Sweet sorghum stalk juice, its preparation method and application in regulating corn growth and development

Abstract

The application belongs to the technical field of plant source biological stimulant preparation and crop planting, and particularly relates to sweet sorghum stem juice, a preparation method thereof and application thereof in regulating corn growth and development. It is found in the application that the component characteristics of the sweet sorghum stem juice are highly consistent with the core substance basis of plant source biological stimulants, the biological active components such as sugar, organic acid, amino acid and flavonoid contained in the sweet sorghum stem juice are synergized through multiple functions such as energy supply, signal regulation, nutrition activation and antioxidant defense, and the core potential of the sweet sorghum stem juice as a new plant source biological stimulant is constructed. Moreover, the sweet sorghum stem juice can be applied to corn through seed soaking, foliar spraying and root irrigation, and obvious concentration effect and application mode specificity are embodied, so that the effects of promoting corn germination, growth and nutrient absorption are achieved, and corn growth and development are effectively regulated.

Description

Technical Field

[0001] This invention belongs to the field of plant-derived biostimulant preparation and crop cultivation technology, specifically relating to a sweet sorghum stalk juice, its preparation method, and its application in regulating maize growth and development. Background Technology

[0002] Corn plays an irreplaceable role in food security, feed production, and industrial processing; however, soil phosphorus deficiency is a key adverse factor limiting its yield. Most phosphorus in the soil exists in an insoluble form, with very low levels of available phosphorus that crops can directly absorb, leading to stunted corn growth, insufficient biomass accumulation, and decreased nutrient use efficiency. Traditionally, excessive application of phosphate fertilizers has brought about many drawbacks, mainly manifested in low phosphorus use efficiency, soil phosphorus accumulation, environmental pollution, and potential negative impacts on crop absorption efficiency. Therefore, there is an urgent need to develop efficient and environmentally friendly green regulation technologies to improve corn's growth adaptability and phosphorus absorption efficiency under low-phosphorus conditions.

[0003] Higher plant-derived biostimulants (hPDBs) are complex mixtures derived from natural extracts or controlled processing of higher plant tissues or their metabolites. Their core function is not to directly provide plant nutrients, but to regulate the plant's own physiological and biochemical processes at extremely low application rates (far below fertilizer levels), thereby enhancing its tolerance and resilience to abiotic stresses (such as drought, salinity, extreme temperatures, and heavy metal pollution), and simultaneously improving nutrient use efficiency, photosynthetic performance, growth and development dynamics, and ultimately, yield and quality. It is noteworthy that widely recognized plant-derived biostimulants are mostly derived from plant materials rich in diverse bioactive components. For example, seaweed is rich in protein, vitamins, fiber, minerals, carbohydrates, and essential fatty acids, and its extracts have identified several key active compounds such as carrageenan, polyphenols, polyunsaturated fatty acids, and fucoidan. The synergistic effect of these components is the core basis for their biostimulatory function.

[0004] Sweet sorghum stalk juice is a sugar-rich liquid obtained by pressing sweet sorghum stalks. It is mainly used as a raw material for the production of biofuels (such as ethanol and butanol), feed protein, plant honey and other products. It can also be used directly as food or beverage. However, there are no reports on its use in regulating the growth and development of corn. Summary of the Invention

[0005] The purpose of this invention is to provide a sweet sorghum stalk juice, its preparation method, and its application in regulating maize growth and development. The sweet sorghum stalk juice can be used as a plant-derived biostimulant to effectively regulate maize growth and development.

[0006] This invention provides a method for preparing sweet sorghum stalk juice, comprising the following steps: Select sweet sorghum plants, cut stems more than 10 cm above the ground, remove leaves and leaf sheaths, and then use a squeeze juicer to press the sweet sorghum stem juice.

[0007] Preferably, the sweet sorghum plant is a plant in the milk stage.

[0008] The present invention also provides a sweet sorghum stalk juice, prepared by the method described in the above technical solution; the sweet sorghum stalk juice comprises the following percentages of metabolites: organic acids and their derivatives 51.08%, lipids and lipid-like molecules 20.05%, organic heterocyclic compounds 8.85%, organic oxygen-containing compounds 7.27%, phenylpropane and polyketide compounds 5.12%, benzene compounds 3.73%, organic nitrogen-containing compounds 3.15%, nucleosides, nucleotides and their analogues 0.36%, lignans, neolignans and related compounds 0.28%, organic sulfur-containing compounds 0.04%, alkaloids and their derivatives 0.03%, benzothiazole compounds 0.02%, hydrocarbon derivatives 0.01%, and other compounds 0.01%.

[0009] The present invention also provides the application of the sweet sorghum stalk juice described in the above technical solution as a plant-derived biostimulant.

[0010] Preferably, the plant-derived biostimulant is a plant-derived biostimulant that regulates the growth and development of maize.

[0011] Preferably, the regulation of maize growth and development includes promoting one or more of maize germination, growth and phosphorus absorption.

[0012] Preferably, the application method of the sweet sorghum stalk juice includes one or more of the following: seed soaking, foliar spraying, and root irrigation.

[0013] This invention also provides a method for promoting the growth and development of maize, comprising one or more of the following three methods: 1) Soak corn seeds in sweet sorghum stalk juice, and then sow the soaked corn seeds for planting management; 2) Foliar spray corn seedlings with sweet sorghum stalk juice; 3) Apply sweet sorghum stalk juice to the roots of corn seedlings; The sweet sorghum stalk juice mentioned in 1) to 3) is the sweet sorghum stalk juice as described in claim 3.

[0014] Preferably, in step 1), the concentration of the sweet sorghum stalk juice is 4.48%~8.96%, and the soaking time is 12 h; In 2), the corn seedlings are corn seedlings that have grown to the stage of 2 leaves and 1 heart, and the sweet sorghum stalk juice is sweet sorghum stalk juice that has been pressed and diluted 110 to 130 times. The volume of each foliar spray is 2.72 mL, and it is sprayed once every 3 days for a total of 4 to 6 times. In 3), the corn seedlings are corn seedlings that have grown to the stage of 2 leaves and 1 heart, the concentration of the sweet sorghum stalk juice is 4.48%~8.96%, the volume of each application is 4~6 mL, spray once every 3 days, and spray a total of 4~6 times.

[0015] Preferably, in the method, the corn is planted hydroponically, and the hydroponics method includes seedling roll hydroponics.

[0016] Beneficial effects: This invention provides a sweet sorghum stalk juice, its preparation method, and its application in regulating maize growth and development. The invention reveals that the compositional characteristics of the sweet sorghum stalk juice highly correlate with the core material basis of plant-derived biostimulants. Its rich content of sugars, organic acids, amino acids, and flavonoids, among other bioactive components, synergistically contribute to its potential as a novel plant-derived biostimulant through multiple functions, including energy supply, signal regulation, nutrient activation, and antioxidant defense. Furthermore, the sweet sorghum stalk juice described in this invention can be applied to maize through seed soaking, foliar spraying, and root irrigation to promote maize germination, growth, stress resistance, and nutrient absorption, effectively regulating maize growth and development. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 This is a distribution diagram of metabolites in the juice of sweet sorghum stalks in Example 2; Figure 2 The distribution and content percentage of the two core metabolites, organic oxygen-containing compounds (A) and organic acids and their derivatives (B), in the sweet sorghum stalk juice in Example 2 are shown. Figure 3 The effects of soaking sweet sorghum stalk juice at different concentrations on (A) plant height, (B) fresh weight, (C) aboveground fresh weight, (D) root fresh weight, (E) dry weight, (F) aboveground dry weight, (G) root dry weight, (H) root-to-shoot ratio, (I) number of lateral roots, (J) total root length, (K) root surface area, and (L) average root diameter in Example 3 were investigated. Figure 4 The effects of different concentrations of sweet sorghum stalk juice soaking treatment (C0 and C4) in Example 3 on the root length (A) of different diameters and the proportion of root length of different diameters to the total root length (B); Figure 5 This is a comprehensive analysis of the effects of different concentrations of sweet sorghum stalk juice soaking treatment on maize growth and root morphology indicators in Example 3; Figure 6 The effects of foliar spraying of sweet sorghum stalk juice under different phosphorus concentrations on (A) leaf SOD enzyme, (B) leaf POD enzyme, (C) leaf APX enzyme, (D) leaf soluble protein content, (E) leaf soluble sugar content, and (F) leaf proline content were investigated in Example 3. Figure 7 This is a heatmap showing the correlation between plant growth / physiological indicators and metabolites under sweet sorghum stem soaking treatments at C3 (A) and C4 (B) concentrations in Example 3. Figure 8 The effects of foliar spraying of sweet sorghum stem juice at different concentrations in Example 4 on (A) plant height, (B) SPAD, (C) stem diameter, (D) fresh weight, (E) aboveground fresh weight, (F) root fresh weight, (G) dry weight, (H) aboveground dry weight, (I) root dry weight, (J) root-to-shoot ratio, (K) total root length, (L) root surface area, (M) average root diameter, (N) aboveground phosphorus concentration, (O) root phosphorus concentration, (P) aboveground phosphorus content, and (Q) aboveground phosphorus content were investigated. Figure 9 This is a comprehensive analysis of the effects of foliar spraying of different concentrations of sweet sorghum stalk juice on maize growth and root morphology in Example 4. Figure 10 The effects of foliar spraying of sweet sorghum stalk juice under different phosphorus concentrations on (A) plant height, (B) SPAD, (C) stem diameter, (D) fresh weight, (E) aboveground fresh weight, (F) root fresh weight, (G) dry weight, (H) aboveground dry weight, (I) root dry weight, (J) root-to-shoot ratio, (K) total root length, (L) root surface area, (M) average root diameter, (N) aboveground phosphorus concentration, (O) root phosphorus concentration, (P) aboveground phosphorus content, and (Q) aboveground phosphorus content were investigated in Example 4. Figure 11 The effects of foliar spraying of sweet sorghum stalk juice under different phosphorus concentrations on (A) leaf SOD enzyme, (B) leaf POD enzyme, (C) leaf APX enzyme, (D) leaf soluble protein content, (E) leaf soluble sugar content, and (F) leaf proline content were investigated in Example 4. Figure 12 The heatmap shows the correlation between plant growth / physiological indicators and metabolites in the low phosphorus (A) and high phosphorus (B) treatments under the condition of foliar spraying of sweet sorghum stalk juice at concentration M in Example 4. Figure 13In Example 5, under low phosphorus conditions, the effects of exogenous addition (root irrigation) of different concentrations of sweet sorghum stalk juice on (A) plant height, (B) SPAD, (C) fresh weight, (D) aboveground fresh weight, (E) root fresh weight, (F) dry weight, (G) aboveground dry weight, (H) root dry weight, (I) root-to-shoot ratio, (J) total root length, (K) root surface area, (L) average root diameter, (M) aboveground phosphorus concentration, (N) aboveground phosphorus content, (O) root phosphorus concentration, and (P) root phosphorus content were investigated. Figure 14 This is a comprehensive analysis of the effects of different concentrations of exogenous addition (root irrigation) of sweet sorghum stalk juice on maize growth and root morphology indicators under low phosphorus conditions in Example 5. Figure 15 The effects of exogenous addition (root irrigation) of sweet sorghum stalk juice at concentrations of C3 and C4 under low phosphorus conditions in Example 5 on (A) plant height, (B) SPAD, (C) stem diameter, (D) fresh weight, (E) aboveground fresh weight, (F) root fresh weight, (G) dry weight, (H) aboveground dry weight, (I) root dry weight, (J) root-to-shoot ratio, (K) aboveground phosphorus concentration, (L) aboveground phosphorus content, (M) root phosphorus concentration, (N) root phosphorus content, (O) root SOD enzyme, (P) root POD enzyme, (Q) root APX enzyme, (R) root soluble protein content, (S) root soluble sugar content, and (T) root proline content were investigated. Figure 16 The effects of exogenous addition (root irrigation) of different concentrations of sweet sorghum stalk juice on (A) plant height, (B) SPAD, (C) stem diameter, (D) fresh weight, (E) aboveground fresh weight, (F) root fresh weight, (G) dry weight, (H) aboveground dry weight, (I) root dry weight, (J) root-to-shoot ratio, (K) total root length, (L) root surface area, (M) average root diameter, (N) aboveground phosphorus concentration, (O) root phosphorus concentration, (P) aboveground phosphorus content, and (Q) aboveground phosphorus content were investigated under normal phosphorus conditions in Example 5. Figure 17 This is a comprehensive analysis of the effects of different concentrations of exogenous addition (root irrigation) of sweet sorghum stalk juice on maize growth and root morphology indicators under normal phosphorus conditions in Example 5. Figure 18 This is a heatmap showing the correlation between plant growth / physiological indicators and metabolites under low and normal phosphorus levels when sweet sorghum stalk juice is applied through root irrigation in Example 5. In this heatmap, A represents low phosphorus + C3 concentration, and B represents low phosphorus + C4 concentration. Figure 19 This is a heatmap showing the correlation between plant growth / physiological indicators and metabolites under low and normal phosphorus levels when sweet sorghum stalk juice is applied through root irrigation in Example 5. A: Normal phosphorus + C2 concentration; D: Normal phosphorus + C3 concentration. Detailed Implementation

[0019] This invention provides a method for preparing sweet sorghum stalk juice, comprising the following steps: Select sweet sorghum plants, cut stems more than 10 cm above the ground, remove leaves and leaf sheaths, and then use a squeeze juicer to press the sweet sorghum stem juice.

[0020] In one embodiment, the sweet sorghum plant described in this invention is a plant in the milk-ripe stage. This invention does not have a particular limitation on the pressing method of the extrusion juicer; conventional pressing methods in the art are acceptable.

[0021] The present invention also provides a sweet sorghum stalk juice, prepared by the method described in the above technical solution; the sweet sorghum stalk juice comprises the following percentages of metabolites: organic acids and their derivatives 51.08%, lipids and lipid-like molecules 20.05%, organic heterocyclic compounds 8.85%, organic oxygen-containing compounds 7.27%, phenylpropane and polyketide compounds 5.12%, benzene compounds 3.73%, organic nitrogen-containing compounds 3.15%, nucleosides, nucleotides and their analogues 0.36%, lignans, neolignans and related compounds 0.28%, organic sulfur-containing compounds 0.04%, alkaloids and their derivatives 0.03%, benzothiazole compounds 0.02%, hydrocarbon derivatives 0.01%, and other compounds 0.01%.

[0022] The sweet sorghum stalk juice described in this invention is rich in bioactive components such as sugars, organic acids, amino acids, and flavonoids, which are highly compatible with the core material basis of known plant-derived biostimulants. It can synergistically construct the core potential of a novel plant-derived biostimulant through multiple functions including energy supply, signal regulation, nutrient activation, and antioxidant defense. Based on this, this invention also provides the application of the sweet sorghum stalk juice described above as a plant-derived biostimulant.

[0023] In one embodiment, the plant-derived biostimulant is a plant-derived biostimulant for regulating maize growth and development. In another embodiment, regulating maize growth and development includes promoting one or more of maize germination, growth, and phosphorus absorption. In one embodiment, the application method of the sweet sorghum stalk juice includes one or more of seed soaking, foliar spraying, and root irrigation. In one embodiment, the stress resistance mentioned in this invention refers to... This invention also provides a method for promoting the growth and development of maize, comprising one or more of the following three methods: 1) Soak corn seeds in sweet sorghum stalk juice, and then sow the soaked corn seeds for planting management; 2) Foliar spray corn seedlings with sweet sorghum stalk juice; 3) Apply sweet sorghum stalk juice to the roots of corn seedlings; The sweet sorghum stalk juice mentioned in 1) to 3) is the sweet sorghum stalk juice as described in claim 3.

[0024] In one embodiment, in step 1), the concentration of the sweet sorghum stalk juice is 4.48% to 8.96%, and the soaking time is 12 hours.

[0025] In one implementation, in step 2), the corn seedlings are corn seedlings that have grown to the stage of 2 leaves and 1 bud, and the sweet sorghum stalk juice is sweet sorghum stalk juice diluted 110-130 times after pressing, and further diluted 120 times; the volume of each foliar spray is 2.72 mL, sprayed once every 3 days, for a total of 4-6 sprays, or up to 5 sprays; in another implementation, the 2.72 mL spray volume is the total amount sprayed on both sides of the leaf. In one implementation, the foliar spray is performed simultaneously on both the front and back sides of the leaf.

[0026] In one implementation, in 3), the corn seedlings are corn seedlings that have grown to the stage of 2 leaves and 1 heart, the concentration of the sweet sorghum stalk juice is 4.48%~8.96%, the volume of each application is 4~6 mL, more preferably 5 mL, sprayed once every 3 days, for a total of 4~6 times, more preferably 5 times.

[0027] This invention does not have any special limitations on other planting management methods after foliar spraying or root irrigation treatment in 2) to 3) above. Conventional planting management methods in this field can be used.

[0028] In one embodiment, the corn is grown hydroponically, including the seedling roll method. In one embodiment, the culture medium used in the seedling roll hydroponics process has the following formula: Ca(NO3)2 (2000 μmol / L). -1 ), MgSO4 (650 μmol L) -1 H3BO3 (1 μmol L) -1 K2SO4 (750 μmol L) -1 ), KCl (100 μmol L -1 ZnSO4 (1 μmol L) -1 MnSO4 (1 μmol L) -1 CuSO4 (0.1 μmol L) -1 (NH4)Mo7O 24 (0.005 μmol L) -1 ), EDTA-FeNa (100 μmol L)-1 KH₂PO₄, 2.5 μmol L -1 Or 150 μmol L -1 .

[0029] In this invention, seed soaking, foliar spraying, and root irrigation show significant differences in their effects on regulating maize growth. Seed soaking is suitable for maize seedlings to resist stress, while foliar spraying and root irrigation are suitable for improving nutrient absorption during the growth period, demonstrating obvious concentration effects and application method specificity.

[0030] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0031] The hydroponic experiments involved in the following examples: Hydroponic experiments were conducted in the artificial climate chamber of Zone 4, College of Resources and Environment, China Agricultural University, with preset daytime hours of sunshine, daytime temperature of 25±1℃, and light intensity of 300 μmol·m⁻¹. -2 ·s -1 The darkness lasted 10 hours, and the nighttime temperature was 18±1℃. The nutrient solution formula was as follows: Ca(NO3)2 (2000 μmol / L). -1 ), MgSO4 (650 μmol L) -1 H3BO3 (1 μmol L) -1 K2SO4 (750 μmol L) -1 ), KCl (100 μmol L -1 ZnSO4 (1 μmol L) -1 MnSO4 (1 μmol L) -1 CuSO4 (0.1 μmol L) -1 (NH4)Mo7O 24 (0.005 μmol L) -1 ), EDTA-FeNa (100 μmol L) -1 KH2PO4 (low phosphorus, 2.5 μmol L) -1 ) / KH2PO4 (high phosphorus, 150 μmol L) -1 ).

[0032] Example 1 A method for preparing sweet sorghum juice, comprising the following steps: The tested sweet sorghum variety was "Zhongke Sweet 968," planted at the Hainan Academy of Agricultural Sciences' Nanfan Breeding Research Center (18.52°N, 108.88°E), using conventional field management (row spacing 50 cm, plant spacing 20 cm). At the milk stage, uniformly grown plants (280 cm tall) were selected, and the middle section of the stem (10 cm from the ground) was cut. After removing leaves and leaf sheaths, the juice was extracted using a squeeze-type juicer, and then filtered to remove residue. Figure 1 ), to obtain sweet sorghum juice.

[0033] The sweet sorghum stalk juice was then aliquoted into 50 mL centrifuge tubes and stored in ultra-low temperature freezers at -80℃ and -20℃ for subsequent metabolomics analysis (two parallel samples were prepared for each biological replicate, for a total of three biological replicates) and experimental applications. This was denoted as sweet sorghum extrusion stock solution (Cmax).

[0034] Example 2 The composition determination of sweet sorghum stalk juice in Example 1 was performed as follows: 1. Determination of non-targeting substance types Untargeted metabolomics analysis was performed using UPLC-MS / MS technology: 1) Sample pretreatment The sweet sorghum squeezed liquid sample was slowly thawed at 4℃. An appropriate volume of sample (0.5~0 mL) was transferred to a centrifuge tube, and 2 times the volume of extraction buffer (methanol / acetonitrile, 1:1, v / v) was added. The sample was vortexed for 60 s and then subjected to low-temperature ultrasonic extraction for 30 min. After centrifugation at 12000 rpm for 10 min at 4℃, the supernatant was collected and placed at -20℃ for 1 h to precipitate the protein. The sample was then centrifuged again at 12000 rpm for 10 min at 4℃. The supernatant was vacuum dried and reconstituted with 100 μL of 30% acetonitrile solution. After vortexing, the sample was centrifuged at 12000 rpm for 10 min at 4℃. The supernatant was then used for instrumental analysis.

[0035] 2) On-machine testing Chromatographic conditions: Waters HSS T3 (100 × 2.1 mm, 1.8 μm); mobile phase: Phase A was ultrapure aqueous solution (containing 0.1% formic acid), and Phase B was acetonitrile solution (containing 0.1% formic acid); flow rate: 0.3 mL / min; column temperature: 40℃; injection volume: 2 μL; elution gradient: 0 min Phase A / Phase B (100:0, v / v), 1 min Phase A / Phase B (100:0, v / v), 4 min Phase A / Phase B (40:60, v / v), 6.5 min Phase A / Phase B (5:95, v / v), 6.6 min Phase A / Phase B (100:0, v / v), 8.0 min Phase A / Phase B (100:0, v / v). The sample was placed in an autosampler at 4℃ throughout the analysis. Mass spectrometry conditions: Electrospray ionization (ESI) source, sheath gas 40 arb, auxiliary gas 10 arb, ion spray voltage +3000 V / -2800 V, temperature 350℃, ion transmission tube temperature 320℃. Scan mode: Full-ms-ddMS2, scan mode: positive ion / negative ion. Primary mass spectrometry scan range: 70-1050 Da, primary resolution: 70000, secondary resolution: 17500.

[0036] 3) Data Analysis The raw data were first preprocessed using Progenesis QI (Waters Corporation, Milford, USA) software, including baseline filtering, peak identification, peak matching, retention time correction, and peak alignment, resulting in a data matrix containing retention time, mass-to-charge ratio, and peak intensity. Peaks containing secondary mass spectrometry data were identified using commercial databases and Sanshu Bio's self-built metabolite secondary mass spectrometry database, along with corresponding fragmentation patterns. The results are as follows: Figure 1 As shown.

[0037] Depend on Figure 1 The results show that, through UHPLC-MS / MS non-targeted metabolomics analysis, combined with database matching and standard validation, a total of 1611 metabolites were identified in sweet sorghum stalk juice, which were classified into 15 major categories based on their structure and function. Figure 1The top five categories accounted for over 80% of metabolites, forming the core components of metabolites: Organic acids and derivatives had the highest percentage at 26.01%; followed by Organic heterocyclic compounds at 19.37%; Lipids and lipid-like molecules, Organic oxygen compounds, and Phenylpropanoids and polyketides accounted for 17.44%, 11.79%, and 10.18%, respectively.

[0038] In addition, Benzenoids and Organic nitrogen compounds accounted for 6.64% and 3.6% respectively, belonging to minor categories in the composition of metabolites. The enlarged area on the right side of the pie chart shows trace categories with a proportion of less than 5%, including 10 categories such as Nucleosides, nucleotides, and analogues (1.43%) and Lignans, neolignans and related compounds (1.24%). Among them, Hydrocarbons and Benzothiazoles accounted for less than 0.1%, reflecting the richness of metabolite composition and the diversity of low-abundance components.

[0039] This distribution characteristic intuitively reflects that the metabolite composition of sweet sorghum stalk juice is mainly composed of categories with high metabolic correlation, such as organic acids and their derivatives, and organic heterocyclic compounds. At the same time, it also covers a variety of low-abundance functional metabolite categories, providing a taxonomic basis for subsequent analysis of its metabolic network and functional characteristics.

[0040] 2. Determination of the content of targeted components Based on the non-targeted detection results, sugars (13 sugars), organic acids (26 organic acids), and amino acids (25 amino acids) were selected for targeted determination using HPLC. Standard curve construction: Standard solutions of different concentrations were prepared and detected under the following chromatographic conditions. A standard curve was plotted with peak area on the ordinate and concentration on the abscissa.

[0041] Chromatographic conditions: Carbohydrate detection was performed using an electrochemical detector. The column was a CarboPac™ PA1 (250 × 4.0 mm), the mobile phase was a CarboPac™ PA1 (250 × 4.0 mm) HPLC column and 100 mM sodium hydroxide solution, the flow rate was 1.0 mL / min, and the column temperature was 30 °C. Organic acid detection was performed using ultra-high performance liquid chromatography (Vanquish, UPLC, Thermo, USA) and high-resolution mass spectrometry (Q Exactive, Thermo, USA). The column was a Waters BEH C18 (50 × 2.1 mm, 1.8 μm), the mobile phase was ultrapure aqueous solution (containing 0.1% formic acid) and acetonitrile solution (containing 0.1% formic acid), the flow rate was 0.35 mL / min, and the column temperature was 40 °C. Amino acid detection was performed using ultra-high performance liquid chromatography (Vanquish, UPLC, Thermo, USA) and high-resolution mass spectrometry (Q Exactive, Thermo, USA). The column was a ChromCore. The column was 120 C18 (100×4.6 mm, 3 μm), with an ultrapure aqueous solution (containing 0.1% formic acid) and acetonitrile solution (containing 0.1% formic acid) as the mobile phase, a flow rate of 0.5 mL / min, and a column temperature of 50℃. Sample detection methods were the same as for standards; the content of each component was calculated based on the standard curve.

[0042] Core functional categories based on non-targeted metabolomics screening ( Figure 2 This embodiment establishes a targeted quantitative method for three major categories of components: sugars, organic acids, and amino acids. A total of 63 target components in the three categories were accurately quantified (Table 1), covering key components in sweet sorghum stalk juice that are closely related to nutrient supply and metabolic activity, providing accurate quantitative basis for elucidating their functional material basis.

[0043] Table 1. Types and contents of different sugars, organic acids, and amino acids in sweet sorghum stalk juice.

[0044] Among them, 12 carbohydrates were detected in the sugar component. Only glucose, fructose, and sucrose were effectively detected, while the remaining 9 (such as rhamnose and arabinose) did not reach the detection threshold. The total content reached 196,830 μg / mL. This component showed the characteristic of "absolute dominance of a single component": the sucrose content was as high as 177,910 μg / mL, accounting for 90.39% of the total sugar content, which is the core carrier of the high sugar properties of the juice; the contents of glucose (11,110 μg / mL) and fructose (7,810 μg / mL) were relatively low, accounting for 9.61% of the total sugar, which helped to form the basis of sugar metabolism in the juice.

[0045] A total of 26 organic acid components were detected, with a total content of 7279.85 μg / mL. Their distribution showed a pattern of "focused core components and dispersed secondary components": citric acid (3888.25 μg / mL) and malic acid (2106.13 μg / mL) were the main components, accounting for 82.34% of the total organic acids; lactic acid (607.43 μg / mL), oxalic acid (168.18 μg / mL), and 19 other components were secondary components, accounting for 17.63% of the total; valeric acid (0.86 μg / mL), salicylic acid (0.61 μg / mL), isobutyric acid (0.60 μg / mL), and 4 other trace components accounted for 0.03% of the total. The high proportion of core components reflects the functional characteristics of the juice in carbon metabolism and rhizosphere microenvironment regulation.

[0046] A total of 25 protein amino acids were detected, with a total content of 12665.91 μg / mL. The nitrogen composition showed a "high content of amides dominating" characteristic: L-asparagine had the highest content (9671.45 μg / mL), accounting for 76.35% of the total amino acid content; L-glutamine was the second highest (1654.6 μg / mL), accounting for 13.06%; the two together accounted for 89.41% of the total amino acids, which is the main form of nitrogen storage and supply in the juice; the contents of other amino acids (such as serine, aspartic acid, etc.) were all below 250 μg / mL, accounting for 10.59% of the total amino acids, providing a diverse trace nitrogen source for crop growth.

[0047] In summary, the targeted quantitative results of sweet sorghum stalk juice clarified the distribution patterns of its core functional components: sucrose forms the absolute core of high-energy carbohydrates, citric acid and malic acid are the main organic acids involved in metabolic regulation, and amides provide a high-quality nitrogen source. These results not only validate the category distribution conclusions of non-targeted metabolomics but also provide detailed quantitative data support for the subsequent development of juice biostimulants.

[0048] Example 3 The effect of soaking corn seeds in sweet sorghum stalk juice on corn growth regulation. 1. The experimental steps are as follows: The sweet sorghum extrusion liquor (Cmax) prepared in Example 1 was diluted with deionized water, and nine concentration gradients were set up: C0 (deionized water, control), C1 (Cmax×1.12%), C2 (Cmax×2.24%), C3 (Cmax×4.48%), C4 (Cmax×8.96%), C5 (Cmax×17.92%), C6 (Cmax×39.44%), C7 (Cmax×71.68%), and C8 (Cmax×100%).

[0049] Select uniform, plump, and undamaged 'Zhengdan 958' corn seeds, disinfect the surface with 10% (v / v) H2O2 for about 10 min, rinse with deionized water 10 times, and then soak them in juices of different concentrations for 12 h. After soaking, rinse with deionized water 3 times.

[0050] Hydroponics using the seedling roll method: Soaked seeds were evenly placed in petri dishes lined with moist filter paper, rolled into a cylindrical shape, and placed in an artificial climate chamber for cultivation. Deionized water was changed every 2 days. Each treatment had 5 replicates, with 15 seeds per replicate. Germination rate was recorded on days 3, 4, and 8 (germination criteria: radicle length ≥ seed length; seedlings were used to determine soluble sugar content (anthrone colorimetric method), catalase (CAT) activity (UV spectrophotometry), and proline content (acid ninhydrin colorimetric method).

[0051] 2. The test results are as follows: (1) Effect of sweet sorghum stalk juice soaking on maize germination Table 2 shows that soaking seeds in different concentrations of sweet sorghum stalk juice had varying degrees of effect on the germination rate, germination index, and vigor index of maize seeds. Compared with the control (C0, 0%), the germination rates of C1 (1.12%), C2 (2.24%), C3 (4.48%), and C6 (35.84%) treatments increased by 8.25%, 8.25%, 8.25%, and 8.50% on day 3, respectively; by 13.02%, 6.39%, 9.83%, and 12.78% on day 4; and by 11.65%, 8.25%, 8.50%, and 12.86% on day 8, respectively. After day 8, there was no significant difference in germination rate between the high-concentration treatments (such as C7 and C8) and the control. In terms of germination index, treatments C1, C2, C3, and C6 showed increases of 11.07%, 8.31%, 9.47%, and 11.28% compared to the control, respectively. Regarding vigor index, treatments C2, C3, and C6 showed increases of 33.01%, 29.76%, and 33.91% compared to the control, respectively. However, high-concentration treatments (such as C8, 100%) showed decreases in germination rate, germination index, and vigor index compared to the control. Specifically, the germination rate on day 3 decreased by 6.75%, and the germination index decreased by 4.84%. This indicates that soaking seeds in appropriate concentrations of sweet sorghum stalk juice can significantly improve the germination rate, germination index, and vigor index of maize seeds, promoting earlier germination, while high concentrations (such as 100%) inhibit seed germination.

[0052] Table 2. Effects of sweet sorghum stalk sap on maize seed germination indices

[0053] Note: Different lowercase letters in the same column indicate significant differences between treatments. P<0.05), the same applies below.

[0054] In summary, as the concentration of sweet sorghum stalk juice increases, the germination rate, germination index, and vigor index of maize seeds show a trend of first increasing and then decreasing. Soaking treatment within the concentration range of 1.12% to 35.84% has a significant promoting effect on seed germination, while excessively high concentrations have an inhibitory effect and are not conducive to seed germination.

[0055] (2) Effects of sweet sorghum stalk juice soaking on maize morphological indicators The effects of soaking sweet sorghum stalks in different concentrations (C1~C8) on plant growth were significantly different. Figure 3 Compared with the control group (C0), the C2-C7 treatments all showed a positive effect on maize plant height, with the C2 and C5 treatments significantly increasing it by 14.41% and 11.70%, respectively. Figure 3 (A). Biomass accumulation showed significant differentiation, with C2, C4, C6, and C8 treatments significantly increasing maize fresh weight. Specifically, C2 treatment increased aboveground fresh weight by 25.26% and root fresh weight by 14.69% compared to the control; C4 treatment significantly increased aboveground fresh weight by 23.73% and root fresh weight by 24.34%; C6 treatment increased aboveground fresh weight by 28.48% and root fresh weight by 8.98%; and C8 treatment increased aboveground fresh weight by 28.59% and root fresh weight by 8.43%. Figure 3 (C2-C7 treatments). Regarding dry weight, the total dry weight of plants under treatments C2-C7 all showed a significant increasing trend, with an increase ranging from 29.10% to 49.56%. Figure 3 (E). The aboveground dry weight showed a consistent trend with the total dry weight. Treatments C2 through C7 all showed a significant increase in total dry weight, ranging from 31.83% to 67.89%, with treatments C5, C6, and C7 showing the best results. Figure 3 (Middle F). Conversely, for root dry weight, treatments C5, C6, and C7 showed no significant difference from the control group, while treatments C2, C3, and C4 significantly increased root dry weight by 32.08%, 37.74%, and 50.94%, respectively. Figure 3 (G). Treatments C2, C5, C6, C7, and C8 significantly reduced the root-to-shoot ratio. Root-to-shoot ratio analysis further indicated that high-concentration sweet sorghum stalk juice soaking inhibited the accumulation of maize root biomass. Figure 3 (H).

[0056] This trend in relative root growth is further reflected in specific root morphology indicators. Regarding the number of lateral roots, there was no significant difference among different concentrations of sweet sorghum stalk juice soaking, but treatments C2, C3, C4, and C6 showed an increasing trend compared to treatment C0. Figure 3(I). A consistent trend was observed in the total root length and root surface area. Treatments C2, C3, and C4 significantly increased the total root length by 35.88%, 49.74%, and 49.74%, and the root surface area by 23.91%, 31.77%, and 31.87%, respectively, while treatments C5 and C7 significantly decreased (…). Figure 3 (J and K). Compared with the control, the C4 treatment significantly reduced the average diameter of maize roots, indicating that exogenous C4 addition increased the growth of fine maize roots. Figure 3 (L). Meanwhile, compared to the control, the C4 treatment significantly increased the length of roots with diameters <0.1 mm and <0.2 mm, and increased the proportion of roots with diameters <0.1 mm and <0.2 mm in the total root length, further demonstrating that the C4 treatment can significantly promote the growth and development of maize fine roots. Figure 4 (A, B)

[0057] Based on the comprehensive analysis of various growth and morphological indicators, treatments C3 and C4 showed the most significant growth-promoting effects on maize seedlings, both superior to the control and other concentration treatments. Figure 5 Specifically, the plant height, fresh weight, and dry weight were all significantly increased compared to the C0 control, and the root morphology indicators (total root length, root surface area, and number of lateral roots) also showed significant optimization, indicating that this concentration range not only promoted the accumulation and distribution of photosynthetic products, but also significantly activated the elongation growth and branching of the root system.

[0058] (3) Effects of soaking seeds in sweet sorghum stalk juice on antioxidant enzymes and metabolites in maize roots The effects of different concentrations (C1~C8) of sweet sorghum stalk juice on the antioxidant defense system and osmotic regulation capacity of maize roots ( Figure 6 Comprehensive analysis of various physiological and biochemical indicators showed that treatments C3 and C4 had the most significant activation effects on the antioxidant defense system and osmotic regulation capacity of maize roots, exhibiting an overall optimization pattern highly synergistic with growth indicators. Specifically, treatments C3 and C4 increased the activity of root antioxidant enzymes, with SOD and APX enzyme activities significantly increasing by 91.92% and 75.91% respectively under C3 treatment, reaching peak values, while POD enzyme activity showed no significant difference. Figure 6 (A~C). This indicates that soaking seeds at this concentration effectively enhances the root system's ability to scavenge reactive oxygen species (ROS), providing a favorable redox environment for root cell division and elongation. Simultaneously, the accumulation of root osmotic regulators shows a significant advantage. Compared to the C0 treatment, the C3 treatment increased soluble protein content by 24.13% and proline content by 46.08%; the C4 treatment significantly increased soluble protein content by 24.13% and proline content by 46.08%. Figure 6 (D~F). However, there was no significant difference in the content of soluble sugars in the roots under different concentration conditions (D~F). Figure 6 (E).

[0059] The results showed that C3 and C4 treatments not only promoted nitrogen metabolism and protein synthesis, but also enhanced the cells' buffering capacity against osmotic stress through proline accumulation. Notably, this physiological optimization effect closely matched the peak values ​​of growth indicators, suggesting that C3 and C4 concentrations may promote the growth and development of maize's aboveground parts and roots through a synergistic mechanism of enhancing antioxidant enzyme activity and improving osmotic regulation.

[0060] (4) Component-effect correlation analysis Treatment at C3 concentration ( Figure 7 In the study of maize root growth, aboveground dry weight, root dry weight, and plant height, carbohydrates and amino acids (excluding L-Ornithine) showed a positive correlation. Ten out of 25 organic acids also showed a positive correlation, indicating they are the core metabolites driving biomass accumulation and plant height growth at these concentrations. Regarding root growth and development, maize root surface area showed a significant positive correlation with multiple metabolites. Specifically, 66.67% of sugars (sucrose and glucose), 72.00% of amino acids, and 8.00% of organic acids were significantly positively correlated with root surface area, while 32% of organic acids showed a significant negative correlation. Furthermore, total root length, lateral root number, and average root diameter were positively correlated with benzoic acid, maleic acid, DL-isocitrate, citric acid, and hexanoic acid, but negatively correlated with quinic acid, propionic acid, butyric acid, valericacid, and salicylic acid, suggesting that specific organic acid components may play a role in regulating root elongation growth.

[0061] Compared to the C3 treatment, the component-effect relationship pattern changed significantly at the C4 concentration. Figure 7(Center B). Under the C4 concentration treatment, the correlations between carbohydrates and amino acids and aboveground dry weight, root dry weight, and plant height were reversed. In the C3 treatment, carbohydrates and amino acids were mainly positively correlated with aboveground dry weight, while at the C4 concentration, they shifted to a positive correlation with root dry weight, indicating that the regulatory targets of these two types of metabolites shifted from aboveground growth to root development. Specifically, carbohydrates and amino acids were negatively correlated with maize plant height, and 28.00% of organic acids were also negatively correlated with maize plant height. Conversely, carbohydrates and amino acids were positively correlated with maize root dry weight, while 28.00% of organic acids were positively correlated with maize root dry weight. Aboveground dry weight was significantly positively correlated with Benzoic acid, Maleic acid, DL-isocitrate, Citric acid, and Hexanoic acid among organic acids, while Butyric acid and Valericacid were significantly negatively correlated. For root growth and development, the correlation between lateral root number and metabolites at the C4 concentration showed the same characteristics as at the C3 concentration. The amino acid L-Omithine and the organic acids Quinic acid, Lactic acid, Propionicacid, Butyric acid, Valeric acid, and Salicylic acid were positively correlated with total root length, root surface area, and average root diameter. Furthermore, the correlation between maize root surface area and metabolites in the C4 treatment showed a significant divergence from that in the C3 treatment.

[0062] Example 4 The effect of foliar spraying of sweet sorghum stalk juice on maize growth regulation. 1. The experimental steps are as follows: Concentration screening stage: Hydroponics using a phosphorus-reduced nutrient solution (KH2PO4, 2.5 μmol / L) was employed. -1 After conventional germination of maize seeds, seedlings with uniform growth were transplanted into hydroponic boxes, 3 seedlings per box, with 1.5 L of phosphorus-reduced nutrient solution per box. The nutrient solution was changed every 3 days. Foliar spraying treatments were initiated when seedlings reached the 2-leaf, 1-heart stage, with 5 concentration gradients: CK (deionized water, control), L (low concentration, Cmax / 200, diluted 200 times), M (medium concentration, Cmax / 120, diluted 120 times), H (high concentration, Cmax / 70, diluted 70 times), and CH (ultra-high concentration, Cmax×30, diluted 30 times). Spraying was performed every 3 days, with approximately 2.72 mL applied per plant each time, evenly sprayed on both sides of the leaves. Each treatment was replicated in triplicate. After 15 days of cultivation, the plants were harvested, and biomass, root structure, phosphorus content (ammonium vanadomolybdate colorimetric method), and soluble sugar content were measured. The optimal concentration (M concentration) was selected based on the measurement results.

[0063] Optimal concentration verification and mechanism investigation phase: Phosphorus-reducing KH₂PO₄, 2.5 μmol / L -1 ) and phosphorus (KH2PO4, 75 μmol L) -1 Two nutrient levels were used, with two treatments at each level: CK (deionized water) and M (medium concentration, Cmax / 120). Each treatment had three replicates, with three seedlings per replicate. Hydroponic conditions and spraying methods were the same as in the concentration screening stage. After 15 days of cultivation, the seedlings were harvested, and biomass, root structure, and phosphorus content were measured.

[0064] 2. Analysis of Experimental Results: (1) Concentration screening results under phosphorus reduction conditions The effects of foliar spraying of different concentrations of sweet sorghum stalk juice on maize growth indicators, phosphorus nutrition indicators, and root morphology indicators showed significant differences. Figure 8 Regarding aboveground growth indicators, compared with the control (CK), the M treatment significantly increased maize plant height by 7.48% and stem diameter by 14.17%, the L treatment significantly increased maize plant height by 8.53%, and there were no significant differences under other treatments. Figure 8 (A, C). For SPAD, M treatment, H treatment, and CH treatment all significantly reduced leaf chlorophyll content (A, C). Figure 8 (B) Biomass accumulation showed significant differentiation. Compared with the control (CK), different concentrations of treatment had no significant difference in the fresh weight of the aboveground parts, while spraying with M, H, and CH treatments significantly increased the fresh weight of the roots, and the fresh weight of the roots increased with increasing treatment concentration. Figure 8 (D~F). Regarding dry weight, the trend was consistent with fresh weight: compared to the control (CK), the M treatment significantly increased the total dry weight of maize by 24.85%, including an increase of 25.35% in aboveground dry weight and 23.93% in root dry weight. Figure 8 Root-to-shoot ratio analysis showed no significant difference between each spraying treatment and the control (CK). Figure 8 (J).

[0065] This significant increase in root biomass is further reflected in specific root morphology indicators. Regarding total root length and root surface area, both showed an increasing trend with increasing spray concentration. The L, M, H, and CH treatments significantly increased total root length and root surface area, increasing total root length by approximately 50.92%, 64.95%, 77.38%, and 120.54% respectively, and root surface area by 33.55%, 34.66%, 52.81%, and 62.05% respectively compared to the control (CK). Figure 8 (Medium GI). Compared with the control (CK), the average diameter of maize roots decreased significantly with increasing spray concentration. Figure 8(M). This indicates that high-concentration foliar spraying promotes the growth and development of fine roots, making the root morphology tend towards a slender and elongated structure.

[0066] From the perspective of phosphorus nutrition indicators, compared with the control (CK), different concentrations of foliar spraying did not show significant differences in phosphorus concentration in the aboveground parts, but the phosphorus concentration in the roots was significantly reduced. Figure 8 In terms of phosphorus accumulation, the M treatment significantly increased the aboveground phosphorus content by 42.10%, while there was no significant difference in root phosphorus content among the treatments. Figure 8 (P, Q).

[0067] Based on the comprehensive analysis of various growth and morphological indicators, the M treatment had the most significant effect on promoting biomass accumulation in maize seedlings, while the H and CH treatments had a more prominent effect on promoting root elongation and surface area expansion. Figure 9 This result indicates that medium-concentration foliar spraying may achieve optimal biomass effects by promoting the accumulation of photosynthetic products and nutrient absorption, while excessively high concentrations, although promoting root morphology (slender roots), may lead to weakened aboveground growth or an imbalance in resource allocation.

[0068] (2) Effects of foliar spraying of sweet sorghum stalk juice on maize morphological indicators The growth-promoting effects of M (medium concentration) treatment under different phosphorus levels were compared in this embodiment. Two phosphorus nutrient levels, LP and HP, were further set up to systematically compare the effects of foliar spraying of sweet sorghum stalk juice on maize morphogenesis. Figure 10 ).

[0069] The results showed that under LP conditions, the growth-promoting effect of M treatment was highly consistent with the concentration screening results, while under HP conditions the effect was significantly weakened, verifying the specific growth-promoting advantage of sweet sorghum stalk juice under phosphorus-limited conditions.

[0070] Under LP conditions, the effect of M treatment on the aboveground morphological indicators of maize was consistent with the concentration screening results: compared with CK, M treatment significantly increased maize plant height and stem diameter ( Figure 10 (A, C), but had no significant effect on SPAD values ​​( Figure 10 In terms of biomass accumulation, the M treatment significantly increased fresh weight by 35.17%, aboveground fresh weight by 37.68%, and root fresh weight by 32.68% under LP conditions. Figure 10 (D~F), and significantly increased the total dry weight of the plant by 22.41%, the aboveground dry weight by 24.64%, and the root dry weight by 19.15% ( Figure 10 The results (1) show that the M treatment significantly promoted biomass accumulation. Meanwhile, under LP conditions, the M treatment did not show a significant difference in the root-to-shoot ratio, which is also consistent with the concentration screening results. Figure 10(J). However, under HP conditions, the M treatment had no significant promoting effect on plant height, SPAD, and stem diameter, and also had no significant effect on the root-to-shoot ratio. Figure 10 (A~C, J). Under HP conditions, compared with CK, the M treatment significantly increased the total fresh weight of maize by 28.76% and the total dry weight by 27.96%; this increase was mainly due to the significant increase in aboveground fresh weight (32.45% increase compared to CK) and aboveground dry weight (32.99% increase compared to CK). Figure 10 D, E, G, H).

[0071] The response patterns of root morphology indices further validated the above conclusions. Under LP conditions, the M treatment significantly increased total root length and root surface area compared to the CK treatment. Figure 10 The treatments K and L showed no significant difference in average root diameter. This is consistent with the M treatment's effect of promoting root elongation and increasing root surface area under reduced phosphorus conditions. Conversely, under HP conditions, the M treatment significantly promoted 49.32% of maize root elongation, increased root surface area by 23.64%, and significantly reduced average root diameter. Figure 10 (Middle K~M).

[0072] Under LP conditions, the effect of treatment M on phosphorus nutrition was generally consistent with the concentration screening results. Compared with CK, treatment M showed no significant difference in phosphorus concentration in the aboveground parts and roots. Figure 10 In terms of phosphorus accumulation, the M treatment significantly increased the phosphorus content in the aboveground parts, but had no significant effect on the phosphorus content in the roots. Figure 10 (P, Q). However, under HP conditions, the effect of the M treatment on phosphorus nutrition indicators was significantly weakened. Compared with the CK, the M treatment had no significant effect on phosphorus concentrations in both shoots and roots, and also had no significant promoting effect on phosphorus content in both shoots and roots. Figure 10 (N~Q). This result indicates that under normal phosphorus supply conditions, sufficient phosphorus allows maize to meet its own growth requirements, and exogenous application of sweet sorghum stalk juice no longer produces a significant phosphorus nutrient regulation effect.

[0073] The study confirmed that foliar spraying of M concentration significantly promoted maize plant height, stem diameter, biomass accumulation, and root morphogenesis under LP conditions, while this effect was weakened under HP conditions, revealing that the growth-promoting effect of foliar spraying of sweet sorghum stem juice has a phosphorus nutrition-dependent characteristic.

[0074] (3) Effects of foliar spraying of sweet sorghum stalk juice on antioxidant enzymes and metabolites in maize roots To further investigate the regulatory mechanism of foliar spraying of sweet sorghum stalk juice on the adaptation of maize to phosphorus stress and physiological metabolism, the results showed that under different phosphorus concentrations, spraying sweet sorghum stalk juice had significant differences in the activity of antioxidant enzymes and the content of osmotic regulators in the aboveground parts of the plant. Figure 11 Under LP conditions, M treatment significantly activated the antioxidant defense system and osmotic regulation mechanism of maize: compared with CK, M treatment significantly increased the activity of POD enzymes and APX enzymes in the aboveground parts. Figure 11 (B, C), while significantly increasing the content of soluble protein and proline ( Figure 11 The results (D, F) indicate that medium-concentration foliar spraying alleviated oxidative damage induced by low phosphorus stress by enhancing ROS scavenging and cell osmotic regulation. However, SOD enzyme activity was not significantly affected under LP conditions, and soluble sugar content was not significantly different from CK. Figure 11 A, E).

[0075] Under HP conditions, M treatment exhibited a distinctly different physiological regulatory pattern compared to LP conditions: compared to CK, M treatment significantly reduced SOD enzyme activity in the aerial parts, and APX enzyme activity also showed a decreasing trend, while POD activity was not significantly different from CK. Figure 11 (A~C). Meanwhile, under treatment M, there were no significant differences in the contents of soluble protein, soluble sugar, and proline ( ). Figure 11 (Middle D~F).

[0076] (4) Component-effect correlation analysis Under low phosphorus (LP) conditions ( Figure 12Organic acids are the core metabolites driving biomass accumulation and plant height growth at this phosphorus level. Of the 25 organic acids, 12 were positively correlated with plant height, with Benzoic acid, Maleic acid, DL-isocitrate, and Hexanoic acid showing significant positive correlations. Five organic acids (e.g., Quinic acid, Propionic acid) were positively correlated with stem diameter; 12 organic acids (e.g., Quinic acid, Lactic acid, Propionic acid) were positively correlated with aboveground biomass; and 15 organic acids (e.g., Lactic acid, D(-)-Tartaric acid, Isobutyric acid, Butyric acid) were positively correlated with root biomass. Similarly, regarding root growth and development, total root length and surface area were positively correlated with 52% organic acid content, but negatively correlated with sugars and amino acids. Sugars, amino acids (except Gamma-Aminobutyric acid), and 40% organic acids were all positively correlated with average root diameter. This suggests that specific organic acid components may play a role in regulating root elongation growth. 33.33% of fructose, 95.83% of amino acids (except L-citrulline), and 20% of organic acids were positively correlated with aboveground phosphorus content; while only 64% of organic acids were positively correlated with root phosphorus content. Furthermore, SPAD values ​​were positively correlated with 72% of organic acids (such as fumaric acid and oxalic acid), indicating that these metabolites may synergistically maintain leaf photosynthetic function.

[0077] Compared to low phosphorus conditions, high phosphorus (HP) conditions ( Figure 12In the high-phosphorus condition (B), the correlations between carbohydrates, amino acids, and organic acids and aboveground dry weight, root dry weight, and plant height were high compared to low-phosphorus conditions, while carbohydrates, amino acids, and organic acids showed the opposite trend. Regarding root growth and development, the correlations between total root length, root surface area, and metabolites under high-phosphorus conditions differed significantly from those under low-phosphorus conditions: sugars (fructose) and amino acids (except L-citrulline and Gamma-Aminobutyric acid) showed significant positive correlations with total root length and root surface area, while 36% of organic acids showed significant negative correlations. Similarly, the average root diameter showed the same trend. Under high-phosphorus conditions, carbohydrates and amino acids (except Gamma-Aminobutyric acid) were all positively correlated. Furthermore, sugars, amino acids, and organic acids showed relatively low correlations with aboveground phosphorus content, while 66.66% of sugars (Glucose, Sucrose) and 52% of organic acids (e.g., Benzoic acid, Maleic acid) were positively correlated with root phosphorus content.

[0078] Example 5 The regulatory effect of exogenous addition (root irrigation) of sweet sorghum stalk juice on maize growth and development. 1. The test method is as follows: Screening for low phosphorus concentrations: Hydroponics using a phosphorus-reduced nutrient solution (KH2PO4, 2.5 μmol / L) was employed. -1 After conventional germination of maize seeds, seedlings with uniform growth were selected and transplanted into hydroponic boxes, 4 seedlings per box, with 1.5 L of phosphorus-reduced nutrient solution per box. The nutrient solution was changed every 3 days. When the seedlings reached the 2-leaf and 1-heart stage, root irrigation treatment began. The sorghum extract was diluted to eight concentration gradients from C0 to C7 (C0 (deionized water, control), C1 (Cmax×1.12%), C2 (Cmax×2.24%), C3 (Cmax×4.48%), C4 (Cmax×8.96%), C5 (Cmax×17.92%), C6 (Cmax×39.44%), C7 (Cmax×71.68%)). Root irrigation was performed every 3 days, with 7.5 mL injected directly into the nutrient solution each time. After 12 days of cultivation, the seedlings were harvested, and biomass, root structure, phosphorus content, and soluble sugar content were measured. The optimal concentrations (C3 and C4) were selected based on the measurement results.

[0079] Optimal concentration verification and mechanism investigation phase: Setting up phosphorus reduction (KH2PO4, 2.5 μmol L) -1 ) and phosphorus (KH2PO4, 150 μmol L) -1Two nutrient levels were used, with three treatments at each level: CK (deionized water), C3 concentration, and C4 concentration. Each treatment had three replicates, and each replicate had five seedlings. Hydroponic conditions and root irrigation methods were the same as in the concentration screening stage. After 15 days of cultivation, the seedlings were harvested, and biomass, root structure, and phosphorus content were measured.

[0080] 2. Analysis of Experimental Results (1) Effects of exogenous addition of sweet sorghum stalk juice on maize growth and phosphorus absorption under phosphorus reduction conditions Under reduced phosphorus conditions, different concentration treatments had significant effects on maize growth indicators, root morphology indicators, and phosphorus nutrition indicators. Figure 13 Compared with the C0 treatment, the C1 and C2 treatments significantly increased maize plant height, while the SPAD values ​​showed no significant difference. Figure 13 (A, B). Biomass accumulation showed significant differentiation. The total fresh weight of treatments C3 and C4 was significantly higher than that of treatment C0, increasing by approximately 23.83% and 16.98% respectively. Specifically, compared with treatment C0, treatment C3 showed a significant increase of 26.25% and 20.44% in aboveground and root fresh weight, respectively. Compared with treatment C0, treatment C4 showed a significant increase of 19.69% in aboveground fresh weight, while root fresh weight showed no significant difference, increasing by 13.19%. Figure 13 (C~E); Meanwhile, the trends of total dry weight, aboveground dry weight, and root dry weight were consistent under treatments C3 and C4, with no significant difference between treatments C3 and C4. Treatment C3 significantly increased total dry weight by 31.21%, with aboveground dry weight increasing by 21.11% and root dry weight increasing by 62.33%, while treatment C4 significantly increased total dry weight by 34.00%, with aboveground dry weight increasing by 29.33% and root dry weight increasing by 50.33%. Figure 13 (F~H). Compared with the C0 treatment, the C2, C3, and C4 treatments increased the root-to-shoot ratio and promoted the relative accumulation of root biomass. Among them, the root-to-shoot ratio increased significantly by 34.65% under the C3 treatment. Figure 13 Middle I).

[0081] This relative growth advantage of the root system is further reflected in specific root morphology indicators. The C3 and C4 treatments showed the most significant increases in total root length and root surface area. Specifically, the total root length of the C3 and C4 treatments increased by 32.68% and 30.98% respectively compared to C0, and the root surface area increased by 31.75% and 29.38% respectively compared to C0. Figure 13 (J, K). Compared with the control, the average root diameter increased under treatments C3, C4, and C5, with treatment C4 showing a significant increase of 4.35% (J, K). Figure 13 (L).

[0082] Regarding phosphorus concentration and content, compared with C0, treatments C3 and C4 showed no significant difference in aboveground and root phosphorus concentrations, but increased the accumulation of phosphorus content in both aboveground and roots. Specifically, treatment C3 increased aboveground phosphorus content by 11.30% and root phosphorus content by 61.04%, while treatment C4 significantly increased aboveground phosphorus content by 18.36% and root phosphorus content by 64.12%. Figure 13 (M~P).

[0083] The radar chart visually illustrates the comprehensive performance of maize under different concentrations of sweet sorghum stalk juice soaking treatments in multiple dimensions, including plant height, biomass, root morphology, and phosphorus nutrition. Figure 14 Among them, C4 and C3 had the largest polygon coverage area and the most balanced distribution, and were at or near the highest level on the periphery in key dimensions such as total root length, root surface area, fresh weight, dry weight, plant height, and phosphorus content. In contrast, the C0 treatment showed a significant inward contraction of the polygon, while the high-concentration treatments (C5-C7) showed a decreasing trend in area with increasing concentration, and all indicators gradually declined. Based on the comprehensive analysis of various growth and morphological indicators, C3 and C4 treatments had the most significant growth-promoting effect on maize seedlings, both superior to the control and other concentration treatments. To further verify this conclusion, the C3 and C4 concentrations were repeatedly tested under the same phosphorus level. The results showed that the trends of plant height, biomass, phosphorus content, and other growth and phosphorus nutrition indicators were highly consistent. Furthermore, the C3 treatment significantly increased the activities of root POD, CAT, and APX, while the C4 treatment significantly increased the content of soluble protein, soluble sugar, and proline. This further confirms the core role of C3 and C4 concentrations in promoting growth under phosphorus reduction stress by synergistically promoting phosphorus absorption and activating stress metabolism. Figure 15 ).

[0084] (2) Effects of exogenous addition of sweet sorghum stalk juice on maize growth and phosphorus uptake speciation under normal phosphorus conditions Under normal phosphorus conditions, different concentrations of phosphorus treatments had significant effects on maize growth indicators, root morphology indicators, and phosphorus nutrition indicators. Figure 16 Compared with the C0 treatment, there was no significant difference in maize plant height under treatments C1 to C5, while treatments C6, C7, and C8 significantly reduced plant height, with treatments C7 and C8 showing the largest decreases. Figure 16 (A). SPAD values ​​showed a trend of first increasing and then decreasing. Compared with the C0 treatment, the C2, C3, C4, and C5 treatments showed a significant increase ( Figure 16 (C0, B). Regarding stem diameter, compared to the C0 treatment, the C2 and C3 treatments significantly increased maize stem diameter by 20.26% and 18.54%, respectively. Figure 16 (C)

[0085] Biomass accumulation showed significant differentiation. The total fresh weight of the C2 treatment was significantly higher than that of the C0 treatment, increasing by approximately 28.81% compared to C0. Specifically, the fresh weight of the aboveground parts increased by 27.29% and the fresh weight of the roots increased significantly by 32.68% in the C2 treatment. In contrast, the total fresh weight of the C3 treatment increased significantly by 38.18%, the fresh weight of the aboveground parts increased significantly by 34.80%, and the fresh weight of the roots increased significantly by 46.77%. Figure 16 (D~F). The dry weight trend was consistent with the fresh weight. Compared with the C0 treatment, the C2 and C3 treatments significantly increased the total dry weight by 51.06% and 47.35%, respectively. Among them, the dry weight of the aboveground parts increased significantly by 48.84% and 67.92%, and the dry weight of the roots increased significantly by 60.76% and 102.53%. Figure 16 (G~I). Root-to-shoot ratio analysis showed that the root-to-shoot ratios of treatments C3 and C4 were significantly higher than those of treatment C0, indicating that this concentration significantly promoted the relative accumulation of root biomass. Figure 16 (J).

[0086] This relative growth advantage of the root system is further reflected in specific root morphology indicators. The C2 and C3 treatments showed the most significant increases in total root length and root surface area. Specifically, the total root length of the C2 and C3 treatments increased by 82.17% and 80.07% respectively compared to C0, and the root surface area increased by 59.04% and 65.64% respectively compared to C0. Figure 16 (K, L). Compared with the control, there was no significant difference in the average root diameter between the C2 and C3 treatments. Figure 16 (M).

[0087] In terms of phosphorus concentration and content, the phosphorus concentration in the aboveground parts of the C1-C5 treatments was significantly lower than that of the C0 treatment, with a reduction range of 11.11%-41.67%, while there was no significant difference in phosphorus concentration in the roots. Figure 16 (N, O). Meanwhile, there was no significant difference in aboveground phosphorus content between treatments C1 to C5 and treatment C0, with treatments C2 and C3 increasing by 10.88% and 22.21%, respectively. Figure 16 (P). Regarding root phosphorus content, the C2 and C3 treatments increased by 24.23% and 66.62% respectively compared to the C0 treatment. Figure 16 (Q).

[0088] The radar chart visually illustrates the comprehensive performance of maize under different concentrations of sweet sorghum stalk juice soaking treatments in multiple dimensions, including plant height, stalk diameter, SPAD / biomass, root morphology, and phosphorus nutrition. Figure 17Among the treatments, C2 and C3 showed the largest and most evenly distributed polygon coverage area. Key dimensions such as total root length, root surface area, fresh weight, dry weight, plant height, and SPAD value were at or near the highest levels on the periphery, and phosphorus content remained at a high level. In contrast, the C0 treatment showed a significant inward contraction of the polygon area, while the high-concentration treatments (C6-C8) exhibited a decreasing area trend with increasing concentration, and all indicators gradually declined. The C8 treatment had the smallest polygon area, with plant height, stem diameter, biomass, and root morphology indicators at the lowest levels. Notably, the C7 and C8 treatments showed an abnormal outward extension in the phosphorus concentration dimension, indicating that while high-concentration treatments inhibited plant growth, they relatively increased root phosphorus concentration. Based on the comprehensive analysis of various growth and morphological indicators, the C2 and C3 treatments had the most significant growth-promoting effect on maize seedlings, both superior to the control and other concentration treatments, while excessively high concentrations (C7 and C8) had a significant inhibitory effect on maize growth.

[0089] (3) Component-effect correlation analysis Under low phosphorus (LP) + C3 concentration treatment ( Figure 18 In the study of plant height, carbohydrates and amino acids (except L-Ornithine) showed a negative correlation, while butyric acid and valeric acid (organic acids) showed a significant positive correlation. Sugars and amino acids were positively correlated with aboveground biomass, but their correlation with aboveground phosphorus content was weak. Among organic acids, valeric acid showed a significant positive correlation with aboveground phosphorus content, while benzoic acid, malic acid, DL-isocitrate, citric acid, and hexanoic acid showed significant negative correlations. Regarding root growth and development, carbohydrates and amino acids (except for a few amino acids) were generally positively correlated with root dry weight, total root length, root surface area, average root diameter, and aboveground phosphorus content. Specifically, 59.26% of metabolites showed a significant positive correlation with root biomass; 3.70% of metabolites showed a significant positive correlation with total root length; 11.11% of metabolites showed a significant positive correlation with root surface area; and 77.78% of metabolites showed a significant positive correlation with root phosphorus content. Among organic acids, 4% of the metabolites were positively correlated with root biomass, and 28% were negatively correlated; 12% of the metabolites were positively correlated with total root length and surface area; 8% of the metabolites were positively correlated with average root diameter; and 32% of the metabolites were negatively correlated with root phosphorus content. Furthermore, the SPAD value was positively correlated with 25% of the organic compounds, indicating that these metabolites may synergistically maintain leaf photosynthetic function.

[0090] Compared to the low phosphorus + C3 treatment, the low phosphorus + C4 concentration ( Figure 18In the B group, the component-effect relationship pattern underwent a significant shift. Under the low-phosphorus + C4 treatment, the correlations between carbohydrates and amino acids and plant height, aboveground / root biomass, and average root diameter were reversed. At the low-phosphorus + C4 concentration, carbohydrates and amino acids were negatively correlated with plant height, aboveground / root biomass, average root diameter, and aboveground phosphorus content, but positively correlated with plant height. Simultaneously, the correlations between carbohydrates and amino acids and total root length, root surface area, and root phosphorus content were weaker at the low-phosphorus + C4 concentration compared to the low-phosphorus + C4 concentration. 36%–64% of organic acid metabolites were positively correlated with aboveground / root biomass, total root length, root surface area, average root diameter, and aboveground / root phosphorus content. Furthermore, 28% of organic acid metabolites were significantly positively correlated with SPAD values.

[0091] Under normal phosphorus (NP) + C2 concentration treatment ( Figure 19 In the middle (A) category, 76% of organic acid metabolites were positively correlated with plant height. Carbohydrates and amino acids were positively correlated with SPAD value, maize stem diameter, aboveground / root biomass, total root length, root surface area, and aboveground / root phosphorus content. Specifically, 100% of carbohydrates and 54.17% of amino acids were significantly positively correlated with aboveground biomass, and 66.67% of carbohydrates and 80% of amino acids were significantly positively correlated with aboveground phosphorus content. Meanwhile, among organic acid metabolites, licorice, isobutyric acid, adipic acid, pimelic acid, and octanoic acid were significantly negatively correlated with aboveground biomass, total root length, root surface area, and aboveground phosphorus content. Shikimic acid, malic acid, itaconic acid, and isovaleric acid were significantly positively correlated with aboveground biomass, total root length, and root surface area.

[0092] Compared to the normal phosphorus + C2 treatment, at the normal phosphorus + C3 concentration ( Figure 19 In the normal phosphorus + C3 treatment, the component-effect correlation pattern showed no significant change. Under normal phosphorus + C3 treatment, carbohydrates and amino acids were positively correlated with plant height, stem diameter, aboveground / root biomass, total root length, root surface area, and aboveground / root phosphorus content. Specifically, 66.67% of carbohydrates and 64.00% of amino acids were significantly positively correlated with root biomass. Meanwhile, under normal phosphorus + C3 treatment, the correlation between carbohydrates and amino acids and SPAD values ​​weakened. Furthermore, consistent with normal phosphorus + C2 treatment, shikimic acid, malic acid, itaconic acid, and isovaleric acid were significantly correlated with root length.

[0093] Based on the above embodiments, it can be concluded that: 1. The compositional characteristics of sweet sorghum stalk juice are highly consistent with the core material basis of known plant-derived biostimulants (hPDBs). Its rich content of sugars, organic acids, amino acids and flavonoids and other bioactive components, through multiple functions such as energy supply, signal regulation, nutrient activation and antioxidant defense, constitute the core potential as a new type of plant-derived biostimulant.

[0094] 2. Differences in the regulatory mechanisms of different application methods on maize growth 1) Mechanism of action of seed soaking treatment: The juice components are absorbed by the seeds, activate the activity of germination-related enzymes, accumulate soluble sugars and proline, and enhance the seedling's resistance to stress, which explains the concentration effect.

[0095] 2) Mechanism of action of foliar spraying: M-concentration sap is absorbed through leaf stomata, directly participates in the metabolic process, promotes the accumulation of photosynthetic products, regulates root morphology and improves phosphorus absorption efficiency; compare the effects under phosphorus reduction and phosphorus addition conditions, and analyze the influence of nutrient environment on the regulatory effect.

[0096] 3) Mechanism of action of root irrigation treatment: C3 and C4 concentration sap acts directly on the rhizosphere, improves root structure, increases absorption area, and at the same time, dissolves insoluble phosphorus in the soil through organic acids, thereby improving phosphorus utilization.

[0097] In summary, the optimal concentrations of M, C3, and C4 are found to be due to insufficient components at low concentrations, while high concentrations may cause osmotic stress and inhibit growth; the appropriate concentration maximizes the effect. Therefore, in actual agricultural production, seed soaking is suitable for the seedling stage, while foliar spraying and root irrigation are suitable for the growing season.

[0098] 3. Seed soaking treatment can significantly improve the germination rate and seedling resistance of maize; foliar spraying with M concentration has the best effect, and can promote maize biomass accumulation and phosphorus absorption under both phosphorus reduction and phosphorus addition conditions; C3 and C4 concentrations performed best in root irrigation treatment, and can effectively improve root structure and increase phosphorus content.

[0099] The above three application methods have different effects on the regulation of maize growth. Seed soaking is suitable for stress resistance in the seedling stage, while foliar spraying and root irrigation are suitable for improving nutrient absorption during the growth period, showing obvious concentration effects and application method specificity.

[0100] It is evident that the sap from sweet sorghum stalks exhibits significant potential as a biostimulant by regulating processes such as corn germination, growth, stress resistance, and nutrient absorption, providing a new avenue for the resource utilization of agricultural waste and the development of green agriculture.

[0101] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing sweet sorghum stalk juice, characterized in that, Includes the following steps: Select sweet sorghum plants, cut stems more than 10 cm above the ground, remove leaves and leaf sheaths, and then use a squeeze juicer to press the sweet sorghum stem juice.

2. The preparation method according to claim 1, characterized in that, The sweet sorghum plants mentioned are those in the milk stage.

3. A sweet sorghum stalk juice, characterized in that, The sweet sorghum stalk juice is prepared by the method according to claim 1 or 2, comprising the following percentages of metabolites: organic acids and their derivatives 51.08%, lipids and lipid-like molecules 20.05%, organic heterocyclic compounds 8.85%, organic oxygen-containing compounds 7.27%, phenylpropanes and polyketides 5.12%, benzene compounds 3.73%, organic nitrogen-containing compounds 3.15%, nucleosides, nucleotides and their analogues 0.36%, lignans, neolignans and related compounds 0.28%, organic sulfur-containing compounds 0.04%, alkaloids and their derivatives 0.03%, benzothiazoles 0.02%, hydrocarbon derivatives 0.01%, and other compounds 0.01%.

4. The use of the sweet sorghum stalk juice as described in claim 3 as a plant-derived biostimulant.

5. The application according to claim 4, characterized in that, The plant-derived biostimulant is a plant-derived biostimulant that regulates the growth and development of maize.

6. The application according to claim 5, characterized in that, The regulation of maize growth and development includes promoting one or more of the following: germination, growth, and phosphorus absorption.

7. The application according to claim 5 or 6, characterized in that, The application methods of the sweet sorghum stalk juice include one or more of the following: seed soaking, foliar spraying, and root irrigation.

8. A method for promoting the growth and development of maize, characterized in that, Includes one or more of the following three methods: 1) Soak corn seeds in sweet sorghum stalk juice, and then sow the soaked corn seeds for planting management; 2) Foliar spray corn seedlings with sweet sorghum stalk juice; 3) Apply sweet sorghum stalk juice to the roots of corn seedlings; The sweet sorghum stalk juice mentioned in 1) to 3) is the sweet sorghum stalk juice as described in claim 3.

9. The method according to claim 8, characterized in that, In step 1), the concentration of the sweet sorghum stalk juice is 4.48%~8.96%, and the soaking time is 12 h; In 2), the corn seedlings are corn seedlings that have grown to the stage of 2 leaves and 1 heart, and the sweet sorghum stalk juice is sweet sorghum stalk juice that has been pressed and diluted 110 to 130 times. The volume of each foliar spray is 2.72 mL, and it is sprayed once every 3 days for a total of 4 to 6 times. In 3), the corn seedlings are corn seedlings that have grown to the stage of 2 leaves and 1 heart, the concentration of the sweet sorghum stalk juice is 4.48%~8.96%, the volume of each application is 4~6 mL, spray once every 3 days, and spray a total of 4~6 times.

10. The method according to claim 8, characterized in that, In the method described above, the corn is grown hydroponically, and the hydroponic method includes seedling roll hydroponics.

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