Growth promoting bacterial fertilizer for pandan leaves and application of growth promoting bacterial fertilizer

Abstract

The invention discloses a growth-promoting bacterial fertilizer for pandan leaves and application of the growth-promoting bacterial fertilizer, and relates to the technical field of biology. Active components of the growth-promoting bacterial fertilizer for the pandan leaves comprise a microorganism combination, the microbial composition comprises bacillus altitudinis, bacillus megatherium, trichoderma longibrachiatum and aspergillus parasuis. Through the synergistic effect of the four strains, the growth-promoting bacterial fertilizer can effectively improve the soil environment of the saline-alkali soil, reduce the pH value of the soil and improve the enzyme activity of the soil and the nutrient effectiveness of the soil; meanwhile, growth and development of pandan leaves can be remarkably promoted, and plant height, stem diameter, fresh weight of single plant and acre yield are increased; the quality of pandan leaves can be improved, and the content of a characteristic aroma substance 2-acetyl-1-pyrroline is increased. The growth-promoting bacterial fertilizer is simple in preparation process, relatively low in cost, environment-friendly and particularly suitable for popularization and application in planting of pandan leaves in saline-alkali soil, and has important agricultural application value and market prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a pandanus leaf growth-promoting bacterial fertilizer and its application. Background Technology

[0002] Pandanus leaves (scientific name: Pandanus amaryllifolius Pandanus (Roxb.), also known as fragrant pandanus or variegated pandanus leaf, is a perennial herbaceous tropical spice plant belonging to the genus Pandanus in the family Pandanaceae. Pandanus leaves are emerald green and emit a unique, sweet aroma. Pandanus leaves are widely used in the food industry, used in the production of pastries, beverages, and condiments, and also have potential applications in cosmetics and health products. In addition, pandanus leaves possess certain medicinal properties, including clearing heat and moistening the lungs, increasing appetite, aiding digestion, enhancing immunity, and improving metabolism. They are rich in nutrients such as linoleic acid, squalene, vitamin K3, phytol, and artemisia argyi, and market demand is steadily increasing.

[0003] Saline-alkali soils are characterized by low organic matter content, high pH (usually above 8.5), high salt content, poor soil structure, and poor water and fertilizer retention capacity. These harsh soil conditions severely restrict the normal growth and development of plants. As a tropical plant, *Pandanus orchid* has high requirements for its growing environment and thrives in fertile, loose, well-drained, slightly acidic to neutral soils with a pH of 5.5-7.0. While *Pandanus orchid* exhibits some tolerance to saline-alkali soils, the high salinity makes it difficult for its roots to absorb water and nutrients, leading to physiological drought and nutrient deficiencies. High soil pH affects the availability of nutrients such as phosphorus, iron, and zinc, resulting in slow growth, stunted plants, and yellowing leaves. Furthermore, saline-alkali stress inhibits the synthesis of secondary metabolites in *Pandanus orchid*, leading to a decrease in the content of the characteristic aroma compound 2-acetyl-1-pyrrolline, resulting in a weaker aroma and ultimately reduced yield, lower quality, and poorer economic benefits.

[0004] Currently, technologies for improving saline-alkali land and increasing crop yields mainly include physical, chemical, and biological methods. Physical methods, such as deep plowing and drainage leaching, involve large-scale engineering projects and are costly, and they are difficult to fundamentally improve soil structure. Chemical methods, such as applying gypsum and humic acid amendments, can adjust soil pH in the short term, but long-term use may cause secondary soil pollution and disrupt the soil microbial ecological balance. Biological methods have advantages such as being environmentally friendly and highly sustainable. Among them, the use of beneficial microbial agents to improve saline-alkali land and promote crop growth has become a research hotspot in recent years.

[0005] In existing technologies, some microbial fertilizers have been used for crop cultivation in saline-alkali land, but there are no reports of specialized growth-promoting microbial fertilizers for pandan leaves grown in saline-alkali land. Conventional microbial fertilizers often use single strains or have unreasonable combinations, making it difficult to exert a stable growth-promoting effect under saline-alkali stress, and they cannot simultaneously meet the dual needs of increasing pandan leaf yield and improving aroma quality. Therefore, developing a specialized growth-promoting microbial fertilizer that is highly targeted, has a significant growth-promoting effect, and can simultaneously increase pandan leaf yield and the content of characteristic aroma substances is of great practical significance for fully utilizing saline-alkali land resources, expanding the planting area of ​​pandan leaves, and improving the yield and quality of pandan leaves. Summary of the Invention

[0006] The purpose of this invention is to provide a growth-promoting microbial fertilizer for pandanus leaves and its application, thereby solving the problems existing in the prior art. This growth-promoting microbial fertilizer, through the synergistic effect of four bacterial strains, can effectively improve the soil environment of saline-alkali land, reduce soil pH, increase soil enzyme activity, and enhance soil nutrient availability; simultaneously, it can significantly promote the growth and development of pandanus leaves, increasing plant height, stem diameter, single plant fresh weight, and yield per acre; it can also improve the quality of pandanus leaves, increasing the content of the characteristic aroma substance 2-acetyl-1-pyrrolline.

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides a microbial ensemble for improving the yield and aroma quality of pandanus leaves grown in saline-alkali land, including Bacillus hygroscopicus (B. hygroscopicus). Altitude Bacillus ), Bacillus megaterium ( Bacillus megaterium Trichoderma longifolia ( Trichoderma longibrachiatum ) and Aspergillus viride ( Aspergillus subolivaceus ).

[0008] Furthermore, the ratio of viable bacteria of the highland Bacillus, the megaterium, the long-branched Trichoderma, and the near-green Aspergillus is (1.5-2):1:1:(1.5-2).

[0009] Preferably, the ratio of viable bacteria of Bacillus hygroscopicus, Bacillus megaterium, Trichoderma longifolia, and Aspergillus viride is 2:1:1:2.

[0010] The present invention also provides the application of the above-mentioned microbial combination in the preparation of pandanus leaf growth-promoting bacterial fertilizer.

[0011] Furthermore, the pandan leaf growth-promoting microbial fertilizer can improve the yield and / or aroma quality of pandan leaves grown in saline-alkali land.

[0012] The present invention also provides a pandanus leaf growth-promoting microbial fertilizer, the active ingredients of which include the above-mentioned microbial combination.

[0013] Furthermore, the pandanus leaf growth-promoting microbial fertilizer also includes a fertilizer carrier.

[0014] Furthermore, the fertilizer carrier includes crop straw, biochar, and kaolin.

[0015] The present invention also provides the application of the above-mentioned microbial combination or pandan leaf growth-promoting bacterial fertilizer in improving the yield and / or aroma quality of pandan leaves grown in saline-alkali land.

[0016] The present invention also provides a method for improving the yield and / or aroma quality of pandan leaves grown in saline-alkali land, comprising the step of applying the above-mentioned microbial combination or pandan leaf growth-promoting bacterial fertilizer to the pandan leaves grown in saline-alkali land.

[0017] The present invention discloses the following technical effects: The four strains screened in this invention have different functional characteristics and synergistic effects with each other. They can work together to improve the soil environment, provide nutrients, promote plant growth, enhance stress resistance, and promote secondary metabolism, significantly improving the growth performance of pandanus leaves in saline-alkali land. Compared with single strains or other combinations, the growth-promoting effect and aroma-enhancing effect are better.

[0018] The growth-promoting microbial fertilizer of this invention improves the soil environment and nutrient supply, promoting root growth, tillering, and leaf development in pandanus leaves, thereby increasing biomass and yield per plant. Experimental results show that after applying the growth-promoting microbial fertilizer of this invention, the fresh weight of pandanus leaves planted in saline-alkali land can increase by 49.0%, and the yield per acre can increase by 43.2%.

[0019] The growth-promoting microbial fertilizer of this invention can promote the activation of secondary metabolic pathways in pandanus leaves, providing precursors and energy for the synthesis of the characteristic aroma compound 2-acetyl-1-pyrrolidine, and significantly increasing its content. Experimental results show that after applying the growth-promoting microbial fertilizer of this invention, the content of 2-acetyl-1-pyrrolidine in pandanus leaves can reach 58.5 μg / g, an increase of 47.7% compared to the control, and the aroma is more concentrated and pure.

[0020] The growth-promoting microbial fertilizer of this invention uses beneficial microorganisms and agricultural waste (crop straw) as the main raw materials. The preparation process is environmentally friendly, and it will not cause soil pollution after application. It can also improve the soil microbial community structure, increase soil fertility, realize the sustainable use of saline-alkali land, and conform to the concept of green agricultural development.

[0021] The preparation method of this invention uses conventional fermentation equipment and granulation process, which is simple to operate, low in cost, suitable for large-scale production, and the microbial fertilizer has good stability, long storage period, and is easy to promote and apply. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] Terminology Explanation: Saline-alkali land refers to a type of land where the salt content and alkalinity of the soil exceed the tolerance range for normal crop growth. It mainly consists of saline soil and alkaline soil and is widely distributed in arid, semi-arid, and coastal areas. Its core characteristics include: high soil salt content (usually exceeding 0.3%), with soluble salts mainly consisting of sodium chloride, sodium sulfate, and sodium carbonate; high soil pH, mostly above 8.5, with some extreme saline-alkali lands reaching pH values ​​above 10; low soil organic matter content, generally below 10 g / kg; poor soil structure, often in a compacted state, with poor aeration and permeability; weak water and fertilizer retention capacity; low nutrient availability, severely inhibiting plant growth and development. The harm of saline-alkali land to plants is mainly manifested in three aspects: First, osmotic stress, high soil salinity leads to an increase in soil solution osmotic pressure, making it difficult for plant roots to absorb water and causing physiological drought. Even if the soil moisture content is sufficient, plants will still show symptoms of water shortage and wilting. Second, ion toxicity, excessive sodium ions, chloride ions, etc. will damage plant cell structure, affect enzyme activity and metabolic processes, and lead to abnormal plant growth. Third, nutrient imbalance, high pH value will reduce the availability of elements such as phosphorus, iron, zinc, and manganese in the soil, leading to nutrient deficiency in plants, while inhibiting the absorption of elements such as nitrogen and potassium by the roots.

[0028] Soil enzymes are a class of catalytic proteins produced in soil by microorganisms, plant roots, and the decomposition of plant and animal remains. They participate in key processes such as the decomposition of organic matter and nutrient transformation in the soil, and their activity levels are important indicators reflecting soil fertility and microbial community function. This invention focuses on soil enzymes including urease and phosphatase. Urease primarily catalyzes the hydrolysis of nitrogen-containing organic matter such as urea in the soil to produce ammoniacal nitrogen. Its activity directly affects the soil nitrogen conversion efficiency and the absorption and utilization of nitrogen nutrients by plants; higher urease activity indicates a stronger soil nitrogen supply capacity. Phosphatase is classified into acid phosphatase, neutral phosphatase, and alkaline phosphatase. It primarily catalyzes the hydrolysis of organic phosphorus compounds in the soil into inorganic phosphorus, increasing the availability of phosphorus and providing absorbable phosphorus nutrients for plant growth. Its activity level is closely related to the soil phosphorus supply capacity.

[0029] In saline-alkali lands, the high pH and salinity inhibit soil enzyme activity, leading to slow nutrient transformation. This is a significant reason for the low soil fertility in saline-alkali lands. Improving soil enzyme activity through amendment measures is a key approach to enhancing the nutrient supply capacity of saline-alkali lands.

[0030] Soil pH is a core indicator for measuring soil acidity and alkalinity. It reflects the negative logarithm of the hydrogen ion concentration in the soil solution, and its value typically ranges from 1 to 14. A pH of 7.0 is neutral, a pH < 7.0 is acidic, and a pH > 7.0 is alkaline. Soil pH directly affects the form and availability of nutrients in the soil and the survival activities of microorganisms, making it one of the key soil factors determining the suitability for plant growth.

[0031] Different plants have specific pH tolerance ranges. Pandanus orchids, as typical acid-neutral plants, thrive in soils with a pH of 5.5-7.0. When the soil pH exceeds 7.5, nutrients such as phosphorus, iron, and zinc form insoluble compounds, reducing their availability. Simultaneously, high pH affects the permeability of root cell membranes, inhibiting root absorption of water and nutrients, leading to poor plant growth. Saline-alkali soils often have pH values ​​above 8.5, significantly exceeding the suitable growth range for pandanus orchids. Therefore, adjusting soil pH is one of the core improvement goals for cultivating pandanus orchids in saline-alkali soils.

[0032] Plant growth-promoting microbial fertilizers are an important type of bio-fertilizer. They refer to fertilizer products made with beneficial microorganisms that promote plant growth, improve soil environment, and enhance crop quality as the core active ingredient, supplemented by a suitable carrier. Compared with traditional chemical fertilizers, plant growth-promoting microbial fertilizers have advantages such as being environmentally friendly, highly sustainable, and having a significant effect on soil improvement. They not only provide indirect nutrition to plants but also improve soil structure, regulate soil pH, and inhibit pathogen reproduction through the metabolic activities of microorganisms, achieving the dual goals of increasing crop yield and quality and improving soil fertility.

[0033] Fertilizer carriers are inert or functional substances used in microbial fertilizer production to carry, immobilize, and protect beneficial microorganisms, improve the physical properties of the fertilizer, and facilitate storage, transportation, and application. High-quality fertilizer carriers should possess the following characteristics: good adsorption properties and porous structure to effectively immobilize microbial cells or spores and reduce the impact of environmental factors on microbial activity; chemical stability, without antagonistic effects on microorganisms or altering soil pH; a certain water and fertilizer retention capacity to provide a suitable microenvironment for microbial growth and reproduction; and wide availability, low cost, and compliance with environmental protection requirements.

[0034] The fertilizer carrier selected in this invention is a compound of crop straw, biochar and kaolin. Crop straw and biochar are organic carriers, while kaolin is an inorganic carrier. The three work synergistically to give full play to the advantages of the organic carrier, which is rich in nutrients and has strong adsorption capacity, and to take advantage of the inorganic carrier, which has good stability and is easy to granulate, thus significantly improving the physicochemical properties and application effect of the microbial fertilizer.

[0035] In this invention, high-performance liquid chromatography (HPLC) is used to determine the content of 2-acetyl-1-pyrrololine, a characteristic aroma compound in pandanus leaves. The detection process mainly includes: sample pretreatment (crushing the pandanus leaf sample, extracting and purifying it with a solvent to obtain the sample solution to be tested); chromatographic condition setting (selecting appropriate parameters such as chromatographic column, mobile phase composition, flow rate, column temperature, and detection wavelength); sample detection (injecting the sample solution into the chromatograph, separating it through the chromatographic column, with each component sequentially entering the detector to generate signals and forming chromatographic peaks); and quantitative analysis (qualitative analysis based on the retention time of the chromatographic peaks of the standard, and quantitative calculation based on the linear relationship between peak area or peak height and the concentration of the standard to obtain the content of 2-acetyl-1-pyrrololine in the sample). This method can accurately and rapidly determine the content of the target 2-acetyl-1-pyrrololine, providing a scientific basis for the quality evaluation of pandanus leaves.

[0036] The indophenol blue colorimetric method is a colorimetric analysis method based on chemical reaction color development. It is mainly used to determine the activity of soil urease. The principle is that urease catalyzes the hydrolysis of urea in the soil to produce ammonia. Ammonia reacts with sodium hypochlorite and phenol under alkaline conditions to produce indophenol blue. The color intensity of indophenol blue is directly proportional to the amount of ammonia produced. The urease activity can be calculated by measuring the absorbance of the colorimetric solution at a specific wavelength using a spectrophotometer.

[0037] The disodium phenyl phosphate colorimetric method is a commonly used colorimetric analysis method for determining soil phosphatase activity. The principle is that soil phosphatase can catalyze the hydrolysis of disodium phenyl phosphate to produce phenol and disodium hydrogen phosphate. Under alkaline conditions, phenol reacts with 4-aminoantipyrine to produce a red quinone compound. The color intensity of this compound is directly proportional to the amount of phenol produced. The phosphatase activity can be calculated by measuring the absorbance with a spectrophotometer.

[0038] The strains involved in this invention are as follows: Highland Bacillus ( Altitude Bacillus Purchased from the China Industrial Microbial Culture Collection Center (CICC), strain number CICC 23264; Bacillus megaterium ( Bacillus megaterium Purchased from the China Agricultural Microbial Culture Collection Center (ACCC), strain number ACCC 04390; Trichoderma longifolia ( Trichoderma long-armed Purchased from the China Agricultural Microbial Culture Collection Center, strain number ACCC 32584; Bacillus subtilis ( Bacillus subtilis Purchased from Shandong Dayi Biotechnology Group Co., Ltd.; Aspergillus aeruginosa ( Aspergillus subolivaceus Purchased from the China Industrial Microbial Culture Collection Center, strain number CICC2307; Aspergillus niger ( Aspergillus niger Purchased from the China Industrial Microbial Culture Collection Center, strain number CICC 41274.

[0039] The culture media involved in the following examples are as follows: LB liquid medium: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, pH 7.2; LB solid medium: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, agar powder 15 g / L, pH 7.2; PDA liquid medium: 200 g / L potato, 20 g / L glucose, natural pH; PDA solid medium: 200 g / L potato, 20 g / L glucose, 15 g / L agar powder, natural pH; Bacterial fermentation medium: glucose 1.5%, peptone 1%, yeast extract 0.6%, potassium dihydrogen phosphate 0.2%, ferrous sulfate heptahydrate 0.002%, zinc sulfate heptahydrate 0.002%, manganese sulfate heptahydrate 0.05%, magnesium sulfate heptahydrate 0.03%, balance water, pH 7.2; Fungal fermentation medium: 3% soluble starch, 1% peptone, 1.5% soybean meal, 0.2% dipotassium hydrogen phosphate, 0.05% potassium dihydrogen phosphate, 0.05% magnesium sulfate, with the remainder being water, pH 6.5.

[0040] Example 1 Preparation of growth-promoting microbial fertilizer: 1. Preparation of Bacillus hygroscopicus fermentation broth (1) Inoculate Bacillus hygroscopicus into LB liquid medium and culture at 37°C and 180 r / min for 16 h to obtain Bacillus hygroscopicus bacterial solution; (2) Streak the Bacillus hygroscopicus culture onto LB solid medium plates and incubate at 37°C for 48 hours; (3) Pick a single colony from the plate and inoculate it into an Erlenmeyer flask containing LB liquid medium. Incubate at 37°C and 180 r / min for 16 h with shaking to obtain seed culture. (4) The seed liquid was transferred to a fermenter containing bacterial fermentation medium at an inoculation rate of 5%, and cultured continuously at 37°C, 200 r / min and 0.05 MPa for 24 h to obtain Bacillus hygroscopicus fermentation broth; (5) Centrifuge the Bacillus hygroscopicus fermentation broth, resuspend it in sterile water, and obtain a viable cell count of 10. 9 Prepare a bacterial suspension of CFU / mL for later use.

[0041] 2. Preparation of Bacillus megaterium fermentation broth Following the fermentation process of Bacillus hygroscopicus, Bacillus megaterium fermentation broth was prepared, and then centrifuged and resuspended until the viable cell count was 10. 9 CFU / mL, for later use.

[0042] 3. Preparation of Trichoderma longifolia fermentation broth (1) Pick a piece of long-branch Trichoderma and inoculate it into PDA liquid culture medium. Incubate at 28℃ and 180r / min for 72h to obtain the culture solution; (2) Dilute the culture medium and spread it onto PDA solid culture medium, and incubate it in an incubator at 28°C for 5 days; (3) Pick a single colony from the plate and inoculate it into an Erlenmeyer flask containing PDA liquid medium. Incubate at 28°C and 200 r / min for 48 h with shaking to obtain seed culture. (4) The seed liquid was transferred to the mold fermentation medium and cultured continuously at 28℃, 220r / min and 0.05Mpa for 48h to obtain the fermentation liquid of Trichoderma longifolia; (5) Centrifuge the fermentation broth of Trichoderma longifolia, resuspend it in sterile water, and obtain an active spore content of 10. 9 Prepare a bacterial suspension of CFU / mL for later use.

[0043] 4. Preparation of Aspergillus viride fermentation broth Following the fermentation process of *Trichoderma longifolia*, a suspension of *Aspergillus viride* was prepared, with an active spore content of 10%. 9 CFU / mL, for later use.

[0044] 5. Preparation of mixed bacterial solution The concentrated suspensions of Bacillus hygroscopicus, Bacillus megaterium, Trichoderma longifolia, and Aspergillus viride were mixed evenly at a live bacteria ratio of 2:1:1:2 to obtain a mixed bacterial solution.

[0045] 6. Preparation of microbial fertilizer Rice straw is placed in a pyrolysis furnace and pyrolyzed at 500°C for 1.5 hours under anaerobic conditions. After pyrolysis, it is cooled to room temperature to obtain biochar.

[0046] Rice straw powder, biochar, and kaolin were mixed evenly in a mass ratio of 7:1:2 to obtain a fertilizer carrier.

[0047] The fertilizer carrier and mixed bacterial solution are mixed evenly at a mass ratio of 5:1, granulated by an extrusion granulator (particle size 3mm), and dried under natural ventilation conditions to obtain the growth-promoting bacterial fertilizer.

[0048] Example 2 Preparation of growth-promoting microbial fertilizer: 1. Preparation of Bacillus hygroscopicus fermentation broth (1) Inoculate Bacillus hygroscopicus into LB liquid medium and culture at 37°C and 180 r / min for 16 h to obtain Bacillus hygroscopicus bacterial solution; (2) Streak the Bacillus hygroscopicus culture onto LB solid medium plates and incubate at 37°C for 48 hours; (3) Pick a single colony from the plate and inoculate it into an Erlenmeyer flask containing LB liquid medium. Incubate at 37°C and 180 r / min for 16 h with shaking to obtain seed culture. (4) The seed liquid was transferred to a fermenter containing bacterial fermentation medium at an inoculation rate of 5%, and cultured continuously at 37°C, 180 r / min and 0.05 MPa for 24 h to obtain Bacillus hygroscopicus fermentation broth; (5) Centrifuge the Bacillus hygroscopicus fermentation broth, resuspend it in sterile water, and obtain a viable cell count of 10. 9 Prepare a bacterial suspension of CFU / mL for later use.

[0049] 2. Preparation of Bacillus megaterium fermentation broth Following the fermentation process of Bacillus hygroscopicus, Bacillus megaterium fermentation broth was prepared, and then centrifuged and resuspended until the viable cell count was 10. 9 CFU / mL, for later use.

[0050] 3. Preparation of Trichoderma longifolia fermentation broth (1) Pick a piece of long-branch Trichoderma and inoculate it into PDA liquid culture medium. Incubate at 28℃ and 180r / min for 72h to obtain the culture solution; (2) Dilute the culture medium and spread it onto PDA solid culture medium, and incubate it in an incubator at 28°C for 5 days; (3) Pick a single colony from the plate and inoculate it into an Erlenmeyer flask containing PDA liquid medium. Incubate at 28°C and 200 r / min for 48 h with shaking to obtain seed culture. (4) The seed liquid was transferred to the mold fermentation medium and cultured continuously at 28℃, 200r / min and 0.05Mpa for 48h to obtain the fermentation liquid of Trichoderma longifolia; (5) Centrifuge the fermentation broth of Trichoderma longifolia, resuspend it in sterile water, and obtain an active spore content of 10. 9 Prepare a bacterial suspension of CFU / mL for later use.

[0051] 4. Preparation of Aspergillus viride fermentation broth Following the fermentation process of *Trichoderma longifolia*, a suspension of *Aspergillus viride* was prepared, with an active spore content of 10%. 9 CFU / mL, for later use.

[0052] 5. Preparation of mixed bacterial solution The concentrated suspensions of Bacillus hygroscopicus, Bacillus megaterium, Trichoderma longifolia, and Aspergillus viride were mixed evenly at a live bacteria ratio of 1.5:1:1:1.5 to obtain a mixed bacterial solution.

[0053] 6. Preparation of microbial fertilizer Rice straw was placed in a pyrolysis furnace and pyrolyzed at 480°C for 2 hours under anaerobic conditions. After pyrolysis, it was cooled to room temperature to obtain biochar.

[0054] Rice straw powder, biochar, and kaolin were mixed evenly in a mass ratio of 7:1:2 to obtain a fertilizer carrier.

[0055] The fertilizer carrier and mixed bacterial solution are mixed evenly at a mass ratio of 4:1, granulated by an extrusion granulator (particle size 3mm), and dried under natural ventilation conditions to obtain the growth-promoting bacterial fertilizer.

[0056] Example 3 Preparation of growth-promoting microbial fertilizer: 1. Preparation of Bacillus hygroscopicus fermentation broth (1) Inoculate Bacillus hygroscopicus into LB liquid medium and culture at 37°C and 180 r / min for 16 h to obtain Bacillus hygroscopicus bacterial solution; (2) Streak the Bacillus hygroscopicus culture onto LB solid medium plates and incubate at 37°C for 48 hours; (3) Pick a single colony from the plate and inoculate it into an Erlenmeyer flask containing LB liquid medium. Incubate at 37°C and 180 r / min for 16 h with shaking to obtain seed culture. (4) The seed liquid was transferred to a fermenter containing bacterial fermentation medium at an inoculation rate of 5%, and cultured continuously at 37°C, 220 r / min and 0.05 MPa for 24 h to obtain Bacillus hygroscopicus fermentation broth; (5) Centrifuge the Bacillus hygroscopicus fermentation broth, resuspend it in sterile water, and obtain a viable cell count of 10. 9 Prepare a bacterial suspension of CFU / mL for later use.

[0057] 2. Preparation of Bacillus megaterium fermentation broth Following the fermentation process of Bacillus hygroscopicus, Bacillus megaterium fermentation broth was prepared, and then centrifuged and resuspended until the viable cell count was 10. 9 CFU / mL, for later use.

[0058] 3. Preparation of Trichoderma longifolia fermentation broth (1) Pick a piece of long-branch Trichoderma and inoculate it into PDA liquid culture medium. Incubate at 28℃ and 180r / min for 72h to obtain the culture solution; (2) Dilute the culture medium and spread it onto PDA solid culture medium, and incubate it in an incubator at 28°C for 5 days; (3) Pick a single colony from the plate and inoculate it into an Erlenmeyer flask containing PDA liquid medium. Incubate at 28°C and 200 r / min for 48 h with shaking to obtain seed culture. (4) The seed liquid was transferred to the mold fermentation medium and cultured continuously at 28℃, 210r / min and 0.05Mpa for 48h to obtain the fermentation liquid of Trichoderma longifolia; (5) Centrifuge the fermentation broth of Trichoderma longifolia, resuspend it in sterile water, and obtain an active spore content of 10. 9 Prepare a bacterial suspension of CFU / mL for later use.

[0059] 4. Preparation of Aspergillus viride fermentation broth Following the fermentation process of *Trichoderma longifolia*, a suspension of *Aspergillus viride* was prepared, with an active spore content of 10%. 9 CFU / mL, for later use.

[0060] 5. Preparation of mixed bacterial solution The concentrated suspensions of Bacillus hygroscopicus, Bacillus megaterium, Trichoderma longifolia, and Aspergillus viride were mixed evenly at a live bacteria ratio of 1.8:1:1:1.8 to obtain a mixed bacterial solution.

[0061] 6. Preparation of microbial fertilizer Rice straw was placed in a pyrolysis furnace and pyrolyzed at 520°C for 1.5 hours under anaerobic conditions. After pyrolysis, it was cooled to room temperature to obtain biochar.

[0062] Rice straw powder, biochar, and kaolin were mixed evenly in a mass ratio of 7:1:2 to obtain a fertilizer carrier.

[0063] The fertilizer carrier and mixed bacterial solution are mixed evenly at a mass ratio of 6:1, granulated by an extrusion granulator (particle size 3mm), and dried under natural ventilation conditions to obtain the growth-promoting bacterial fertilizer.

[0064] Comparative Example 1 Same as Example 1, except that Bacillus hygroscopicus is replaced with Bacillus subtilis.

[0065] Comparative Example 2 Same as Example 1, except that Aspergillus viride is replaced with Aspergillus niger.

[0066] Comparative Example 3 Same as Example 1, except that Bacillus hygroscopicus is removed when preparing the mixed bacterial solution.

[0067] Comparative Example 4 Same as Example 1, except that Bacillus megaterium is removed when preparing the mixed bacterial solution.

[0068] Comparative Example 5 Same as Example 1, except that Trichoderma longifolia is removed when preparing the mixed bacterial solution.

[0069] Comparative Example 6 Same as Example 1, except that Aspergillus viride is removed when preparing the mixed bacterial solution.

[0070] Example 1 of effect verification 1. Experimental Design The experimental field is located at the Zhanjiang Experimental Station of the Chinese Academy of Tropical Agricultural Sciences. The soil texture is saline-alkali soil with a salt content of 0.55%, an alkalinity of 18.5%, a pH value of 8.8, and a soil organic matter content of 7.9 g / kg.

[0071] Test material: 25cm tall Pansy seedlings.

[0072] The experiment consisted of eight treatment groups, with 30 *Pandanus orchid* plants planted in each group. A randomized block design with three replicates was used. The plot area was 15 m² (3 m × 5 m), with a row spacing of 50 cm and a plant spacing of 30 cm. The types and amounts of fertilizer applied to each treatment group are shown in Table 1. During the experiment, field management (irrigation, weeding, etc.) remained consistent across all treatment groups. Drip irrigation was used to avoid excessive watering that could lead to soil salinization.

[0073] Table 1 Experimental Groups Note: The compound fertilizer used is a 15-15-15 NPK compound fertilizer.

[0074] 2. Indicator Testing (1) Growth indicators: 180 days after the Pandanus leaves were planted (harvest period), the plant height, stem diameter, fresh weight of a single plant and dry weight of a single plant of Pandanus leaves in each treatment group were measured, and the yield per mu was calculated.

[0075] (2) Quality indicators: During the harvest period, 20 mature leaves were randomly collected from each treatment group and the content of 2-acetyl-1-pyrrolidone was determined (high performance liquid chromatography, HPLC).

[0076] (3) Soil indicators: Soil samples from the 0-20cm soil layer of each treatment group were collected before planting and after harvest, and soil pH, urease activity (indophenol blue colorimetric method) and phosphatase activity (sodium phenyl phosphate colorimetric method) were measured.

[0077] 3. Experimental Results (1) Growth index results The growth indicators of Panax notoginseng leaves in each treatment group are shown in Table 2.

[0078] Table 2. Results of growth index measurements of Panax notoginseng leaves in each treatment group The results show that treatment group 1 (applied with the growth-promoting microbial fertilizer of this invention) exhibited significantly higher plant height, stem diameter, single-plant fresh weight, single-plant dry weight, and yield per mu (667 square meters) than other treatment groups. The single-plant fresh weight of treatment group 1 reached 426.8 g, an increase of 49.0% compared to the control treatment group 8 (blank fertilizer carrier); the yield per mu reached 1568.3 kg, an increase of 43.2% compared to treatment group 8. Through strain replacement comparison, it was found that the selected Bacillus megaterium and Aspergillus viride have unique functional characteristics and stronger synergistic effects with other strains; replacing them could not achieve the same growth-promoting effect. Through strain type deficiency comparison, it was found that the absence of any one strain significantly reduced the growth-promoting effect, indicating that all four strains are indispensable, and only through their combined action can the optimal growth-promoting effect be achieved.

[0079] (2) Results of quality indicators The quality indicators of Panax notoginseng leaves in each treatment group are shown in Table 3.

[0080] Table 3. Results of quality index determination of Panax notoginseng leaves in each treatment group The quality index test results show that the content of 2-acetyl-1-pyrrolidone in treatment group 1 was significantly higher than that in other treatment groups. As a characteristic aroma substance of pandanus leaves, the content of 2-acetyl-1-pyrrolidone in treatment group 1 reached 58.5 μg / g, which was 47.7% higher than that in control treatment group 8, indicating that the growth-promoting bacterial fertilizer of the present invention can significantly improve the aroma quality of pandanus leaves.

[0081] Comparative Examples 1 and 2 had their strains replaced, while Comparative Examples 3-7 had one strain missing. The content of 2-acetyl-1-pyrrolidone was lower than that of treatment group 1. In particular, the content of Comparative Example 6, which lacked Aspergillus viride, was the most significantly reduced. This indicates that Aspergillus viride plays an important role in promoting the synthesis of characteristic aroma substances. The synergistic effect of the four strains can effectively activate the secondary metabolic pathway of Panax notoginseng leaves and increase the content of aroma substances.

[0082] (3) Soil index results The changes in soil parameters for each treatment group are shown in Table 4.

[0083] Table 4. Changes in soil parameters for each treatment group Soil index testing results showed that treatment group 1 exhibited the most significant decrease in soil pH, dropping from 8.8 before planting to 8.0 after harvest, effectively alleviating soil alkalization. Soil urease and phosphatase activities were significantly increased, rising by 74.1% and 121.1% respectively compared to before planting, and by 51.0% and 76.9% compared to the control group 8. Urease and phosphatase are important hydrolytic enzymes in soil, and their activity levels reflect the soil's nitrogen and phosphorus nutrient conversion capacity. This indicates that the growth-promoting microbial fertilizer of this invention can effectively improve the soil microbial community structure, increase soil enzyme activity, promote soil nutrient conversion, and provide sufficient nutrition for the growth of Panax notoginseng leaves. The decrease in soil pH and the increase in enzyme activity in other treatment groups were less than those in treatment group 1, further demonstrating the superiority of the synergistic effect of the four strains combined in this invention in improving the saline-alkali soil environment.

[0084] In summary, the growth-promoting microbial fertilizer of this invention, through the synergistic effect of four strains, can effectively improve the soil environment of saline-alkali land, reduce soil pH, increase soil enzyme activity, and improve soil nutrient availability. Simultaneously, it can significantly promote the growth and development of pandanus leaves, increasing plant height, stem diameter, single plant fresh weight, and yield per acre; it can also improve the quality of pandanus leaves, increasing the content of the characteristic aroma compound 2-acetyl-1-pyrrolline. This growth-promoting microbial fertilizer has a simple preparation process, low cost, and is environmentally friendly, making it suitable for widespread application in pandanus cultivation in saline-alkali land, and possesses significant agricultural application value and market prospects.

[0085] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A microbial combination for improving yield and aroma quality of Zingiber zerumbet planted in saline-alkali soil, characterized in that, Including Bacillus hygroscopicus ( Bacillus altitudinis ), Bacillus megaterium ( Bacillus megaterium Trichoderma longifolia ( Trichoderma longibrachiatum ) and Aspergillus viride ( Aspergillus subolivaceus ).

2. The microbial combination of claim 1, wherein, The viable cell number ratio of the Bacillus altitudinis, the Bacillus megaterium, the Trichoderma longibrachiatum and the Aspergillus subolivaceus is (1.5-2):1:1:(1.5-2).

3. The microbial combination of claim 2, wherein, The viable cell number ratio of the Bacillus altitudinis, the Bacillus megaterium, the Trichoderma longibrachiatum and the Aspergillus subolivaceus is 2:1:1:

2.

4. Use of the microbial combination of any one of claims 1-3 in the preparation of a microbial fertilizer for promoting the growth of Pogostemonis Herba.

5. Use according to claim 4, characterized in that, The microbial fertilizer for promoting the growth of Pogostemonis Herba has the effect of improving the yield and / or aroma quality of Pogostemonis Herba planted in saline-alkali soil.

6. A PLS leaf growth-promoting bacterial fertilizer, characterized by, The active ingredient comprises the microbial combination of any one of claims 1-3.

7. The Pseudomonas putida growth promoting bacterial inoculant of claim 6, wherein, The microbial fertilizer for promoting the growth of Pogostemonis Herba further comprises a fertilizer carrier.

8. The Pseudomonas putida growth promoting bacterial inoculant of claim 7, wherein, The fertilizer carrier comprises crop straw, biochar and kaolin.

9. Use of the microbial combination of any one of claims 1-3 or the microbial fertilizer for promoting the growth of Pogostemonis Herba of any one of claims 6-8 in improving the yield and / or aroma quality of Pogostemonis Herba planted in saline-alkali soil.

10. A method of improving yield and / or aroma quality of a plant of a Zingiber officinale cv. Roscoe grown in a saline-alkali soil, the method comprising, The use comprises the step of applying the microbial combination of any one of claims 1-3 or the microbial fertilizer for promoting the growth of Pogostemonis Herba of any one of claims 6-8 to Pogostemonis Herba planted in saline-alkali soil.

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