A method for preventing low temperature frost injury to sugarcane seedlings

Abstract

The application discloses a method for preventing low-temperature freezing injury of sugarcane seedlings and relates to the technical field of sugarcane planting. The method for preventing freezing injury is that Bacillus amyloliquefaciens is uniformly mixed with base fertilizer and is applied together in the process of sugarcane planting, and a freezing prevention agent is sprayed in the sugarcane seedling stage, wherein the freezing prevention agent comprises 1-5 parts of sodium chloride, 100-200 parts of wheat extract and 2-10 parts of sodium caseinate according to weight fractions. The method for preventing freezing injury can increase the proline content in the cells of the sugarcane, reduce the cell membrane permeability, increase the osmotic pressure in the cells, prevent protoplasm from dehydrating and coagulating, and also increase the CAT, SOD and POD enzyme activities, so that the oxidation damage of oxygen negative ions to the cells and tissues is prevented, and the purpose of relieving the freezing injury of leaf cells is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of sugarcane planting, in particular to a method for preventing sugarcane seedlings from being frozen at low temperatures. Background Art

[0002] Sugarcane, originating in tropical and subtropical regions, is a thermophilic crop that requires suitable temperatures and humidity for normal growth. Every spring, when temperatures drop below 10°C or when late spring frosts occur, sugarcane seedlings often suffer frostbite, resulting in leaf curling, withering, and even death. Furthermore, the frequent occurrence of extreme weather events in recent years has led to an increase in chilling and freezing damage, resulting in significant losses for sugarcane production.

[0003] Existing measures to protect sugarcane from cold and frost include using cold-resistant varieties, improving cultivation and management practices, applying microbial fertilizers, and chemical manipulation. Improved cultivation and management practices primarily involve combining base fertilizer with mulching during sugarcane planting. Chemical manipulation primarily involves applying exogenous hormones such as gibberellins and abscisic acid, and applying chemicals such as MnCl2, CaCl2, KCl, and salicylic acid to mitigate damage to sugarcane seedlings from low temperatures. Both mulching and chemical manipulation create environmental stress.

[0004] Patent application number 201810570448.5, "A microbial fertilizer for enhancing crop cold resistance and its preparation method," discloses the use of Bacillus subtilis, Bacillus gelatinosa, lactic acid bacteria, and Aspergillus oryzae for crop cold resistance and frost protection, achieving certain results. However, there are currently no reports of using Bacillus amyloliquefaciens as a fertilizer for sugarcane frost protection. Bacillus amyloliquefaciens is generally used as a biocontrol agent to control pests and diseases. The applicant has found in production practice that Bacillus amyloliquefaciens has a good preventive effect on frostbite in sugarcane seedlings. Summary of the Invention

[0005] In response to the above problems, the present invention provides a method for preventing sugarcane seedlings from freezing due to low temperatures, which can effectively alleviate the occurrence of freezing in the sugarcane seedling stage and improve the survival rate of the seedlings. The specific technical solution is as follows:

[0006] A method for preventing sugarcane seedlings from being frozen by low temperature, comprising applying Bacillus amyloliquefaciens when planting sugarcane.

[0007] Preferably, the Bacillus amyloliquefaciens is mixed with the base fertilizer and applied together, and the application amount of the Bacillus amyloliquefaciens is 0.5-5 kg / mu.

[0008] Preferably, the method further comprises applying an antifreeze agent during the sugarcane seedling stage, wherein the antifreeze agent comprises 1-5 parts of sodium chloride, 100-200 parts of wheat extract and 2-10 parts of sodium caseinate in parts by weight.

[0009] Preferably, the preparation method of the wheat extract is: taking high-quality wheat seeds and soaking them in water for 2-4 hours, taking them out and spreading them out, placing them in a humid environment at 28-30°C until they germinate, placing the germinated seeds in methanol and crushing them, placing them at 4-6°C for 20-24 hours, filtering, placing the filter residue in methanol for extraction, taking the filtrate, repeating the extraction 3-4 times, combining the filtrates, concentrating under reduced pressure at 40-50°C to half of the original volume, adding an equal volume of petroleum ether, shaking, and standing until layers are separated, taking the lower layer, extracting with an equal volume of ethyl acetate 2-3 times, combining the extracts, evaporating under reduced pressure at 40-50°C, and adding ethanol to dissolve, thereby obtaining the wheat extract.

[0010] Preferably, the volume fraction of methanol is 80-90%.

[0011] Preferably, the volume fraction of ethanol is 90-95%.

[0012] Preferably, the antifreeze is applied by diluting it 100-300 times with water and then spraying it on the whole plant.

[0013] Preferably, the spraying time is morning or evening.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1. The Bacillus amyloliquefaciens of the present invention, when used together with base fertilizer, can improve the enzymatic activities of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) in sugarcane leaves, prevent oxidative damage to cells and tissues by oxygen anions, effectively alleviate the occurrence of frostbite in the sugarcane seedling stage, and improve the survival rate of the seedlings. In addition, the base fertilizer provides a good growth and reproduction environment for Bacillus amyloliquefaciens, further improving the use effect.

[0016] 2. The sodium chloride in the antifreeze of this invention increases intracellular proline content; wheat extract, which primarily contains gibberellins, amylase, and sugars, can enhance superoxide dismutase (SOD) activity, cellular sugar content, and proline content; and sodium caseinate increases amylase activity, promoting the conversion of starch into sugars within the plant, and also serves as an emulsifier. The synergistic effect of sodium chloride, wheat extract, and sodium caseinate increases the proline and sugar content in sugarcane leaves, elevating intracellular osmotic pressure, preventing protoplasm dehydration and coagulation, and inhibiting oxidative damage to cells and tissues caused by negative oxygen ions, thereby alleviating leaf cell frostbite. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0018] Figure 1This is a comparison of the curling of the heart leaves of Example 1 (left) and the blank control group (right) on the 4th day of low temperature treatment.

[0019] Figure 2 This is a graph showing the changes in CAT in sugarcane leaves within 84 hours of applying Bacillus amyloliquefaciens at 5°C.

[0020] Figure 3 This is a graph showing the changes in CAT in sugarcane leaves within 84 hours of applying Bacillus amyloliquefaciens at 10°C.

[0021] Figure 4 This is a graph showing the changes in SOD in sugarcane leaves within 84 hours after application of Bacillus amyloliquefaciens at 5°C.

[0022] Figure 5 This is a graph showing the changes in SOD in sugarcane leaves within 84 hours after application of Bacillus amyloliquefaciens at 10℃.

[0023] Figure 6 This is a graph showing the changes in POD of sugarcane leaves within 84 hours after application of Bacillus amyloliquefaciens at 5°C.

[0024] Figure 7 This is a graph showing the changes in POD of sugarcane leaves within 84 hours after application of Bacillus amyloliquefaciens at 10℃. DETAILED DESCRIPTION

[0025] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0026] It should be noted that the Bacillus amyloliquefaciens and Bacillus subtilis used in the following examples and comparative examples were purchased from Shandong Bilan Biotechnology Co., Ltd., with a content of 1000×10 8 CFU / g.

[0027] Example 1

[0028] A method for preventing sugarcane seedlings from freezing due to low temperatures, wherein Bacillus amyloliquefaciens is mixed with base fertilizer and applied together at a rate of 0.5 kg / mu during sugarcane planting, and the base fertilizer is a decomposed organic fertilizer at a rate of 1000 kg / hm2. 2 Antifreeze should be applied during the sugarcane seedling stage by diluting it 100 times with water and spraying it on the whole plant in the morning.

[0029] The antifreeze comprises, by weight, 1 part sodium chloride, 100 parts wheat extract, and 2 parts sodium caseinate. The wheat extract is prepared by soaking high-quality wheat seeds in water for 2 hours, removing them and spreading them out, placing them in a humid environment at 28°C until they germinate, placing the germinated seeds in 80% methanol by volume, crushing them, placing them at 4°C for 20 hours, filtering them, placing the filter residue in 80% methanol by volume for extraction, taking the filtrate, repeating the extraction three times, combining the filtrates, concentrating under reduced pressure at 40°C to half of the original volume, adding an equal volume of petroleum ether, shaking, and standing until layers are separated, taking the lower layer, extracting them twice with an equal volume of ethyl acetate, combining the extracts, evaporating them to dryness under reduced pressure at 40°C, and adding 90% ethanol by volume for dissolution, thereby obtaining the wheat extract.

[0030] Example 2

[0031] A method for preventing sugarcane seedlings from freezing due to low temperatures, wherein Bacillus amyloliquefaciens is mixed with base fertilizer during sugarcane planting and applied together at a rate of 5 kg / mu, and the base fertilizer is a compound fertilizer at a rate of 800 kg / hm2. 2 Antifreeze should be applied during the sugarcane seedling stage by diluting it 300 times with water and spraying it on the whole plant in the evening.

[0032] The antifreeze comprises, by weight, 5 parts of sodium chloride, 200 parts of wheat extract, and 10 parts of sodium caseinate. The wheat extract is prepared by soaking high-quality wheat seeds in water for 4 hours, removing them and spreading them out, placing them in a humid environment at 30°C until they germinate, placing the germinated seeds in 90% methanol by volume, crushing them, placing them at 6°C for 24 hours, filtering them, placing the filter residue in 90% methanol by volume for extraction, taking the filtrate, repeating the extraction four times, combining the filtrates, concentrating under reduced pressure at 50°C to half of the original volume, adding an equal volume of petroleum ether, shaking, and standing until layers are separated, taking the lower layer, extracting them three times with an equal volume of ethyl acetate, combining the extracts, evaporating them to dryness under reduced pressure at 50°C, and adding 95% ethanol by volume for dissolution, thereby obtaining the wheat extract.

[0033] Example 3

[0034] A method for preventing sugarcane seedlings from freezing due to low temperatures: when planting sugarcane, Bacillus amyloliquefaciens is mixed with base fertilizer and applied together at a rate of 4 kg / mu. The base fertilizer is a bio-organic fertilizer at a rate of 1200 kg / hm2. 2 Antifreeze should be applied during the sugarcane seedling stage by diluting it 200 times with water and spraying it on the whole plant in the morning or evening.

[0035] The antifreeze comprises, by weight, 3 parts of sodium chloride, 150 parts of wheat extract, and 7 parts of sodium caseinate. The wheat extract is prepared by soaking high-quality wheat seeds in water for 3 hours, removing them and spreading them out, placing them in a humid environment at 29°C until they germinate, placing the germinated seeds in 85% methanol by volume, crushing them, placing them at 5°C for 22 hours, filtering them, placing the filter residue in 85% methanol by volume for extraction, taking the filtrate, repeating the extraction four times, combining the filtrates, concentrating under reduced pressure at 45°C to half of the original volume, adding an equal volume of petroleum ether, shaking, and standing until layers separate, taking the lower layer, extracting it three times with an equal volume of ethyl acetate, combining the extracts, evaporating them to dryness under reduced pressure at 45°C, and dissolving them in 93% ethanol by volume to obtain the wheat extract.

[0036] Comparative test

[0037] The applicant selected Guitang No. 42 as the test material and conducted the test in the experimental greenhouse of the Guangxi Academy of Agricultural Sciences from March 2021 to December 2022. The test was divided into 10 groups. The detoxified sugarcane single bud seed stems were planted in nutrient barrels, with one plant planted in each barrel, 30 barrels per group, and 3 replicates. The test environment conditions were: temperature 5°C, relative humidity 60-70%, and light intensity 250-300umolm -2 s -1 , photoperiod 12h. Spraying degree is based on the degree of dripping on the leaves. The treatments of each group are as follows:

[0038] A group of planting was performed in each of Examples 1 to 3, and the steps of each example were followed.

[0039] Group 1: Bacillus subtilis was used instead of Bacillus amyloliquefaciens during planting, and other steps and treatment methods were the same as in Example 1.

[0040] Group 2: During the seedling stage (5-6 leaves), exogenous ABA was used instead of the antifreeze of the present application for spraying. Other steps and treatment methods were the same as those in Example 1.

[0041] Group 3: The antifreeze sprayed at the seedling stage (5-6 leaves) did not contain sodium chloride. Other steps and treatment methods were the same as those in Example 1.

[0042] Group 4: The antifreeze sprayed at the seedling stage (5-6 leaves) did not contain wheat extract. Other steps and treatment methods were the same as those in Example 1.

[0043] Group 5: The antifreeze sprayed at the seedling stage (5-6 leaves stage) did not contain sodium caseinate. Other steps and treatment methods were the same as those in Example 1.

[0044] Group 6: The antifreeze sprayed at the seedling stage (5-6 leaves) did not contain sodium chloride and sodium caseinate. Other steps and treatment methods were the same as those in Example 1.

[0045] CK group (blank control group): No treatment was done, that is, the sugarcane plants were planted at a low temperature of 5°C without any protective measures.

[0046] On the seventh day of low-temperature treatment, indicators such as cell membrane permeability, proline content, heart leaf normal coefficient, SOD enzyme activity, and POD enzyme activity (mean values) were measured for each example and each group. Cell membrane permeability was measured using the conductivity method, and 6 pieces of +3 leaves from each group (1 / 3 of each piece) were cut for measurement. Proline content was measured using the ninhydrin colorimetric method, and 6 pieces of +2 leaves from each group (1 / 3 of each piece) were cut for measurement. The heart leaf normal coefficient survey method was to define plants with obvious shrinkage and wilting of the heart leaves as wilted plants, and plants with leaves still unfolded as normal plants. The heart leaf normal coefficient was defined as the percentage of normal plants in the total plants. SOD enzyme activity was measured using the nitroblue tetrazolium photoreduction method, and POD enzyme activity was measured using the guaiacol method. 6 pieces of +1 leaves from each group (1 / 3 of each piece) were cut for measurement.

[0047] Note: The top leaf with exposed thick leaves is leaf +1, the one immediately below is leaf +2, and so on.

[0048] The measurement results of each indicator are shown in Table 1:

[0049] Table 1 Measurement results of each group and each embodiment

[0050]

[0051] The data of Group 1, Group 2, Example 1, and Group CK showed that the use of Bacillus amyloliquefaciens was better than that of Bacillus subtilis, and the use of the antifreeze of the present invention was better than that of exogenous ABA (conventional antifreeze). The combined use of Bacillus amyloliquefaciens and the antifreeze of the present invention could improve the normal coefficient of the heart leaves, thereby improving the survival rate of the seedlings.

[0052] The data from Groups 3 to 6 showed that the sodium chloride, wheat extract, and sodium caseinate in the antifreeze of the present invention acted synergistically to increase the proline content in sugarcane leaves, reduce cell membrane permeability, increase intracellular osmotic pressure, and prevent protoplasm dehydration and coagulation. Furthermore, the activities of SOD and POD enzymes were increased, preventing oxidative damage to cells and tissues caused by oxygen anions, thereby alleviating frostbite in leaf cells.

[0053] Figure 1 The results showed that after 4 days of low temperature treatment, the heart leaves of the blank control group began to curl, while the heart leaves of the treatment method of the present invention were normal.

[0054] To illustrate the effect of using Bacillus amyloliquefaciens, the applicant set up the following experiment: applying Bacillus amyloliquefaciens at 0.5 kg / mu at 5°C was set as the 5-0.5 group, applying Bacillus amyloliquefaciens at 1 kg / mu at 5°C was set as the 5-1 group, and no Bacillus amyloliquefaciens was applied at 5°C as the control group, i.e., the 5-CK group; applying Bacillus amyloliquefaciens at 0.5 kg / mu at 10°C was set as the 10-0.5 group, applying Bacillus amyloliquefaciens at 10°C was set as the 10-1 group, and no Bacillus amyloliquefaciens was applied at 10°C as the control group, i.e., the 10-CK group. The changes in the enzyme contents of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) in sugarcane leaves within 84 hours of application in each group were detected. The results are as follows: Figures 2 to 7 show.

[0055] Figures 2 to 7 The results showed that the Bacillus amyloliquefaciens of the present invention can increase the enzyme content of catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) in sugarcane leaves, prevent the oxidative damage of oxygen anions to cells and tissues, effectively alleviate the occurrence of frostbite in sugarcane seedlings, and improve the survival rate of seedlings.

[0056] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for preventing sugarcane seedlings from freezing, characterized in that: Applying Bacillus amyloliquefaciens during sugarcane planting, and also applying antifreeze during the sugarcane seedling stage; The antifreeze agent comprises, by weight, 1-5 parts of sodium chloride, 100-200 parts of wheat extract, and 2-10 parts of sodium caseinate; The preparation method of the wheat extract comprises the following steps: taking high-quality wheat seeds, soaking them in water for 2-4 hours, taking them out and spreading them out, placing them in a humid environment at 28-30°C until they germinate, placing the germinated seeds in methanol, crushing them, placing them at 4-6°C for 20-24 hours, filtering them, placing the filter residue in methanol for extraction, taking the filtrate, repeating the extraction 3-4 times, combining the filtrates, concentrating them under reduced pressure at 40-50°C to half of their original volume, adding an equal volume of petroleum ether, shaking them, and standing them until they separate into layers, taking the lower layer, extracting them with an equal volume of ethyl acetate for 2-3 times, combining the extracts, evaporating them to dryness under reduced pressure at 40-50°C, and adding ethanol to dissolve them, thereby obtaining the wheat extract.

2. The method for preventing sugarcane seedlings from freezing to low temperatures according to claim 1, wherein: The Bacillus amyloliquefaciens is mixed with the base fertilizer and applied together. The application amount of the Bacillus amyloliquefaciens is 0.5-5 kg / mu.

3. The method for preventing sugarcane seedlings from freezing to low temperatures according to claim 1, wherein: The methanol volume fraction is 80-90%.

4. The method for preventing sugarcane seedlings from freezing to death according to claim 1, wherein: The ethanol volume fraction is 90-95%.

5. The method for preventing sugarcane seedlings from freezing to death according to claim 1, wherein: The antifreeze agent is applied by diluting it 100-300 times with water and then spraying it on the whole plant.

6. The method for preventing sugarcane seedlings from freezing to death according to claim 5, characterized in that: The spraying time is in the morning or evening.

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