Compound microbial inoculants for improving watermelon yield and quality and soil nutrients and their application

Abstract

This invention relates to the field of biotechnology, and discloses a compound microbial agent for improving watermelon yield and quality and refining soil nutrients, as well as its application. The compound microbial agent includes *Bacillus belye* D103 and *Panthera pineapple* LAD128; the total viable count is 1 × 10⁻⁶. 5 CFU·mL ‑1 The compound microbial agent of this invention can significantly promote the root and plant growth of watermelon seedlings; promote uniform fruit coloring; increase the content of soluble sugars and lycopene in the fruit, improving color and quality; and simultaneously increase the total and available nitrogen, phosphorus, and potassium content in the soil, promoting nutrient absorption by the plants. This compound microbial agent activates molecular pathways related to watermelon growth and quality improvement by regulating gene expression in plant hormone signal transduction and the MAPK signaling pathway. Through multiple mechanisms, including improving soil fertility, regulating endogenous hormone balance, and optimizing carbohydrate and pigment metabolism, this compound microbial agent achieves a comprehensive effect of increased watermelon yield, improved quality, and enhanced marketability, demonstrating good field application value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to compound microbial agents and their applications for improving watermelon yield and quality and improving soil nutrients. Background Technology

[0002] watermelon( Citrullus lanatus Watermelon (Citrus aurantiacus) is an annual vine-like herbaceous plant belonging to the genus Citrus in the family Cucurbitaceae. Watermelon fruit is not only crisp, juicy, and sweet, but also rich in unique flavor and high levels of nutrients (amino acids, carotenoids, sugars, and organic acids, etc.), which can promote human health. Studies have shown that watermelon is one of the important dietary sources of lycopene. Lycopene, as a highly effective antioxidant, has significant effects in scavenging free radicals and preventing cardiovascular diseases and certain cancers. However, the formation of watermelon quality is subject to the comprehensive regulation of multiple factors. Cultivation management measures such as fertilization methods and ratios, and the types of organic fertilizers directly or indirectly affect fruit quality. In addition, the excessive application of chemical fertilizers and pesticides, and continuous cropping obstacles are important bottlenecks restricting watermelon production. Long-term use of chemical pesticides and continuous cropping lead to soil microbial community imbalance, exacerbation of soil-borne wilt disease, and accumulation of autotoxic effects in root exudates, thereby reducing yield and quality. Therefore, exploring safe and efficient ways to improve quality has become an urgent need for the watermelon industry.

[0003] Rhizosphere growth-promoting bacteria are a class of beneficial microorganisms that colonize the rhizosphere of plants and promote plant growth and improve plant health through various mechanisms. They directly promote growth by assisting plants in acquiring mineral nutrients through nitrogen fixation, phosphorus solubilization, and potassium solubilization; by regulating root development through the secretion of plant hormones such as indoleacetic acid (IAA); and by alleviating abiotic stress by reducing ethylene levels through ACC deaminase. Indirect growth promotion occurs by inhibiting soil-borne pathogens through the production of siderophores, antibiotics, and extracellular hydrolases, thereby inducing systemic resistance (ISR) and exerting a biocontrol function. In recent years, research on *Bacillus belye* has mainly focused on inducing systemic resistance (ISR), promoting plant and animal growth, antagonizing pathogenic microorganisms, elucidating antibacterial active substances, identifying functional gene clusters, and understanding antagonistic mechanisms.

[0004] In watermelon production, the application of PGPR has demonstrated good growth-promoting and disease-preventing effects. For example, *Pyrobacterium polymyxa* (PPM) Paenibacillus polymyxaSQR-21 is not only an effective biocontrol agent against watermelon wilt, but it also promotes growth by remodeling root protein expression. Although there is some research foundation on the application of PGPR in watermelon production, the mechanism by which PGPR improves watermelon fruit quality (sugar content, lycopene content, etc.) is still not well understood. In particular, the causal relationship between PGPR and the physiological and biochemical regulatory network of watermelon fruit quality formation lacks a systematic explanation. Therefore, in-depth exploration of the mechanism of PGPR in promoting growth and improving quality will not only help to improve the theoretical framework of PGPR-plant interaction, but also provide scientific basis and technical support for developing efficient microbial agents and achieving green quality improvement and efficiency enhancement of watermelons. Summary of the Invention

[0005] The purpose of this invention is to provide a compound microbial agent and its application for improving watermelon yield and quality and improving soil nutrients. This compound microbial agent can significantly promote the root and plant growth of watermelon seedlings; promote uniform fruit coloring; increase the content of soluble sugars and lycopene in the fruit, improving color and quality; and simultaneously increase the total and available nitrogen, phosphorus, and potassium content in the soil, promoting nutrient absorption by the plants. This compound microbial agent activates molecular pathways related to watermelon growth and quality improvement by regulating gene expression in plant hormone signal transduction and the MAPK signaling pathway. Through multiple mechanisms such as improving soil fertility, regulating endogenous hormone balance, and optimizing carbohydrate and pigment metabolism, this compound microbial agent achieves a comprehensive effect of increasing watermelon yield, improving quality, and enhancing marketability, and has good field application value.

[0006] To achieve the above objectives, this invention provides a compound microbial agent for improving watermelon yield and quality and refining soil nutrients, wherein the compound microbial agent includes Bacillus belye (… Bacillus velezensis D103 and Pantothecin pineapple ( Pantoea ananatis LAD128; the total viable count in the compound microbial agent is 1×10⁻⁶. 3 -1×10 6 CFU·mL -1 ; Bacillus belesiensis ( Bacillus velezensis D103 was deposited on July 15, 2021, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 22905, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and classified as *Bacillus belye*. Bacillus velezensis ; Pineapple pancreatitis ( Pantoea ananatis LAD128 was deposited on April 27, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 24784, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and classified as Pantotheca pineapple. Pantoea ananatis .

[0007] Furthermore, the total viable count in the compound microbial agent is 1×10⁻⁶. 5 CFU·mL -1 .

[0008] The present invention also provides the application of the above-mentioned compound microbial agent in improving watermelon yield and quality.

[0009] Furthermore, it is applied to: ① Promotes the growth of watermelon seedlings and enhances root development; Increase the total root length, root surface area, root volume, plant height, and aboveground and root biomass of watermelon seedlings; ② Improve watermelon yield and fruit appearance quality; It promotes fruit enlargement and ripening, makes the fruit color uniform and improves the ripeness, and significantly increases the weight of a single fruit, the yield per acre, and the transverse and longitudinal diameter of the fruit. ③ Regulate hormone and carbohydrate metabolism to improve the internal quality of fruit; By regulating plant hormone signal transduction pathways, it promotes the accumulation of auxin, cytokinin, and gibberellin, thereby enhancing fruit growth and development. By regulating the expression of genes related to starch and sucrose metabolism, glycolysis, amino acid metabolism, and lycopene synthesis, it increases the content of glucose, sucrose, fructose, vitamin C, soluble protein, and lycopene, reduces total acid content, and improves sweetness, nutritional quality, color, and flavor.

[0010] The present invention also provides the application of the above-mentioned compound microbial agent in improving soil nutrients.

[0011] Furthermore, it is applied to: ① Improve soil nitrogen supply capacity; It significantly increases the content of total nitrogen, available nitrogen and ammonium nitrogen in the soil, and enhances the soil's nitrogen supply capacity; ② Promote the availability of phosphorus and potassium nutrients in the soil; Increase the content of total phosphorus, available phosphorus, total potassium and available potassium in the soil, and improve the phosphorus and potassium nutrient status of the soil; ③ Enhance plant nutrient accumulation and improve soil-plant nutrient cycle efficiency: It improves the soil nutrient environment, enhances the absorption and translocation efficiency of nitrogen, phosphorus, and potassium by plants, and promotes the transformation of soil nutrients into crop yield and quality.

[0012] The present invention also provides a microbial compound fertilizer for improving watermelon yield and quality. The microbial compound fertilizer comprises the above-mentioned compound microbial agent and auxiliary materials. The auxiliary materials include one or more of the following: peat moss, humic acid, bentonite, diatomaceous earth, zeolite powder, wheat bran, rice bran, molasses, amino acids, trehalose, sodium carboxymethyl cellulose, sodium alginate, potassium humate, potassium fulvate, and trace elements.

[0013] The present invention also provides a microbial compound fertilizer for improving soil nutrients, wherein the microbial compound fertilizer comprises the above-mentioned compound microbial agent and auxiliary materials; the auxiliary materials include one or more of the following: peat moss, humic acid, bentonite, diatomaceous earth, zeolite powder, wheat bran, rice bran, molasses, amino acids, trehalose, sodium carboxymethyl cellulose, sodium alginate, potassium humate, potassium fulvate, and trace elements.

[0014] The advantages and positive effects of the compound microbial agent for improving watermelon yield and quality and refining soil nutrients described in this invention are as follows: Compared with existing technologies, the Bacillus vesiculosus D103+LAD128 compound microbial agent provided by this invention can significantly promote the root and plant growth of watermelon seedlings and increase plant biomass accumulation; it can promote fruit enlargement and uniform coloring, increase single fruit weight and yield per acre; it can increase the content of soluble sugar, vitamin C, soluble protein and lycopene in the fruit, reduce the total acid content, and improve the sugar-acid ratio, color and flavor quality; at the same time, it can increase the content of total and available nitrogen, phosphorus and potassium nutrients in the soil and promote the absorption of mineral nutrients by plants. Transcriptome and qRT-PCR results show that this compound microbial agent can activate molecular pathways related to watermelon growth and quality improvement by regulating the expression of genes related to plant hormone signal transduction, MAPK signaling pathway, photosynthesis, phenylpropanoid biosynthesis, starch and sucrose metabolism, amino acid metabolism and lycopene synthesis. Therefore, this compound microbial agent can achieve the comprehensive effect of increasing watermelon yield, improving quality and improving marketability through multiple mechanisms such as improving soil fertility, regulating endogenous hormone balance and optimizing carbohydrate and pigment metabolism, and has good field application value.

[0015] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0016] Figure 1 The following are examples of watermelon seedling growth under different concentrations of compound bacterial solution treatment in this invention. Figure 2 The figures represent the changes in growth indicators of watermelon seedlings after treatment with different concentrations of compound bacterial solution in this embodiment of the invention. In the figures, A is plant height; B is aboveground fresh weight; C is aboveground dry weight; D is root fresh weight; E is root dry weight; F is total root length; G is root surface area; H is root volume; different letters (ad) indicate the degree of significant difference between different treatments, and the same letter above the bar chart indicates no significant difference. Figure 3 These are phenotypic images of watermelon fruit growth under different treatments (CK group and B group) in this embodiment of the invention. Figure 4The figures show the changes in watermelon fruit growth indicators under different treatments (CK group and B group) in this embodiment of the invention, where A is the weight of a single fruit; B is the yield; and C is the transverse and longitudinal diameter. p<0.05; p<0.01; Figure 5 This is a statistical representation of differentially expressed genes in this embodiment of the invention. In the figure, A represents genes observed in the CK vs B comparison as shown by the Venn diagram; B represents differentially expressed genes as shown by the volcano diagram; red dots represent upregulated genes, and blue dots represent downregulated genes. Figure 6 This is an example of differential gene GO analysis to observe the enrichment pathway in fruits treated with CKvsB in this invention. Figure 7 This invention provides an example of differential gene KEGG analysis to identify the enrichment pathways observed in fruits treated with CKvsB. Figure 8 The figures illustrate the changes in hormone content under different treatments in this invention embodiment, where A is auxin; B is isopentenyl adenine; C is zeatin; and D is gibberellin. p<0.05; p<0.01; Figure 9 This is the qRT-PCR verification result of the hormone metabolism-related DEGs identified by RNA-seq in the embodiments of the present invention; p<0.05; p<0.01; p<0.001; Figure 10 This is the qRT-PCR verification result of the carbohydrate metabolism-related DEGs identified by RNA-seq in the embodiments of the present invention; p<0.05; p<0.01; Figure 11 The figures illustrate the changes in fruit nutrients under different treatments in this invention, where A represents glucose; B represents sucrose; C represents fructose; D represents starch; E represents total sugar; F represents total acid; G represents lycopene; H represents vitamin C; and I represents soluble protein. p<0.05; p<0.01; p<0.001; Figure 12 The figures illustrate the changes in soil physicochemical properties and plant nutrients under different treatments in this invention. In these figures, A represents total soil nitrogen; B represents soil nitrate nitrogen; C represents soil ammonium nitrogen; D represents total soil phosphorus; E represents soil available phosphorus; F represents total soil potassium; G represents soil available potassium; H represents total plant nitrogen; I represents total plant potassium; and J represents total plant phosphorus. p<0.05; p<0.01.

[0017] Information on the preservation of biological materials Bacillus belesiensis ( Bacillus velezensis D103 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 22905, on July 15, 2021, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and is classified as *Bacillus belye*. Bacillus velezensis .

[0018] Pineapple pancreatitis ( Pantoea ananatis LAD 128 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24784, on April 27, 2022, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and is classified as Pantotheca pineapple. Pantoea ananatis . Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0021] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0022] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in the methods of this invention.

[0023] Plant materials and tested strains: Watermelon ( Citrullus lanatus Varieties: Kirin; Bacillus belesii ( Bacillus velezensis D103, Pantothecin pineapple ( Pantoea ananatis LAD 128 was isolated and identified from the rhizosphere of maize.

[0024] Example 1 1. Screening of the optimal concentration for promoting growth of compound microbial agents: Watermelon seeds were disinfected with 3% sodium hypochlorite and 75% anhydrous ethanol for 10 min, rinsed with sterile water, and then germinated in a constant temperature incubator at (28±2) ℃. D103 and LAD 128 glycerol storage tubes were taken from a -80℃ ultra-low temperature freezer and streaked onto LB solid medium, incubated at 37℃ for 24 h. Healthy seedlings with germination of 3 days and radicles of 0.5 cm were selected and transplanted individually into seedling cups, ensuring root spread and soil compaction. Single colonies of D103 and LAD 128 were inoculated into fresh LB liquid medium and cultured at 37℃ with shaking at 180 rpm until the logarithmic growth phase. The bacterial suspension was then adjusted to 1×10⁻⁶. 8 CFU·mL -1 Eight treatment groups were established using a serial dilution method with distilled water. A 20 mL water treatment served as the control. The mixed bacteria were inoculated with a mixture of 10 mL D103 and 10 mL LAD 128. Every week, 20 mL of the corresponding concentration of bacterial solution was applied to the watermelon root soil. Each treatment was replicated in triplicate. Seedlings were cultured in a light-temperature controlled environment for 25 days under the following conditions: light-to-dark ratio 2:1, day / night temperature 25℃ / 16℃, and humidity 50%-60%. Every two days, 100 mL of sterile water was applied, and growth was observed. Each treatment was replicated in triplicate.

[0025] 2. Field Experiment: The field trial included a control group (CK, irrigation treatment) and a treatment group (B, 10). 5 CFU·mL -1 (Mixed bacterial suspension). Watermelons were planted from March to July in watermelon greenhouses in Xinmin City, Shenyang, Liaoning Province. Cultivation conditions included a day / night temperature of 30℃ / 19℃, relative humidity of 40%–60%, and a photoperiod of 12 hours of light / 12 hours of darkness. The greenhouses used potassium sulfate compound fertilizer with integrated fertigation drip irrigation technology. Pruning, pinching, and topping were carried out according to conventional field management practices. Isolation zones and protective rows were set up inside the greenhouses to prevent cross-contamination. Insect-proof nets were installed at the ventilation openings to block insect-borne viruses. Artificial pollination was used instead of bee pollination.

[0026] 3. Transcriptome analysis: Total RNA was extracted from control samples and fruits inoculated with a compound microbial agent in a field experiment according to the instructions of the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit from Tiangen Biotech. The purity and concentration were analyzed using a NanoDrop 2000 spectrophotometer. RNA integrity was assessed using an Agient 2100 / LabChip GX spectrophotometer. mRNA was purified using oligomeric (DT) beads and then fragmented with fragmentation buffer. Using messenger RNA as a template, the first cDNA strand and fragment buffer were synthesized and purified. The purified double-stranded cDNA was then repaired, A-tailed, and ligated for sequencing splicing. Fragment size selection was performed using AMPure XP beads, and the resulting library was amplified by polymerase chain reaction (PCR). After library identification, sequencing was performed using a high-throughput sequencing platform in PE150 mode. Data analysis was performed using the bioinformatics analysis program provided by BMKCloud (www.BioCloud.net). The false detection rate (FDR) was used as the core indicator for differential gene screening. When FDR < 0.05 and |log2(fold change)| ≥ 1, the difference was considered to be statistically significant.

[0027] 4. Determination of plant hormones: All measurements were performed using fully ripe watermelon fruits at a uniform ripening period, characterized by consistent color and firmness. Freeze-dried watermelon fruits were pulverized into powder, accurately weighed, and placed in test tubes. Ten volumes of pre-cooled chromatographic acetonitrile were added, along with an appropriate amount of internal standard stock solution. The mixture was ultrasonically extracted at 4°C for 30 min, followed by centrifugation at 4°C and 5000 rpm for 10 min, and the supernatant was collected. Five volumes of pre-cooled chromatographic acetonitrile were added to the remaining precipitate, and the ultrasonic extraction and centrifugation steps were repeated, collecting the second supernatant. The two supernatants were combined, and appropriate amounts of C18 and GCB adsorbents were added. After shaking for 30 s, the mixture was centrifuged at 4°C and 5000 rpm for 10 min, and the supernatant was collected. The supernatant was dried under nitrogen, reconstituted with a mixture of methanol and ultrapure water, and centrifuged at 4°C and 12000 rpm for 5 min. The supernatant was filtered through a 0.11 μm organic phase filter membrane for instrumental analysis. A Poroshell 120 EC-C18 reversed-phase column (2.1 × 100 mm, 2.7 μm) was used, along with a guard column of the same type (2.1 × 5 mm, 2.7 μm). The column temperature was set at 40 ℃, the flow rate at 0.3 mL / min, and the injection volume at 10 µL. The mobile phase consisted of phase A (water-0.02% formic acid) and phase B (chromatographic methanol). The gradient elution program was as follows: 0–1 min (A 95%, B 5%), 1–9 min (A 95% → 40%, B 5% → 60%), 9–11 min (A 40% → 5%, B 60% → 95%), 11–13 min (A 5%, B 95%), 13–13.2 min (A 5% → 95%, B 95% → 5%), and 13.2–15 min (A 95%, B 5%). Electrospray ionization (ESI) was used with an ionization voltage of +5500 V / -5000 V. Three watermelon fruits were combined to form a representative mixed sample for each experimental group, and three replicates were performed.

[0028] 5. Determination of total starch: The treatment groups (10) were measured separately. 5 CFU·mL -1Total starch content of watermelon fruits treated with mixed bacterial suspension and control group (watering treatment) was compared. Freeze-dried watermelon fruits were ground into powder, and a certain amount was placed in a centrifuge tube. The powder was washed multiple times with petroleum ether to remove fat, followed by multiple washes with ethanol solution to remove the residue. After filtration, the residue was transferred to an Erlenmeyer flask with a small amount of water, hydrochloric acid was added, and the mixture was hydrolyzed in a boiling water bath for 2 hours. After cooling, the pH was adjusted to neutral. Lead acetate and sodium sulfate solutions were added sequentially, and after standing, the liquid was diluted to 50 mL. The initial filtrate was discarded, and the filtrate was retained for analysis. A series of glucose standard solutions were prepared, and DNS reagent was added for color development in a boiling water bath. After cooling, the solution was diluted to volume, and the absorbance was measured at 540 nm using a visible spectrophotometer (Shanghai UNICO Co., Ltd., China). Different known concentrations of glucose solutions (0, 10, 20, 30, 40, and 50 μg·mL) were used for analysis. -1 The standard curve and linear equation were prepared to estimate the total starch content of watermelon fruits. The total starch content was calculated based on reducing sugar content, fixed volume, sampling volume, and sample weight; the result was multiplied by 0.9 to obtain the starch conversion value. Watermelon fruits from the same growth stage that were not inoculated with D103 bacterial suspension served as a control. Three watermelon fruits were combined to form representative mixed samples for each experimental group, and three biological replicates were performed.

[0029] 6. Determination of glucose, fructose, and sucrose content: Standard working solutions for glucose, fructose, and sucrose were prepared separately. 2 mL of water was added to each volume of standard solution, and anthrone reagent was added. The mixture was heated in a boiling water bath for 3.5 min, then rapidly cooled with ice water. The absorbance was measured at 640 nm using a blank as the zero point, and standard curves for each sugar were plotted. Samples were weighed and added to 75% ethanol. Extraction was performed in an 80 °C water bath for 15 min. After cooling and centrifugation, the supernatant was collected, the residue was washed repeatedly, and the volume was adjusted to 25 mL. The filtered extract was the soluble sugar extract. For sucrose determination, the extract was treated with KOH in a boiling water bath, cooled, and the absorbance was measured using the anthrone colorimetric method. The sucrose content was calculated using the reference curve. Glucose and fructose were developed using room temperature and a 50 °C water bath, respectively. The fructose and glucose contents were calculated by the difference between the two absorbance measurements, and finally converted to the sugar content per unit mass of sample. Three watermelon fruits were combined to form representative mixed samples for each experimental group, and three biological replicates were performed.

[0030] 7. Determination of total sugar: Take different volumes of glucose standard solution, add water to make up to 1 mL, add phenol solution and concentrated sulfuric acid sequentially, let stand and mix well, then react in a water bath at 30 ℃, measure the absorbance at 490 nm, and plot a standard curve. Accurately weigh the sample, add water and concentrated hydrochloric acid, hydrolyze in a boiling water bath for 3 h, cool, filter and wash the residue, and make up to volume as the test solution. Take 1 mL of the sample solution and perform colorimetric determination according to the standard curve procedure, and calculate the total sugar content of the sample based on the absorbance. Combine three watermelon fruits to form representative mixed samples for each experimental group, and perform three biological replicates.

[0031] 8. Determination of soluble protein content: Weigh the sample, add a small amount of water, and grind into a homogenate. Transfer to a centrifuge tube, wash the mortar with water, allow to stand at room temperature for extraction, centrifuge, and dilute the supernatant to 10 mL for later use. Prepare a series of bovine serum albumin standard solutions, add Coomassie brilliant blue reagent, mix well, allow to stand, and measure the absorbance at 595 nm to plot a protein standard curve. Take an appropriate amount of sample extract and determine the protein content using the same method. Calculate the protein content based on the curve, and combine the diluted volume and sample weight to calculate the soluble protein content.

[0032] 9. Vitamin C content determination: Add phosphomolybdic acid reagent to volumetric flasks, preheat in a boiling water bath, then add different volumes of vitamin C standard solution, continue boiling in the water bath for 10 min, cool, and dilute to volume with sulfuric acid solution. Measure the absorbance at 820 nm and plot a standard curve. Weigh the sample, homogenize with oxalic acid solution, add zinc sulfate and potassium ferrocyanide to precipitate protein, dilute to volume, filter, and measure the absorbance using the same colorimetric method. Calculate the vitamin C content from the curve, expressing it as milligrams per 100 g of sample.

[0033] 10. Determination of total acid content: Weigh the sample, homogenize it, transfer it to a volumetric flask, add water to make up to volume, and allow it to stand and filter to obtain the sample extract. Take a certain amount of the filtrate, add phenolphthalein indicator, and titrate with sodium hydroxide standard solution until a faint red color appears and does not fade after 30 seconds. Record the volume consumed. Calculate the mass fraction of titratable acid in the sample based on the NaOH concentration, titration volume, final volume of the sample, and the amount of sample weighed.

[0034] 11. Lycopene content determination: Sudan I pigment was weighed and diluted to volume with anhydrous ethanol to prepare a standard stock solution. This stock solution was then serially diluted to prepare a series of gradient standard solutions. The absorbance was measured at 485 nm, and a regression equation was established. A homogenized sample was weighed, and the yellow pigment was removed by repeated washing with methanol. The filtrate was discarded. The residue was then extracted with petroleum ether multiple times until colorless, and all extracts were collected for analysis. The absorbance of the sample extract was measured at the same wavelength. The lycopene concentration was calculated using the regression equation. Combining the extraction volume and sample mass, the lycopene content per unit mass of sample was calculated.

[0035] 12. RT-PCR analysis: Gene expression levels were quantified by RT-qPCR using the StepOne Plus real-time polymerase chain reaction system (Thermo Fisher Science, Waltham, USA). Actin, an internal reference gene, was used for normalization. Three biological replications were performed in the experiment. Two [units / items] were used. -ΔΔ-CT The method determined the relative expression of each target gene.

[0036] 13. Determination of nitrogen, phosphorus, and potassium content in soil and fruits (methods for determining basic physicochemical properties of soil): The methods for determining the physicochemical properties of soil were in accordance with "Soil Agrochemical Analysis" (Bao Shidan, 2000). For elemental analysis: total nitrogen, total phosphorus, and total potassium were determined using the Kjeldahl method, the molybdenum-antimony colorimetric method, and inductively coupled plasma atomic emission spectrometry, respectively; available nitrogen, available potassium, and available phosphorus were determined using the culture-diffusion titration method, ammonium acetate extraction, and sodium bicarbonate extraction-molybdenum-antimony colorimetric method, respectively. Total phosphorus (TP) and total potassium (TK) in plants were analyzed using inductively coupled plasma optical emission spectrometry (ICP-OES). Accurately weigh 0.01–0.5 g of dried sample or 0.2–5.0 g of fresh sample (weighing accuracy 0.0001 g) and place them in a polytetrafluoroethylene (PTFE) digestion vessel. Add 5 mL of analytical grade nitric acid and let it soak overnight at room temperature. Seal the digestion vessel, tighten the stainless steel pressure-resistant outer casing, and place it in a constant temperature drying oven for gradient temperature digestion: maintain a constant temperature of 80℃ for 2 h, increase to 120℃ for 2 h, and finally increase to 160℃ for 4 h. After digestion, place the vessel in an oven to cool naturally to room temperature, slowly open the lid, and heat on a hot plate at a low temperature to remove acid until the solution is nearly dry. Transfer the digestion solution to 10 mL or 25 mL of liquid. In a volumetric flask, the inner vessel and inner cap were washed three times with 1% nitric acid solution, and all washing solutions were combined into the volumetric flask. The volume was then adjusted to the mark with 1% nitric acid aqueous solution, shaken well and allowed to stand for analysis. A blank sample was prepared simultaneously to eliminate interference between reagents and the environmental matrix. The working parameters of the ICP-MS instrument were adjusted, and the blank solution, standard series solutions and sample solution were introduced in sequence to measure the mass spectrometry signal values ​​of each target element. The total nitrogen determination method employs an elemental analyzer combustion method: an appropriate amount of the sample to be tested is accurately weighed, wrapped in a tin bag, and placed in a combustion tube filled with tungsten oxide packing at 950℃ for complete combustion. The gas produced by combustion enters a reduction tube filled with granular copper and silver wire at 800℃ for a reduction reaction, during which all nitrogen is converted into nitrogen gas. The gas then passes through an adsorption column to complete adsorption and desorption separation sequentially, and finally enters a thermal conductivity detector for detection. By comparing and correcting the data with the determination data of standard samples, the total nitrogen content in the sample is calculated.

[0037] 14. Statistical Analysis: Statistical analysis software was used to create graphs. Data are expressed as mean ± standard deviation. Tukey's HSD was used for multiple comparisons among three or more groups, while the t-test was used for comparisons between two groups. All data were statistically analyzed using SPSS Statistical Analysis 27.0 software (IBM, Armonk, New York), and statistical significance was considered to be ≤0.05.

[0038] Results and Analysis of Example 2 1. Compound microbial agent treatment affects the growth and development of watermelon seedlings and fruits: Watermelon seedlings were treated with compound bacterial solutions of varying concentrations to promote growth. After 25 days of seedling growth, aboveground biomass, root length, root surface area, and other growth indicators were collected to determine the optimal concentration of the compound bacterial agent for promoting watermelon growth. Results showed that, compared to the control, when the total bacterial count was around 10... 5 CFU·mL -1 At this time, the effect of promoting growth is the best. Figure 1 Under this bacterial concentration, watermelon seedlings showed an 83.69% increase in total root length, a 37.06% increase in plant height, a 58.76% increase in above-ground fresh weight, a 75.98% increase in root fresh weight, a 67% increase in above-ground dry weight, a 48.46% increase in root dry weight, a 54.41% increase in root surface area, and a 46.1% increase in root volume. Figure 2 ).

[0039] The effects of compound bacterial solution on the growth and quality of watermelon fruit were studied by observing and analyzing relevant indicators. Compared with the control group CK (watering treatment), treatment group B (10... 5 CFU·mL -1 Fruits treated with compound bacterial solution showed uniform coloring and significantly improved maturity. Figure 3 Furthermore, under this concentration treatment, the weight of a single watermelon fruit, yield, and fruit diameter (both transverse and longitudinal) increased by 37.91%, 41.8%, 18.35%, and 16.16%, respectively, compared to the control. Figure 4 This indicates that the compound bacterial solution treatment can effectively promote watermelon fruit growth and improve fruit size and appearance quality.

[0040] 2. Transcriptome analysis revealed the regulation of D103 on watermelon fruit: Using watermelon fruits from treatments CK and B as the source of total RNA, libraries were constructed and RNA-seq analysis was performed. A total of 10 RNA samples were obtained from treatment group B (10...). 5 CFU·mL -1 Clean reads were obtained from the mixed bacterial suspension treatment library and the control group CK (watering treatment) library. After filtering, the total clean reads reached 39.54 GB. The clean reads were mapped to the watermelon reference genome. The alignment rates for all samples ranged from 97.53% to 98.95%, with Q30 values ​​all above 97.32%, and GC content ranging from 43.72% to 44.51%. A total of 2942 DEGs were identified (1089 upregulated and 1853 downregulated), and related charts are provided for further analysis. Figure 5 (A, B)

[0041] These DEGs are mainly enriched in the KEGG pathway, including phenylpropanoid biosynthesis, photosynthesis, plant hormone signal transduction, plant pathogen-pathogen interaction, flavonoid biosynthesis, and plant MAPK signaling pathway, etc. Figure 7 Regarding GO annotation, DEGs were significantly enriched in cellular processes, metabolic processes, and biological regulation. Figure 6 KEGG enrichment analysis showed that DEGs were enriched in multiple key KEGG pathways, which jointly participate in regulating physiological processes such as watermelon growth and development, stress resistance, and antioxidant activity, providing a foundation for further elucidating the growth-promoting mechanism of Bacillus belyssus D103.

[0042] The above results indicate that D103+LAD 128 can significantly regulate the physiological and biochemical processes of watermelon fruit, activate growth and development-related responses, and regulate processes such as stress resistance, antioxidant activity, transcription factor expression, and nutrient allocation. Differential genes are enriched in core life activities and key pathways, providing material and energy support for watermelon growth and enhancing the plant's stress resistance.

[0043] 3. Compound microbial agents promote watermelon fruit growth by regulating plant hormone pathways: During the growth-promoting process of D103+LAD 128, the plant hormone synthesis and signal transduction pathways in watermelon fruit underwent significant reprogramming. Key differentially expressed genes in the auxin, cytokinin, and gibberellin pathways were specifically detected using qRT-PCR, and their functions in the growth-promoting effect of the compound microbial agent were analyzed.

[0044] RT-qPCR analysis confirmed that the expression changes of all detected genes were consistent with the RNA-seq results. Figure 9 In plants with upregulated expression of the auxin-responsive protein SAUR71 (Cla013463), protein 45 containing the NAC domain (Cla016169), and adenylate isopentenyltransferase 3 (Cla014749) genes, the accumulation levels of auxin, isopentenyl adenine, and zeatin in watermelon fruits significantly increased by 15.19%, 34.91%, and 6.05%, respectively. Figure 8 (AC). However, in watermelon fruits with downregulated expression of transcription factors MYC2 (Cla008890) and DELLA protein (Cla019248) and the control, the accumulation of gibberellin GA3 was significantly increased by 19.56% (AC). Figure 8 (D).

[0045] 4. Compound microbial agents improve watermelon fruit quality by regulating the carbohydrate pathway: Studies have shown that after treatment with D103+LAD 128, the expression of key genes involved in starch and sucrose metabolism, such as α-galactosidase precursor, galactitol synthase 2, β-fructofuranosidase, insoluble isoenzymes CWINV1, β-amylase 3, sugar transporter 13, WRKY transcription factor 65, and basic leucine zipper 43, was upregulated, while the expression of hexokinase, sucrose synthase 5, and L-ascorbic acid oxidase was downregulated. In the glycolysis pathway, hexokinase and ATP-dependent 6-phosphofructokinase 6 were downregulated, while LOC103501223 and phosphoenolpyruvate carboxylkinase were upregulated. In amino acid metabolism, glutathione S-transferase and cysteine ​​synthase 1 were upregulated. In lycopene synthesis, ethylene response transcription factor ERF061 was upregulated, while lycopene ε-cyclase was downregulated.

[0046] Six key differentially expressed genes were detected using qRT-PCR to verify their roles in promoting fruit growth and regulating fruit quality using compound microbial agents. The results showed that in mature watermelon fruits, the expression levels of hexokinase 3 (Cla006319), sucrose synthase 5 (Cla009124), and L-ascorbic acid oxidase (Cla014164) were decreased, while the expression levels of β-fructofuranosidase, insoluble isoenzyme CWINV1 (Cla015574), phosphoenolpyruvate carboxylkinase (Cla023084), and cysteine ​​synthase 1 (Cla001238) were significantly increased, which was largely consistent with the RNA-seq sequencing results. Figure 10 ).

[0047] Notably, watermelon plants with upregulated expression of the ethylene response transcription factor ERF061 (Cla010914) and downregulated expression of the lycopene ε-cyclase (Cla016840) gene showed significant improvements in fruit coloring, with lycopene content increasing by 17.6%. Figure 11 (G). Although the soluble protein content in plants with upregulated cysteine ​​synthase 1 gene increased significantly by 14.7% (G). Figure 11 The regulation of hexokinase 3 (HXMT) significantly promoted the accumulation of glucose, sucrose, and fructose, increasing them by 15.14%, 31.44%, and 14.1%, respectively, while reducing the total acid content by 12.4%. Figure 11 The effects of gene expression changes on watermelon fruit quality indicators, including glucose, sucrose, fructose, vitamin C, soluble protein, lycopene, and total acid content, were further determined. The results showed that the effects of different target gene expression changes on fruit quality indicators varied. Downregulation of hexokinase 3 led to a significant increase in soluble sugar content and a significant decrease in total acid content in watermelon fruit, indicating its important role in sugar metabolism and sugar-acid ratio regulation. Figure 11(DF). Furthermore, inhibition of L-ascorbic acid oxidase significantly increased the vitamin C content of watermelon fruit, increasing it by 6.9% ( ). Figure 11 (H).

[0048] 5. Effects of compound microbial agent treatment on soil physicochemical properties and element content in fruits: Compared to the control (CK) treatment, the soil total nitrogen, nitrate nitrogen, and ammonium nitrogen contents increased by 14.08%, 10.62%, and 19.62%, respectively, after treatment with the compound microbial agent (B treatment); the soil total phosphorus and available phosphorus contents increased by 10.55% and 6.11%, respectively; and the soil total potassium and available potassium contents increased by 22.14% and 6.67%, respectively. Meanwhile, the total nitrogen, total phosphorus, and total potassium contents in plants increased significantly by 4.02%, 5.9%, and 6.58%, respectively. Figure 12 ).

[0049] This study systematically investigated the regulatory effects of a Bacillus belyceta var. berberis D103+LAD 128 compound microbial agent on watermelon seedling growth, fruit development, and quality formation. Transcriptome sequencing and physicochemical index determination were used to elucidate its core mechanism of action, ultimately clarifying the optimal concentration and molecular pathway for the compound microbial agent to promote growth and improve quality. The compound microbial agent exhibited a significant concentration-dependent growth-promoting effect on watermelon. 5 CFU·mL -1 This concentration represents the optimal application concentration. Treatment with this microbial agent significantly promotes the root and vegetative growth of watermelon seedlings, substantially increasing key growth indicators such as root length, root surface area, plant height, and above-ground and below-ground fresh and dry weight, effectively strengthening seedling growth and enhancing plant biomass accumulation. Simultaneously, this treatment significantly optimizes the appearance and yield traits of watermelon fruits, promoting uniform fruit coloring, improving maturity, significantly increasing single fruit weight and yield per acre, and enlarging the transverse and longitudinal diameters of the fruits, achieving a dual improvement in watermelon yield and appearance quality.

[0050] Transcriptome sequencing results confirmed that the compound microbial agent can mediate the growth, development, and stress resistance physiological processes of watermelon by regulating a large number of differentially expressed genes. A total of 2942 differentially expressed genes were screened, of which 1089 were upregulated and 1853 were downregulated. These genes were mainly enriched in key KEGG pathways such as plant hormone signal transduction, MAPK signaling pathway, photosynthesis, and phenylpropanoid biosynthesis, as well as GO functional items such as cell metabolism and biological regulation. They participate in regulating core physiological processes such as watermelon growth metabolism, stress resistance, antioxidant capacity, and nutrient allocation, providing molecular-level theoretical support for the growth-promoting mechanism of the compound microbial agent.

[0051] Plant hormone pathways are one of the core pathways for compound microbial agents to regulate watermelon fruit growth. The compound microbial agent can reprogram the hormone synthesis and signal transduction processes in watermelon fruit, significantly upregulating the expression of genes related to auxin, isopentenyladenine, and gibberellin GA3 synthesis, while inhibiting the expression of auxin degradation genes. This effectively promotes the accumulation of auxin, cytokinin, and gibberellin within the fruit. By regulating hormone metabolism balance and signal transduction, it accelerates watermelon fruit growth and development. Furthermore, the qRT-PCR verification results are highly consistent with the transcriptome sequencing results, confirming the accuracy and reliability of gene expression regulation.

[0052] Carbohydrate, amino acid, and pigment metabolism pathways are key mechanisms by which compound microbial agents improve watermelon fruit quality. These agents can optimize the intrinsic quality of the fruit by regulating the differential expression of genes related to starch and sucrose metabolism, glycolysis, amino acid synthesis, and lycopene synthesis. Specifically, downregulating the expression of genes such as hexokinase, sucrose synthase 5, and L-ascorbic acid oxidase significantly increases the content of soluble sugars (glucose, sucrose, fructose, etc.) and vitamin C in watermelon fruits, while reducing total acid content and optimizing the sugar-acid ratio. Upregulating the expression of genes such as the ethylene response transcription factor ERF0611 and cysteine ​​synthase 1 promotes the accumulation of lycopene and soluble proteins, effectively improving fruit color and flavor quality. Furthermore, the synergistic effect of these gene regulatory pathways has no significant negative impact on quality.

[0053] Compound microbial agents can effectively improve the physical and chemical properties of soil, promote the activation of soil mineral nutrients and the absorption of nutrients by plants. After application of compound microbial agents, the total and available nitrogen, phosphorus, and potassium content in the soil are significantly increased, with ammonium nitrogen and available nitrogen showing the most significant increases, greatly improving soil fertility. At the same time, microbial treatment can promote the absorption and accumulation of nitrogen, phosphorus, and potassium macroelements by watermelon plants, increase the nutrient content of fruits, and provide a good soil environment and material basis for watermelon growth, development, and quality improvement by improving soil fertility and enhancing plant nutrient absorption.

[0054] In summary, the D103+LAD 128 compound microbial agent can achieve comprehensive effects of promoting watermelon growth, increasing yield, and optimizing fruit quality through multiple regulatory mechanisms, including improving soil fertility, activating hormone-induced growth pathways, regulating carbohydrate and pigment metabolism, and mediating multi-pathway gene expression. It has good value for field application and promotion.

[0055] Therefore, the compound microbial agent of this invention, which enhances watermelon yield and quality and improves soil nutrients, can significantly promote the root and plant growth of watermelon seedlings and increase plant biomass accumulation; it can promote fruit enlargement and uniform coloring, increasing single fruit weight and yield per acre; it can increase the content of soluble sugar, vitamin C, soluble protein, and lycopene in the fruit, reduce the total acid content, and improve the sugar-acid ratio, color, and flavor quality; at the same time, it can increase the total and available nitrogen, phosphorus, and potassium nutrient content in the soil and promote the absorption of mineral nutrients by plants. Transcriptome and qRT-PCR results show that this compound microbial agent can activate molecular pathways related to watermelon growth and quality improvement by regulating the expression of genes related to plant hormone signal transduction, MAPK signaling pathway, photosynthesis, phenylpropanoid biosynthesis, starch and sucrose metabolism, amino acid metabolism, and lycopene synthesis. Thus, this compound microbial agent can achieve comprehensive effects of increasing watermelon yield, improving quality, and enhancing marketability through multiple mechanisms such as improving soil fertility, regulating endogenous hormone balance, and optimizing carbohydrate and pigment metabolism, and has good field application value.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A compound microbial agent for improving watermelon yield and quality and improving soil nutrients, characterized by: The compound microbial agent includes Bacillus vesiculosus (B. vesiculosus) Bacillus velezensis D103 and Pantothecin pineapple ( Pantoea ananatis LAD128; the total viable count in the compound microbial agent is 1×10⁻⁶. 3 -1×10 6 CFU·mL -1 ; Bacillus belesiensis ( Bacillus velezensis D103 was deposited on July 15, 2021, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 22905, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and classified as *Bacillus belye*. Bacillus velezensis ; Pineapple pancreatitis ( Pantoea ananatis LAD128 was deposited on April 27, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 24784, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and classified as Pantotheca pineapple. Pantoea ananatis .

2. The compound microbial agent for improving watermelon yield and quality and improving soil nutrients according to claim 1, characterized in that: The total viable count in the compound microbial agent is 1×10⁻⁶. 5 CFU·mL -1 .

3. The application of the compound microbial agent according to claim 1 in improving watermelon yield and quality.

4. The application according to claim 3, characterized in that, Applied to: ① Promotes the growth of watermelon seedlings and enhances root development; Increase the total root length, root surface area, root volume, plant height, and aboveground and root biomass of watermelon seedlings; ② Improve watermelon yield and fruit appearance quality; It promotes fruit enlargement and ripening, makes the fruit color uniform and improves the ripeness, and significantly increases the weight of a single fruit, the yield per acre, and the transverse and longitudinal diameter of the fruit. ③ Regulate hormone and carbohydrate metabolism to improve the internal quality of fruit; By regulating plant hormone signal transduction pathways, it promotes the accumulation of auxin, cytokinin, and gibberellin, thereby enhancing fruit growth and development. By regulating the expression of genes related to starch and sucrose metabolism, glycolysis, amino acid metabolism, and lycopene synthesis, it increases the content of glucose, sucrose, fructose, vitamin C, soluble protein, and lycopene, reduces total acid content, and improves sweetness, nutritional quality, color, and flavor.

5. The application of the compound microbial agent according to claim 1 in improving soil nutrients.

6. The application according to claim 5, characterized in that, Applied to: ① Improve soil nitrogen supply capacity; It significantly increases the content of total nitrogen, available nitrogen and ammonium nitrogen in the soil, and enhances the soil's nitrogen supply capacity; ② Promote the availability of phosphorus and potassium nutrients in the soil; Increase the content of total phosphorus, available phosphorus, total potassium and available potassium in the soil, and improve the phosphorus and potassium nutrient status of the soil; ③ Enhance plant nutrient accumulation and improve soil-plant nutrient cycle efficiency: It improves the soil nutrient environment, enhances the absorption and translocation efficiency of nitrogen, phosphorus, and potassium by plants, and promotes the transformation of soil nutrients into crop yield and quality.

7. A microbial compound fertilizer for improving watermelon yield and quality, characterized in that: The microbial compound fertilizer comprises the compound microbial agent and excipients as described in claim 1; the excipients include one or more of the following: peat moss, humic acid, bentonite, diatomaceous earth, zeolite powder, wheat bran, rice bran, molasses, amino acids, trehalose, sodium carboxymethyl cellulose, sodium alginate, potassium humate, potassium fulvate, and trace elements.

8. A microbial compound fertilizer for improving soil nutrients, characterized in that: The microbial compound fertilizer comprises the compound microbial agent and excipients as described in claim 1; the excipients include one or more of the following: peat moss, humic acid, bentonite, diatomaceous earth, zeolite powder, wheat bran, rice bran, molasses, amino acids, trehalose, sodium carboxymethyl cellulose, sodium alginate, potassium humate, potassium fulvate, and trace elements.

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