rpm for 24 h to obtain a fermentation broth. Subsequently, use high-speed centrifugation (10,000 rpm, 15 min) to remove the bacteria, and collect the sterile fermentation broth;

[0049] (3) Use concentrated H2SO4 (or concentrated HCl) to adjust the pH of the sterile fermentation broth obtained in step (2) to 1.0 - 2.0, and let it stand at 2 - 5 °C for 16 h. Subsequently, obtain a precipitate again by high-speed centrifugation (10,000 rpm, 30 min), wash it out with as little sterile water as possible, and adjust the pH to 7 - 7.5 with NaOH to obtain a paste, which is B-MES;

[0050] (4) Freeze-dry the paste-like B-MES obtained in step (3) to obtain B-MES powder.

[0051] The evaluation and grading of the apparent chilling injury index of the plants (0 - 7) are shown in Table 1.

[0052] Table 1

[0053] 0 The branches and leaves are basically undamaged, without obvious changes, and without wilting l The leaves are basically undamaged, only the top part wilts or the leaves of 1 / 4 of the branches wilt 3 The leaves of 1 / 4 - 1 / 2 of the branches wilt, and the leaves are partially brown and have brownish spots 5 The leaves of 1 / 2 - 3 / 4 of the branches wilt, and the leaves have brownish spots 7 All the branches and leaves wilt and die

[0054] Example 1 Screening and Optimization of a Special Cold Resistance Inducer for "Yan 3" Apple Trees

[0055] In the laboratory, screen and optimize the special cold resistance inducer for "Yan 3" apple trees through in vitro shoot drug treatment and artificial simulation of low temperature stress. The in vitro shoots are cultured for preservation in 1 / 5 MS liquid medium (artificial climate chamber: 25 °C, 60% light for 14 h + no light for 10 h, humidity 50%). One day before cold stress, the in vitro shoots are treated by spraying once on the leaf surface. Artificial simulation of low temperature stress (artificial climate chamber: 0 °C, 60% light for 14 h + no light for 10 h, humidity 50%), and record and measure relevant indicators.

[0056] First, screen the special cold resistance inducer for "Yan 3" apple trees with different ratios, and set the drug concentrations of each treatment group (as shown in Table 2). The results are as Figure 2 shown. In the treatment group with a drug concentration ratio of B-MES 12.0 - 16.0 mg / L, trehalose 4.0 - 5.0 g / L, CaCl2·2H2O 5.0 g / L, ZnSO4 2.0 g / L, Na2B4O7·10H2O 5.0 g / L, and FeSO4·7H2O 1.0 g / L, the malondialdehyde content and the apparent index are better than those of other treatment groups. There are no obvious wilting signs on the leaf surface, and no freezing injury symptoms such as black spots appear, and the whole remains intact.

[0057] Table 2

[0058]

[0059] Secondly, according to the screening results, optimize the cold-resistant inducer specific to "Yan 3" apple trees, and set the drug concentrations of each treatment group (as shown in Table 3). The results are as Figure 3 shown. The treatment group with the drug concentration ratio of B-MES 14.0 mg / L, trehalose 4.0 g / L, CaCl2·2H2O 5.0 g / L, ZnSO4 2.0 g / L, Na2B4O7·10H2O 5.0 g / L, and FeSO4·7H2O 1.0 g / L has the best effect. Compared with treatment group 1 (the blank group), its relative conductivity is reduced by about 60%. There are no obvious signs of wilting on the leaf surface. The leaves remain fresh green and upright, without black spots or other morbid symptoms caused by freezing injury, and the overall remains in a healthy state without damage, showing strong cold resistance and effectively ensuring the healthy growth of fruit trees in low-temperature environments.

[0060] Table 3

[0061]

[0062] Example 2 Verification of the Cold-Resistant Inducer Specific to "Yan 3" Apple Trees in Field Plants

[0063] Conduct experiments in the apple plantations of the Xinjiang Academy of Forestry Sciences and other places. Verify the effect of the cold-resistant inducer specific to "Yan 3" apple trees by spraying drugs on branches and artificially simulating low-temperature stress on detached branches. The artificial simulation of low-temperature stress, drug spraying methods, etc. refer to Example 1, and relevant physiological indexes are measured. Set the following 5 treatment groups:

[0064] ① Control group: water;

[0065] ② E6 group: 14.0 mg / L B-MES + 4.0 g / L trehalose + 5.0 g / L CaCl2·2H2O + 2.0 g / L ZnSO4 + 5.0 g / L Na2B4O7·10H2O + 1.0 g / L FeSO4·7H2O;

[0066] ③ ZnSO4 group: 2.0 g / L ZnSO4;

[0067] ④ B-MES: 14.0 mg / L B-MES;

[0068] ⑤ B-MES + ZnSO4 group: 14.0 mg / L B-MES + 2 g / L ZnSO4;

[0069] The experimental results are as Figure 4As shown, there was no significant change in the chlorophyll content of each treatment group. Compared with the Control group, the relative conductivity of each drug treatment group decreased, and the E6 treatment group was the most significant, indicating that the integrity of the leaf cell membrane was effectively maintained. At the same time, from the apparent morphology, there were no obvious wilting signs on the leaf surface of the E6 treatment group. The leaves remained fresh green and upright, and the overall remained in a healthy state without damage, showing significant cold resistance, verifying again the preventive effect of the specific cold-resistant inducer on low-temperature stress damage, and effectively ensuring the healthy growth of "Yan 3" apple trees in the severe cold environment.

[0070] Example 3 Development of a Specific Cold-Resistant Inducer for "Xinxin 2" Walnut Trees

[0071] Experiments were carried out in the Xinjiang Academy of Forestry Sciences and other places. Based on the specific cold-resistant inducer for "Yan 3" apple trees in Examples 1-2, a specific cold-resistant inducer for "Xinxin 2" walnut trees was developed by spraying drugs on detached branches, artificially simulating low-temperature stress. The artificial simulation of low-temperature stress, drug spraying methods, etc. were referred to Example 1, and relevant physiological indexes were measured. The following 5 treatment groups were set:

[0072] ① Control group: water;

[0073] ② E6 group: 14.0 mg / L B-MES + 4.0 g / L trehalose + 5.0 g / L CaCl2·2H2O + 2.0 g / L ZnSO4 + 5.0 g / L Na2B4O7·10H2O + 1.0 g / L FeSO4·7H2O;

[0074] ③ CaCl2·2H2O group: 5.0 g / L CaCl2·2H2O;

[0075] ④ B-MES: 14.0 mg / L B-MES;

[0076] ⑤ B-MES + CaCl2·2H2O group: 14.0 mg / L B-MES + 5.0 g / L CaCl2·2H2O;

[0077] The experimental results are as Figure 5As shown, compared with the Control group, the chlorophyll content and relative conductivity of each drug treatment group decreased. However, the decrease in chlorophyll content in the E6 treatment group was not significant, indicating that it had basically no effect on the photosynthetic system. The relative conductivity in the E6 treatment group decreased most significantly, suggesting that the integrity of the leaf cell membrane was effectively maintained. From the observation of the apparent morphology of the leaves, although there were slight signs of wilting in the E6 treatment group, compared with other treatment groups, these signs were not obvious. Overall, the leaves in the E6 treatment group maintained a relatively healthy state, showing good cold resistance, thus effectively ensuring the healthy growth of the "Xinxin 2" walnut tree in a low-temperature environment. The drug concentration ratio in the E6 treatment group can be used as a special cold-resistant inducer for the "Xinxin 2" walnut tree.

[0078] Research and Development of Special Cold-Resistant Inducer for Apricot Trees in Example 3

[0079] Based on the special cold-resistant inducers for the "Yan 3" apple trees in Examples 1 - 2, a special cold-resistant inducer for the "Xinxin 2" walnut tree was developed by spraying drugs on detached branches, artificially simulating low-temperature stress. The artificial simulation of low-temperature stress, drug spraying methods, etc. refer to Example 1, and relevant indicators were measured and recorded. The drug concentrations of each treatment group were set (as shown in Table 4). The results are as Figure 6 shown. The treatment group with a drug concentration ratio of B-MES 14.0 ± 2.0 mg / L had the best effect. Compared with treatment group 1 (blank group), there were no obvious signs of wilting on the leaf surface. The leaves remained fresh green and upright, without black spots or other pathological symptoms caused by freezing injury, and overall remained in a healthy and intact state, showing strong cold resistance and effectively ensuring the healthy growth of apricot trees in a low-temperature environment. It can be used as a special cold-resistant inducer for apricot trees.

[0080] Table 4

[0081]

[0082] Effect Verification of B-MES in Plant Cold Resistance in Example 4

[0083] Using cotton seedlings as experimental materials, treatment groups with B-MES concentrations of 0, 2, 4, 6, 8, and 10 mg / L were set. Potted cotton seedlings were treated by spraying once a day on the leaf surface for 3 days before cold stress. Artificial low-temperature stress was simulated (artificial climate chamber: 0°C, 60% light for 14 h + no light for 10 h, humidity 50%). Relevant indicators were recorded and sampled for measurement (such as Figure 7As shown. The results showed that the application of 6.0 mg / L and 8.0 mg / L B-MES at the cotyledon stage of cotton seedlings significantly improved their cold tolerance. Compared with the blank group, after foliar spraying of 6.0 mg / L and 8.0 mg / L B-MES, the chlorophyll content of cotton seedlings increased significantly, thereby improving the photosynthesis ability, and effectively increasing the dry weight, fresh weight, main root length and plant height of the plants. In addition, the B-MES treatment also significantly reduced the chilling injury index, improved the plant nutritional status, and enhanced the adaptability of the roots to adverse conditions. These changes together promoted the photosynthesis and spatial competition ability of cotton seedlings. Thus, it was shown that B-MES had a significant cold resistance effect. More importantly, these results also showed that B-MES had the potential to increase crop yields, providing a useful reference for agricultural production.

[0084] Example 5 Development of a Specific Cold Resistance Inducer for Cotton Seedlings

[0085] Using cotton seedlings as experimental materials, 10 treatment groups were set up, and the drug ratios of each group were as Figure 8 shown in A, and the experiment was carried out with reference to the treatment method of Example 4. The results were as Figure 8 shown in B - D. Each drug treatment group showed stronger cold tolerance than the blank group, and there was no significant difference in the chilling injury index among different drug treatment groups; it was worth noting that in the drug combination treatment group of B-MES 8.0 mg / L, ZnSO4 1.0 mg / L, and ABA 0.5 mg / L, the malondialdehyde content of the plants was the lowest, indicating that this specific drug ratio had the best effect on improving the cell membrane stability of the plants. In addition, there were no obvious wilting signs on the leaf surface of this treatment group, and the leaves remained fresh green and straight, further confirming the effectiveness of this drug ratio in enhancing the cold resistance of cotton seedlings. Therefore, this drug ratio had the potential to be applied as a specific cold resistance inducer for cotton seedlings and could provide effective low-temperature protection for cotton seedlings.

[0086] The above is only the preferred embodiment of the present invention. The cold resistance inducer in the present invention uses B-MES for the first time and is improved and optimized based on the existing inducer formula. In the present invention, apples, walnuts, apricots, and cotton seedlings are used as examples to illustrate its effects. In addition, the present invention can also be extended to be used in fruit trees such as almonds, apricot plums, jujubes, and other crops such as cotton and wheat. Without departing from the principle of the present invention, several improvements and retouches can be made, and these improvements and retouches should also be regarded as the protection scope of the present invention.

Claims

1. A cold resistance inducer, characterized in that The main components are Bacillus subtilis fermentation extract B-MES and calcium source, zinc source, iron source, boron source, trehalose and abscisic acid; wherein the concentration of B-MES is 4.0-20.0 mg / L, the concentration of calcium source is 0-6.0 g / L, the concentration of zinc source is 0-2.5 g / L, the concentration of iron source is 0-1.5 g / L, the concentration of boron source is 0-6.0 g / L, the concentration of trehalose is 0-6.0 g / L, the concentration of abscisic acid is 0-1.0 mg / L, and the solvent is water.

2. The cold resistance inducer according to claim 1, characterized in that The calcium source is CaCl2, CaCl2·2H2O, Ca(NO3)2 or sugar alcohol chelated calcium; the zinc source is ZnSO4, ZnSO4·7H2O, chelated zinc; the iron source is FeSO4, FeSO4·7H2O, chelated iron; the boron source is Na2B4O7, Na2B4O7·10H2O, H3BO3.

3. A cold resistance inducer for apple trees or walnut trees, characterized in that: The concentration ratio of each raw material is: B-MES 14.0±0.5 mg / L, trehalose 4.0±0.4 g / L, CaCl2·2H2O 5.0±0.5 g / L, ZnSO4 2.0±0.2 g / L, Na2B4O7·10H2O 5.0±0.5 g / L, FeSO4·7H2O 1.0±0.1 g / L, and the solvent is water.

4. A cold resistance inducer for apricot trees, characterized in that: The concentration ratio of each raw material is: B-MES 14.0±2.0 mg / L, and the solvent is water.

5. A special cold resistance inducer for crop seedlings, characterized in that: The concentration ratio of each raw material is: B-MES 6.0±2.0mg / L, ZnSO4 0~1.5mg / L, abscisic acid 0~1.5mg / L, and the solvent is water.

6. The cold resistance inducer according to any one of claims 1 to 5, characterized in that The B-MES is a fermentation product of Bacillus subtilis; the preservation number of the Bacillus subtilis is CCTCC No: M2017309.

7. The cold resistance inducer according to any one of claims 1 to 6, characterized in that The B-MES extraction method specifically comprises the following steps: (1) inoculating Bacillus subtilis with a deposit number of CCTCC No: M2017309 into a culture medium, and culturing at a constant temperature with shaking to obtain a seed solution; (2) taking the seed solution obtained in step (1), inoculating it into a culture medium, and culturing it at a constant temperature with shaking to obtain a fermentation solution; Subsequently, high-speed centrifugation was performed to collect the sterile fermentation broth; (3) adjusting the pH of the sterile fermentation liquid obtained in step (2) with concentrated acid, allowing it to stand, and then again subjecting it to high-speed centrifugation to obtain a precipitate, washing it with sterile water, and adjusting the pH with NaOH to obtain a paste, namely B-MES; (4) The paste B-MES obtained in step (3) is freeze-dried to obtain B-MES powder.

8. The fruit tree cold resistance inducer according to claim 7, characterized in that: In step (1), the constant temperature shaking culture is cultured at 37°C and 180rpm for 16h; in step (2), the volume ratio of the seed solution to the culture medium is 1:100; in step (2), the constant temperature shaking culture is cultured at 37°C and 180rpm for 24h; in steps (1) and (2), the culture medium includes but is not limited to potato glucose culture medium and beef extract peptone culture medium; in step (3), concentrated acid is used to adjust the pH to 1.0-2.0, and then the culture medium is allowed to stand at 2-5°C for 16h; NaOH is used to adjust the pH to 7-7.

5.

9. A method for preparing the cold resistance inducer according to any one of claims 1 to 5, characterized in that: The following steps are involved: Weigh B-MES, calcium source, zinc source, iron source, boron source, trehalose or abscisic acid according to a predetermined ratio, dissolve them in water, and mix them evenly to obtain the cold resistance inducer.

10. Use of the cold resistance inducer according to any one of claims 1 to 5 in improving the cold resistance of fruit trees or crops, characterized in that: The application method includes applying the cold resistance inducer 1 to 7 days before the onset of early spring low temperature, late spring frost and other sudden cold damage in the growth stage, applying 1 to 3 times, and the application interval is 1 to 3 days; the use method is: after preparing the inducer according to a predetermined ratio, apply it to the fruit trees by foliar spraying or root irrigation, so that the leaf surface is evenly covered with a layer of mist droplets; the irrigation is carried out at 40 to 45 L / mu of the agent dissolved in the irrigation water.

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