# Source paper archive - Source title: Organic Fertilizer Substitution for Chemical Fertilizer Improves Yield, Medicinal Active Compounds and Amino-Acid Profiles of Dendrobium officinale with Multi-Index Comprehensive Evaluation - Author(s): Zhou, L., Zhou, W., Zhao, X., Liu, H., Yang, H. - Source landing URL: https://www.biorxiv.org/content/10.64898/2026.10.29.762708v1 - PDF URL: https://www.biorxiv.org/content/10.64898/2026.10.29.762708v1.full.pdf - PDF SHA-256: c802ba38e1a785b6785396afb0ae05837d167fb8c34a765cf035a362121d77c9 - Converter: pymupdf4llm 0.0.27 - Limits: Automated PDF text extraction; reading order, equations, tables and formatting may be incomplete or incorrect. Images are omitted, and no OCR was performed. Markup and links are neutralized for safe downloading. Consult the original PDF for the authoritative version. --- [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.762708; this version posted November 3, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.762708) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 1 # Organic Fertilizer Substitution for Chemical Fertilizer Improves Yield, Medicinal Active Compounds and Amino‑Acid Profiles of Dendrobium 2 officinale with Multi‑Index Comprehensive Evaluation 3 Long Zhou [ 1*], Wenna Zhou [ 1], Xudong Zhao [ 1], Huarong Liu [ 1] and Hongbin Yang [ 2*] 4 1.College of Rural Revitalizing and Education of Yunnan, Yunnan Open University, Kunming 650101, China; 2. Ruili 5 Lingrui Agricultural Development Co., Ltd., Ruili 678600, China; 6 ¤ Current address:No. 113, Xuefu Road, Wuhua District, Kunming City, Yunnan Province, China 7 - liuhuarong@ynou.edu.cn; gjshyfzx@163.com 8 9 **Abstract** 10 _Dendrobium officinale_ ( _D. officinale_ ) is an important medicinal horticultural crop. Its wild resources are threatened 11 by over-exploitation. Fertilization regimes that balance yield and medicinal quality are urgently required for its 12 artificial cultivation. This study aimed to investigate the effects of reduced chemical fertilizer combined with organic 13 fertilizer on the yield and quality of _D. officinale_, and screen the optimal combined fertilization pattern, so as to 14 provide a theoretical basis for standardized fertilization in _D. officinale_ cultivation. A bed-cultivation experiment was 15 carried out in greenhouse. Seven treatments were set up, including no fertilizer application (CK), sole chemical 16 fertilizer (CF), 20% organic fertilizer substitution for chemical fertilizer (T20), 40% organic fertilizer substitution for 17 chemical fertilizer (T40), 60% organic fertilizer substitution for chemical fertilizer (T60), 80% organic fertilizer 18 substitution for chemical fertilizer (T80), and single organic fertilizer application (OM). Plant growth traits, yield, 19 active ingredients, protein and amino acid components of _D. officinale_ were determined. The membership function 20 method was adopted for comprehensive evaluation of all treatments. Fertilization significantly improved the growth 21 traits, yield and quality of _D. officinale_ . Compared with CK, stem length, stem diameter and yield under fertilized 22 treatments increased significantly by 15.5%-60.3%, 34.2%-64.7% and 65%-143.2%, respectively; dendrobine, 23 polyphenols and alcohol-soluble extract contents increased significantly by 21.2%-83.7%, 14.0%-49.1% and 24 10.8%-26.9%, respectively. The highest plant growth and yield were observed under sole chemical fertilizer 25 treatment. With the increase of organic fertilizer substitution ratio, plant growth and yield decreased gradually, 26 while medicinal active ingredients including dendrobine, polysaccharides and flavonoids showed a trend of rising 27 first and then falling. A total of 17 amino acids were detected in all treatments. T40 and T60 treatments significantly 28 increased total amino acids, essential amino acids, as well as contents of medicinal, umami and bitter-tasting 29 amino acids, whereas excessive proportion of organic fertilizer led to the decline of amino acid contents. Most 30 growth, quality and amino acid indicators showed significant or extremely significant positive correlations with each 31 other. The comprehensive evaluation order by membership function method was T40 > T60 > CF > T20 > T80 > 32 OM > CK, and T40 obtained the highest comprehensive evaluation value. Sole chemical fertilizer contributes to 33 yield increase of _D. officinale_, while combined application with organic fertilizer improves its medicinal quality. The 34 40 % organic substitution scheme provides a feasible fertilization strategy for high‑quality greenhouse production 35 of this medicinal horticultural plant. 36 37 **1. Introduction** 38 _D. officinale_, a perennial epiphytic herb belonging to Orchidaceae Dendrobium genus, is a precious medicinal horticultural crop in China. It has attracted extensive attention in pharmaceutical and agricultural 40 research owing to its unique medicinal efficacy and abundant nutritional components[1]. Its medicinal value has 41 been well‑documented in ancient Chinese herbal classics. Compendium of Materia Medica records that it could 42 “replenish yin and essence, and tonify the stomach and intestines upon long‑term consumption”. Ranked first 43 among the legendary “Nine Immortal Herbs” of traditional Chinese medicine and reputed as “life‑saving immortal 44 herb” among folk communities, _D. officinale_ is rich in bioactive substances including polysaccharides, alkaloids, 45 polyphenols and flavonoids. Modern pharmacological studies have verified its multiple pharmacological activities 46 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.762708; this version posted November 3, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.762708) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) such as antioxidant capacity, immunomodulation, anti‑tumor effect and blood‑pressure‑lowering and 47 ‑ ‑ vascular protective functions, endowing it with high medicinal and health care values[2]. 48 _D. officinale_ possesses complex nutritional profiles and outstanding nutritional value, and abundant mineral ‑ elements as well as functional nutrients constitute the material basis for its health promoting effects[3]. 50 Dendrobium polysaccharides, alcohol‑soluble extracts and dendrobine are core quality indicators for evaluating 51 ‑ medicinal materials of _D. officinale_ and play vital roles in medical and health care applications[4]. As the primary 52 functional component, dendrobium polysaccharides can effectively enhance organismal immunity, regulate blood 53 ‑ ‑ ‑ glucose, exert anti oxidation and anti aging effects, and inhibit the proliferation of tumor cells[5]. Alcohol soluble 54 extracts mainly consist of polyphenols, flavonoids, alkaloids and other active compounds. Among them, 55 polyphenols and flavonoids exhibit remarkable free‑radical scavenging capacity to delay cellular senescence, 56 together with physiological functions including anti‑inflammation, anti‑biosis and immunoregulation[6]. 57 ‑ Dendrobine, a characteristic alkaloid specific to _D. officinale_, exerts prominent effects in anti fatigue, memory 58 ‑ improvement, organism development promotion and anti tumor activities[7]. Furthermore, _D. officinale_ contains 59 multiple essential amino acids that human bodies cannot synthesize independently. Components such as 60 glutamic acid, aspartic acid, leucine and alanine serve as critical material carriers for its clinical health‑care 61 efficacy. They participate in diverse human physiological metabolic processes and perform irreplaceable 62 functions in maintaining physical health, facilitating growth and development, and strengthening immune 63 functions[8−10]. Accordingly, systematic investigation into the composition and concentration characteristics of 64 ‑ nutritional and active components in _D. officinale_ can not only clarify its medicinal and health care mechanisms, 65 but also provide theoretical support for the scientific exploitation and efficient utilization of _D. officinale_ 66 resources[11]. 67 Benefiting from its superior medicinal and economic values, the market demand for _D. officinale_ keeps increasing. Nevertheless, long‑term excessive wild collection, destruction of natural habitats and low reproduction 69 coefficient of wild populations have led to sharp decline of its wild natural resources. Wild _D. officinale_ is now 70 listed as critically endangered and can hardly satisfy the requirements of industrial development[12]. Artificial 71 cultivation acts as a core approach for germplasm conservation of wild _D. officinale_ and resolution of market 72 supply‑demand contradiction, and it represents the dominant pattern for current industrial production of 73 Dendrobium. With continuous innovation in cultivation techniques, greenhouse seedling raising and large‑scale 74 planting have been widely popularized, effectively solving industrial bottlenecks such as seedling shortage and 75 ‑ low transplant survival rate, and laying foundations for large scale production of _D. officinale_ [13]. However, 76 practical production has revealed a prevalent problem in artificially‑cultivated _D. officinale_ : yield increases while 77 quality degrades. The content of bioactive components in cultivated medicinal materials is markedly lower than 78 that in wild counterparts, which seriously impairs its commodity value and restricts the high‑quality development 79 of _D. officinale_ industry. Hence, exploring scientific cultivation regulation technologies to realize synergistic 80 ‑ improvement in yield and quality of artificially cultivated _D. officinale_ has become a key and challenging issue in 81 current Dendrobium cultivation research. 82 Diverse artificial cultivation patterns have been developed for _D. officinale_, mainly including greenhouse bed cultivation, under‑forest epiphytic cultivation on living trees, and simulated‑wild epiphytic cultivation on rocks. 84 Different cultivation modes, environmental conditions and management practices significantly affect 85 ‑ ‑ growth metabolism, accumulation of bioactive ingredients and medicinal quality of _D. officinale_ [14 18]. Existing 86 ‑ ‑ studies have demonstrated that _D. officinale_ under under forest tree epiphytic cultivation presents significantly 87 ‑ higher contents of polyphenols, flavonoids, free amino acids and in vitro antioxidant activities than those under 88 greenhouse pot cultivation and board‑attached cultivation[19]. Higher accumulation of flavonoids and flavonols is 89 ‑ ‑ observed under rock epiphytic and tree epiphytic systems compared with greenhouse cultivation, whereas 90 ‑ rock epiphytic samples show advantages in polysaccharide content and antioxidant activity[20]. Meanwhile, 91 researchers have carried out regulatory experiments on environmental factors including cultivation substrate, 92 planting cycle and light intensity, confirming that appropriate cultivation conditions can effectively boost plant 93 growth and development and optimize quality indicators of _D. officinale_ . 94 Apart from environmental factors, exogenous substance regulation and fertilization management are important strategies to promote plant growth and improve quality of _D. officinale_ . Liu et al.[21] reported that 96 exogenous application of 2,3‑butanediol fermentation broth, γ‑aminobutyric acid, melatonin and other substances 97 could simultaneously promote biomass accumulation as well as the synthesis of polysaccharides and alkaloids in 98 protocorms of _D. officinale_ . In terms of fertilization regulation, available researches mainly focus on single 99 ‑ fertilization and foliar fertilization modes. Spraying nano calcium fertilizer facilitates the accumulation of 100 endogenous phytohormones and soluble sugars, and accelerates stem elongation and tillering of _D._ 101 _officinale_ [22]. Application of botanical fertilizer markedly enhances antioxidant capacity and promotes 102 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.762708; this version posted November 3, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.762708) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) polysaccharide accumulation[23]. Special foliar fertilizers and combined organic‑inorganic foliar fertilizers can 103 improve growth parameters and quality traits of _D. officinale_ to varying degrees, and optimal fertilizer 104 concentration maximizes fertilization benefits. Fertilization has been proven as a critical measure regulating yield 105 and quality of _D. officinale_ . However, current researches mostly concentrate on single fertilizer application and 106 foliar fertilization. Systematic studies on chemical fertilizer reduction and scientific combined application of 107 organic and chemical fertilizers remain scarce. Particularly, fertilization ratio schemes balancing high yield and 108 superior quality of _D. officinale_ are insufficient, which cannot meet the production demands for standardized and 109 high‑quality cultivation[24]. 110 Current studies concerning quality improvement of _D. officinale_ predominantly focus on environmental regulation such as cultivation pattern, substrate, light and growth cycle, and relatively sound technical systems for 112 cultivation optimization have been established[25‑26]. Nevertheless, research gaps still exist in fertilization 113 regulation for synergistic promotion of yield and quality. 114 Fertilization constitutes a core field management measure for yield increase and quality optimization of crops. Combined application of organic and inorganic fertilizers is an efficient fertilization mode to stabilize crop 116 yield and optimize quality, which has been validated in multiple cash crops. However, its application in 117 ‑ greenhouse cultivated _D. officinale_ has rarely been reported. Few studies have systematically evaluated how 118 ‑ organic substitution gradients alter not only yield and secondary metabolites, but also comprehensive amino acid 119 ‑ ‑ ‑ profiles (including medicinal and umami taste amino acids) for greenhouse bed cultivated _D. officinale_ . 120 Multi‑indicator comprehensive assessment balancing agronomic yield and nutraceutical quality remains 121 insufficient. 122 ‑ Greenhouse bed cultivated _D. officinale_ was adopted as experimental material in this study. Treatments with different gradients of organic manure substituting chemical fertilizer were set up to systematically investigate the 124 effects of reduced chemical fertilizer combined with organic manure on growth‑yield, medicinal bioactive 125 ‑ components and amino acid profiles of _D. officinale_ . The optimal fertilization ratio was screened out through 126 multi‑indicator comprehensive evaluation. This study aims to clarify the regulatory effects of combined 127 ‑ organic chemical fertilization on yield and quality of _D. officinale_, identify fertilization patterns balancing high yield 128 and good quality, and provide scientific fertilization schemes as well as theoretical foundations for standardized 129 ‑ ‑ high quality greenhouse cultivation of _D. officinale_, so as to contribute to quality benefit improvement and green 130 sustainable development of Dendrobium industry. 131 **2. Materials and Methods** 132 **2.1 Overview of the Experimental Site** 133 ‑ The experiment was carried out in a standardized multi span greenhouse at the National Dendrobium Germplasm Resource Bank in Ruili, Dehong Prefecture, Yunnan Province (23°52′26″ N, 97°56′31″ E). Located in 135 the tropical monsoon climate zone of western Yunnan, the site sits at an altitude of 780 m, with an annual average 136 temperature of 21.3 ℃ and annual precipitation of 1450 mm. It is frost‑free year‑round, with sufficient solar 137 radiation and favorable humidity, representing a genuine producing area suitable for _D. officinale_ cultivation. 138 The greenhouse features controllable environmental conditions: annual indoor temperature ranges from 18 ℃ to 28 ℃, relative air humidity is maintained at 75%‑90%, and the shading rate is set at 70%. With good ventilation, 140 free from industrial pollution and low risk of severe pest and disease outbreaks, the greenhouse meets the 141 standards for germplasm conservation and standardized cultivation trials of Chinese medicinal materials. 142 The cultivation substrate consisted of decomposed pine bark, coconut husk chips and volcanic rock at a volume ratio of 6 ∶ 3 ∶ 1 (v/v). The substrate was fully decomposed, free of insect eggs and mildew, and disinfected by 144 sunlight exposure prior to use. Its basic physicochemical properties were as follows: pH 5.5‑6.2, organic matter 145 42.6 g·kg ⁻ ¹, total nitrogen 1.83 g·kg ⁻ ¹, available phosphorus 36.5 mg·kg ⁻ ¹, and available potassium 128.4 mg·kg ⁻ ¹. 146 Characterized by loose texture, good aeration and favorable water‑fertilizer retention capacity, the substrate 147 received no fertilizer application before transplantation and exhibited uniform fertility, so as to eliminate the 148 interference of background fertility differences across experimental units. 149 **2.2 Experimental Materials** 150 ‑ ‑ ‑ 1 year old acclimated tissue cultured plantlets of _D. officinale_ were obtained from the National Dendrobium Germplasm Resource Bank in Ruili. The plantlets had pure genetic background, uniform growth performance, and 152 ‑ ‑ were free of pests, diseases and mechanical damage. Each seedling was 8 10 cm high with 2 3 valid tillers. 153 ‑ Uniform sized plantlets were selected for transplantation to guarantee consistency of experimental materials. 154 The tested organic fertilizer was a special organic fertilizer independently developed by the base (CN202510102937.8). It was fermented with wormcast, _Pleurotus eryngii_ mushroom residue and mugwort straw 156 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.762708; this version posted November 3, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.762708) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) as raw materials. The fermentation substrate was soaked in aqueous solution containing _D. officinale_ extract and 157 mugwort extract, followed by inoculation with _Bacillus subtilis_ strain H728 for thorough decomposition. This organic 158 fertilizer contained 2.5% total nitrogen with a moisture content of 28.0%. The chemical fertilizer adopted was a 159 ‑ ‑ ‑ ‑ balanced compound fertilizer (N P ₂ O ₅ K ₂ O 15 15 15, total nutrient ≥45%), purchased from Yunnan Yuntianhua 160 Co., Ltd. 161 **2.3 Experimental Design** 162 _D. officinale_ was cultivated on elevated growing beds approximately 60 cm above the ground inside the ‑ greenhouse. Each bed measured 12 m × 1.5 m, with a 60 cm spacing between adjacent beds. A single factor 164 randomized block design was adopted in this experiment, with equivalent nitrogen supply as the core principle for 165 organic fertilizer substitution. Seven treatments of combined organic‑inorganic fertilization were set: no fertilizer 166 application (CK), sole chemical fertilizer (CF), 20% organic fertilizer substitution for chemical fertilizer (T20), 40% 167 organic fertilizer substitution for chemical fertilizer (T40), 60% organic fertilizer substitution for chemical fertilizer 168 (T60), 80% organic fertilizer substitution for chemical fertilizer (T80), and sole organic fertilizer (OM). Each 169 treatment was replicated three times, generating a total of 21 experimental plots. Each plot covered an area of 170 4 m × 1.5 m = 6 m², where 171 clumps of _D. officinale_ were planted at a planting density of 15 cm × 20 cm with 171 consistent planting depth. 172 ‑ The balanced compound fertilizer served as the reference fertilizer for equivalent nitrogen treatment. The application rate of organic fertilizer was calculated based on its total nitrogen content and moisture content. The 174 annual nitrogen application rate for _D. officinale_ was set at 180 kg·ha [-1] . The full amount of organic fertilizer was 175 thoroughly mixed with the cultivation substrate and applied as basal fertilizer by layered broadcasting; 176 transplantation was performed after substrate maturation. Chemical nitrogen fertilizer was applied four times 177 throughout the growing cycle, accounting for 40%, 15%, 30% and 15% of total nitrogen at basal stage, sprouting 178 stage, vigorous‑growth stage and quality‑accumulation stage respectively, and uniform irrigation scheduling was 179 applied for all experimental plots to maintain substrate moisture. 180 ‑ Uniform field management including watering, ventilation, shading, green pest disease control and weeding was implemented for all treatments. No foliar fertilizer or plant growth regulators were sprayed during the whole 182 experimental period. The trial lasted for one complete growth cycle (March to February of the subsequent year). 183 Details of fertilization treatments are shown in Table 1. 184 Table 1 Experimental Treatments 185 Fertilizer application amount(kg·ha [-1] ) Treatment Fertilization mode Compound fertilizer(15-15-15) Organic fertilizer(2.5%N) CK No fertilization 0 0.0 CF Sole chemical fertilizer 1200 0.0 T20 T40 T60 T80 20% organic fertilizer + 80% chemical fertilizer 40% organic fertilizer + 60% chemical fertilizer 60% organic fertilizer + 40% chemical fertilizer 80% organic fertilizer + 20% chemical fertilizer 960 2000.0 720 4000.0 480 6000.0 240 8000.0 OM Sole organic fertilizer 0 10000.0 186 **2.4 Determination of Measurement Indicators** 187 2.4.1 Determination of Growth Indices 188 ‑ Plant growth traits and yield indices were measured after one complete annual planting cycle of _D. officinale_ . ‑ The five point sampling method was adopted. Five sampling points were randomly set within each experimental 190 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.762708; this version posted November 3, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.762708) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) plot, and three clumps of healthy _D. officinale_ plants with uniform growth and free of pests and diseases were 191 selected at each point as test samples. Growth indices including stem length, stem diameter, internode length, 192 ‑ number of current year fresh stems, chlorophyll content and yield were measured and recorded. 193 Specific measurement procedures were as follows: Stem length per clump was measured with a meter ruler from the second internode at the base of pseudobulb to the apex of pseudobulb. Stem diameter was determined at 195 the middle part of the fourth internode upward from the pseudobulb base using a vernier caliper to guarantee 196 consistent measuring position and reliable data. The total number of internodes and leaves per clump were 197 manually counted, and the average internode length was calculated by the formula: 198 Internode length = stem length / number of internodes. ‑ The number of newly emerged stems in the current year was counted and recorded as the number of fresh stems. Chlorophyll content of functional leaves was determined by a SPAD‑502 portable chlorophyll meter, with 201 three replicate measurements per plant for averaging. 202 ‑ After determination of morphological indices, the above ground stems and leaves of _D. officinale_ were harvested. Total fresh weight of samples was accurately weighed and recorded using an electronic balance. Fresh 204 samples were divided into two subsamples for subsequent treatments. The first subsample was cut into pieces 205 and mixed evenly, subjected to fixation at 90 ℃ for 15 min in an oven, and then dried to constant weight at a 206 constant temperature of 60 ℃ . Dry‑sample weight was recorded. Water content was calculated according to the 207 formula: 208 Water content (%) = (fresh weight − dry weight) / fresh weight × 100%. ‑ The dried samples were ground, sieved and stored in sealed containers for subsequent quality index determination. The second set of fresh stem samples were packed into sampling bags, snap‑frozen in liquid 211 nitrogen for 30 min, and preserved in an ultra‑low‑temperature refrigerator for the assay of medicinal bioactive 212 components. 213 2.4.2 Determination of Quality Indices 214 Quality indices were assayed using the two sets of preserved _D. officinale_ samples mentioned above. Three technical replicates were arranged for each index. Detailed determination methods and calculation formulas are 216 described below. 217 ‑ Alcohol soluble extracts (AE) were determined in accordance with the standard method specified in Volume I of _Pharmacopoeia of the People’s Republic of China_ (2020 Edition)[27]. 219 Contents of polysaccharides (Po) and total flavonoids (TF) were measured by ultraviolet spectrophotometry following Wu et al.[28]. The unified calculation formula was: 221 Index content (mg·g [-1] ) = C × V / M, ‑ where C = concentration of sample solution derived from standard curve fitting; V = total volume of sample extract; M = actual weighed mass of sample. Rutin was used as the reference substance for total‑flavonoid 224 determination. 225 ‑ Soluble protein content was determined via Coomassie brilliant blue method described in Volume III of _Pharmacopoeia of the People’s Republic of China_ (2020 Edition)[29]. 227 Total alkaloids (AL, expressed as dendrobine equivalent) were assayed by acid‑dye colorimetry according to Ao et 228 al.[30]. The formula was: 229 Alkaloid content (mg·g [-1] ) = C × (V ÷ V ₁ ) ÷ W, where C = dendrobine concentration in reaction solution (μg); V = total volume of sample extract (mL); V ₁ = volume of reaction solution for measurement (mL); W = mass of tested sample (g). 232 ‑ Crude fiber (CF) was measured following the national standard GB/T 5009.10 2003[31]. The formula was: ‑ Crude fiber content (%) = M / m × 100%, ‑ where m = mass of tested sample (g); M = mass of residue or mass loss after high temperature ashing (g). Total polyphenol content was determined by ultraviolet spectrophotometry with gallic acid as reference substance, as described by Huang et al.[6], with minor optimization of sample pretreatment. The calculation 237 formula was consistent with that for total flavonoids: 238 Polyphenol content (mg/g) = C × V / M, and parameters were defined as above. ‑ ‑ A total of 17 amino acid species were quantified referring to the national food safety standard GB 5009.124‑2016 _National Food Safety Standard_ _‑Determination of Amino Acids in Foods_ [32]. According to 242 biosynthetic origin and human nutritional requirements, the 17 amino acids were classified into essential amino 243 acids (Threonine‑Thr, Phenylalanine‑Phe, Lysine‑Lys, Valine‑Val, Methionine‑Met, Isoleucine‑Ile, Leucine‑Leu) 244 ‑ ‑ ‑ ‑ ‑ ‑ and non essential amino acids (Aspartic acid Asp, Tyrosine Tyr, Serine Ser, Glutamic acid Glu, Glycine Gly, 245 ‑ ‑ ‑ ‑ Alanine Ala, Cystine Cys, Histidine His, Arginine Arg, Proline). Based on physiological properties and functions, 246 amino acids were further divided into umami‑taste amino acids (4 species: Asp, Glu, Gly, Ala), bitter‑taste amino 247 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.762708; this version posted November 3, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.762708) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) acids (8 species: Val, Met, Ile, Leu, Tyr, Phe, His, Arg) and medicinal amino acids (9 species: Asp, Glu, Gly, Arg, 248 Met, Phe, Leu, Lys, Tyr). The nutritional differences in amino‑acid profiles among fertilization treatments were 249 reflected by calculating their respective mass fractions. The formulas were as follows: 250 ‑ ‑ ‑ Essential amino acid fraction (EAA/TAA, %) = content of essential amino acids / total amino acid content ‑ ‑ ‑ ‑ ‑ Non essential amino acid fraction (NEAA/TAA, %) = content of non essential amino acids / total amino acid content 253 ‑ ‑ ‑ Medicinal amino acid fraction (MAA/TAA) = content of medicinal amino acids / total amino acid content **2.5 Comprehensive Evaluation of Growth and Quality of D. officinale** 255 ‑ ‑ The membership function method[33 34] was applied for comprehensive evaluation of growth and quality of _D. officinale_ under different fertilization treatments. 257 ‑ ‑ For positively correlated indices, membership function values { _u_ ( _X_ _j_ )} were calculated using Equation (1). 258 𝜇(𝑋 𝑗 ) = (𝑋(𝑋 𝑚𝑎𝑥𝑗 −𝑋−𝑋 𝑚𝑖𝑛𝑚𝑖𝑛 ) ) (1) ‑ For indices negatively correlated with plant growth and quality, membership function values were calculated using Equation (2). 261 ~~−~~ (𝑋 𝑗 −𝑋 𝑚𝑖𝑛 ) 𝜇(𝑋 𝑗 ) = 1 (𝑋 𝑚𝑎𝑥 −𝑋 𝑚𝑖𝑛 ) (2) Standard coefficients( _V_ _j_ ) and weights( _W_ _j_ ) for each index were computed via Equation (3) and Equation (4). 𝑉 = 𝑗 𝑋 𝑗 (3) 𝑊 𝑗 = ∑ [𝑛] 𝑗=1 [𝑉] 𝑗 (4) The comprehensive evaluation value ( _D_ ) was obtained by Equation (5). 𝐷= ∑ [𝑛] 𝑖=1 [[𝜇(𝑋] [) ∙𝑊] []] (5) 𝑊 = 𝑗 𝑉 𝑗 ∑ [𝑛] 𝐷= ∑ 𝑖=1 [[𝜇(𝑋] 𝑗 [) ∙𝑊] 𝑗 []] (5) Where: ‑ _X_ _j_ represents the measured value of the j th index; ‑ _X_ _min_ represents the minimum value of the j th index; ‑ _X_ _max_ represents the maximum value of the j th index; ‑ ‑ ‑ _X_  _ij_ represents the membership function value of the j th index under the i th treatment; ‑ ‑ _X_ _j_ represents the average membership function value of the j th index across all treatments; _D_ represents the comprehensive evaluation value; higher D values indicate better growth performance and higher medicinal quality of _D. officinale_ ; 275 _n_ represents the renumber of experimental treatments. **2.6 Statistical Analysis and Graphing** 277 ‑ Raw experimental data were preliminarily processed in Excel 2020. One way analysis of variance (ANOVA) ‑ ‑ and correlation analysis computation were performed using IBM SPSS Statistics 27. Duncan’s multiple range test 279 was adopted for post‑hoc comparison. Origin 2021 was used for figure plotting. 280 **3. Results** 281 **3.1. Effects of Different Fertilization Treatments on Growth Traits of** _**D. officinale**_ 282 Different fertilization treatments exerted significant effects on the growth traits of _D. officinale_ (Table 1). Compared with the non‑fertilization control (CK), sole chemical fertilizer application, reduced chemical fertilizer 284 combined with organic manure, and sole organic fertilizer application all significantly modified stem length, stem 285 diameter, internode length, leaf number, chlorophyll content, number of fresh stems and yield of _D. officinale_ . 286 These parameters increased by 15.5%‑60.3%, 34.2%‑64.7%, 10.3%‑59.0%, 59.7%‑110.3%, 6.0%‑36.1%, 287 ‑ ‑ ‑ 20.1% 108.5% and 65.0% 143.2%, respectively, relative to the non fertilization treatment ( _P_ <0.05). Compared 288 with CK, the dry‑matter conversion rate of CF, T20, T60 and OM treatments increased significantly by 7.2%, 3.4%, 289 3.8% and 3.0%, respectively ( _P_ <0.05). 290 ‑ Among all fertilization regimes, the sole chemical fertilizer treatment (CF) achieved the optimal performance ‑ in growth traits and yield of _D. officinale_ . Treatments with chemical fertilizer reduction combined with organic 292 manure (T20, T40, T60, T80) and sole organic manure (OM) reduced plant growth performance and yield. Growth 293 traits and yield exhibited a gradual declining trend as the proportion of chemical‑fertilizer substitution by organic 294 manure increased. Relative to the CF treatment, stem length, internode length and number of fresh stems 295 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.762708; this version posted November 3, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.762708) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) decreased significantly by 9.7%‑27.9%, 14.9%‑30.7% and 18.9%‑42.4% under T40, T60, T80 and OM treatments; 296 stem diameter, leaf number, chlorophyll content, yield and dry‑matter conversion rate also declined significantly by 297 ‑ ‑ ‑ ‑ ‑ 11.5% 18.5%, 7.5% 24.1%, 7.8% 22.1%, 4.2% 32.1% and 3.2% 5.6%, respectively ( _P_ <0.05). 298 Table 1 Effects of different fertilization treatments on growth traits of _D. officinale_ 299 **Tre** **atm** **ent** **Stem length** **(cm)** **Stem** **diameter** **(mm)** **Internode** **length (cm)** **Number of** **fresh shoots** **(Branch** **Cluster** **[-1]** **)** **Number of** **leaves** **(leaves** **Cluster** **[-1]** **)** **Chlorophyll** **(mg kg** **[-1]** **)** **Yield (kg ha** **[-1]** **)** **Dry weight** **ratio (%)** CK 36.23±2.28e 3.16±0.09f 1.33±0.04e 151.5±3.7e 4.25±0.09e 10.5±0.7e 3487.5±130.1f 23.82±0.39c CF 58.1±1.76a 5.2±0.06a 2.12±0.14a 318.6±4.3a 5.79±0.03a 21.9±1.2a 8482.5±170.2a 25.53±0.28a T20 54.57±0.9a 4.6±0.04b 1.92±0.05a 294.7±6.6b 5.34±0.11b 19.5±0.7a 8130±73.2b 24.62±0.16b T40 52.47±0.8b 4.52±0.05bc 1.8±0.03b 275.4±6.6b 5.14±0.1b 17.7±0.5b 7999.5±108.6b 24.4±0.06bc T60 49.43±1.06c 4.43±0.06cd 1.67±0.03c 263.7±4.4c 4.87±0.1c 14.7±0.2c 7427.5±127.8c 24.72±0.25b T80 46.2±0.6c 4.39±0.04d 1.54±0.05d 241.9±7d 4.56±0.11c 14.4±0.6c 6714.5±109.6d 24.09±0.67bc OM 41.87±1.46d 4.24±0.07e 1.47±0.03d 248.5±7.8d 4.51±0.11d 12.6±0.5d 5759.5±148.8e 24.52±0.38b Note: CK: No fertilization; CF: Sole chemical fertilizer; T20: 20% organic fertilizer + 80% chemical fertilizer; T40: 40% organic 300 fertilizer + 60% chemical fertilizer; T60: 60% organic fertilizer + 40% chemical fertilizer; T80: 80% organic fertilizer + 20% chemical 301 fertilizer; OM: Sole organic fertilizer. Values are means ± standard deviation ( _n_ = 3). Values with the same lower-case letters are not 302 significantly different among different treatments at the 5% level by the LSD. The same below. 303 **3.2. Effects of Different Fertilization Treatments on Quality of** _**D. officinale**_ 304 Different fertilization treatments significantly affected the quality of _D. officinale_ . All fertilized treatments ‑ exhibited higher contents of dendrobine, polysaccharides, polyphenols, flavonoids, alcohol soluble extracts and 306 protein compared with the non‑fertilization control (Figure 1). Sole chemical fertilizer application, reduced chemical 307 fertilizer combined with organic manure, and sole organic fertilizer application all significantly altered the contents 308 ‑ ‑ of dendrobine, polyphenols and alcohol soluble extracts. Relative to the non fertilization treatment (CK), these 309 ‑ ‑ ‑ components increased significantly by 21.2% 83.7%, 14.0% 49.1% and 10.8% 26.9%, respectively ( _P_ <0.05) 310 (Figure 1a, b, c). 311 In addition, compared with CK, polysaccharide contents in T20, T40, T60, T80 and OM treatments increased significantly by 25.1%, 32.6%, 54.5%, 34.6% and 26.1% ( _P_ <0.05); flavonoid contents increased significantly by 313 18.1%, 49.1%, 45.6%, 38.6% and 33.8% ( _P_ <0.05). Protein contents in CF, T20, T40, T60 and OM treatments rose 314 ‑ significantly by 44.6%, 36.6%, 33.1%, 13.1% and 12.9%. Crude fiber contents in CF and T20 treatments increased 315 significantly by 31.0% and 21.3% ( _P_ <0.05) (Figure 1). 316 Quality indicators of _D. officinale_ were significantly modified by combined application of reduced chemical fertilizer and organic manure (T20, T40, T60, T80) as well as sole organic manure treatment (OM). As the 318 ‑ proportion of chemical fertilizer reduction increased, the quality of _D. officinale_ showed a trend of first increasing 319 and then decreasing. Compared with the CF treatment, dendrobine, polysaccharide and flavonoid contents in T20, 320 T40, T60, T80 and OM treatments increased significantly by 26.2%‑51.6%, 12.1%‑38.4% and 15.5%‑45.8%, 321 respectively. Polyphenol contents in T40, T60, T80 and OM treatments increased significantly by 24.3%, 16.8%, 322 16.4% and 8.3%, whereas protein contents decreased significantly by 7.9%, 21.8%, 26.5% and 21.9%, and 323 ‑ ‑ crude fiber contents decreased significantly by 30.4%, 30.6%, 23.2% and 20.0%. Notably, alcohol soluble extracts 324 in the T40 treatment increased significantly by 14.5% ( _P_ <0.05). 325 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.762708; this version posted November 3, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.762708) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 326 327 **Figure 1.** Effects of different fertilization treatments on quality of _D. officinale_ . CK: No fertilization; CF: Sole chemical fertilizer; T20: 328 20% organic fertilizer + 80% chemical fertilizer; T40: 40% organic fertilizer + 60% chemical fertilizer; T60: 60% organic fertilizer + 329 40% chemical fertilizer; T80: 80% organic fertilizer + 20% chemical fertilizer; OM: Sole organic fertilizer. Values are means ± 330 standard deviation (n = 3). Values with the same lower-case letters are not significantly different among different treatments at the 331 5% level by the LSD. The same below. 332 **‑** **3.3. Effects of Different Fertilization Treatments on Amino** **Acid Composition of** _**D. officinale**_ 333 Amino acids are fundamental substances for all living organisms. In plants, amino acids are synthesized via biochemical reactions driven by various enzymes and enzyme systems during photosynthesis. For plants 335 themselves, amino acids can promote root development, seedling vigour, enhance leaf photosynthetic capacity 336 ‑ and resistance to pests and diseases, thereby improving yield. For human beings, plant derived amino acids 337 supply essential amino acids that cannot be synthesized de novo by the human body and play an indispensable 338 ‑ role in life activities. The determination results of amino acid composition of _D. officinale_ under different fertilization 339 ‑ treatments are shown in Table 2. As presented in Table 2, all fertilization treatments yielded complete amino acid 340 profiles, with a total of 17 amino acids detected in all samples. 341 ‑ ‑ Different fertilization treatments significantly affected the total amino acid content and individual amino acid concentrations of _D. officinale_ (Table 2). In terms of total content and proportion of amino acids, sole chemical 343 fertilizer application, reduced chemical fertilizer combined with organic manure, and sole organic fertilizer 344 ‑ ‑ ‑ application significantly altered total amino acid content and total non essential amino acid content. Compared 345 ‑ ‑ with the non fertilization control (CK), these two parameters increased significantly by 25.9% 213.8% and 346 ‑ ‑ 39.1% 228.5%, respectively ( _P_ <0.05). Except for the OM treatment, essential amino acid content increased 347 significantly by 58.8%‑197.4%. Regarding the proportional relationships among essential amino acids, 348 non‑essential amino acids and total amino acids, the ratios of EAA/TAA, NEAA/TAA and EAA/NEAA ranged from 349 0.355 to 0.438, 0.562 to 0.665, and 0.504 to 0.778 across fertilization treatments, respectively. Compared with CK, 350 the CF treatment significantly raised EAA/TAA and EAA/NEAA by 9.9% and 17.7%, while significantly decreasing 351 NEAA/TAA by 6.6%. For T40, T60, T80 and OM treatments, EAA/TAA and EAA/NEAA declined significantly by 352 ‑ ‑ ‑ 5.2% 15.8% and 8.3% 23.8%, whereas NEAA/TAA increased significantly by 3.4% 10.5% ( _P_ <0.05). 353 ‑ For amino acid composition, fertilization generally increased the concentrations of most individual amino acids, except for serine and cystine. As chemical‑fertilizer dosage was reduced and organic‑manure proportion 355 increased, the content of each amino‑acid component exhibited a trend of first increasing and then decreasing. 356 Relative to CK, the contents of leucine, aspartic acid, glutamic acid, arginine and proline under all fertilization 357 treatments increased significantly by 62.9%‑277.9%, 100.6%‑547.8%, 101.8%‑458.6%, 16.6%‑111.9% and 358 27.5%‑196.4%, respectively. Furthermore, compared with CK, threonine and alanine contents increased 359 significantly by 67.3%‑187.1% and 32.5%‑111.9% in CF, T20, T40 and T60 treatments. Phenylalanine, lysine, 360 valine, methionine, isoleucine, tyrosine and histidine increased significantly by 22.6%‑148.6%, 77.9%‑142.8%, 361 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.762708; this version posted November 3, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.762708) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 64.4%‑236.3%, 65.7%‑270.0%, 38.9%‑211.8%, 97.8%‑266.2% and 40.0%‑481.6% in CF, T20, T40, T60 and T80 362 treatments, respectively ( _P_ <0.05). In addition, compared with CK, phenylalanine content decreased significantly 363 by 33.1% under OM. Serine content increased significantly by 82.7% and 92.2% in T40 and T60, but decreased 364 significantly by 9.0% and 18.9% in T80 and OM. Glycine content dropped significantly by 84.5% under CF, 365 whereas it increased significantly by 20.7%, 94.4%, 154.2% and 56.4% in T20, T40, T60 and T80. Cystine content 366 decreased significantly by 28.1% under CF and increased significantly by 26.6% under OM ( _P_ <0.05). No 367 significant differences were observed for other treatments. 368 Combined application of reduced chemical fertilizer with organic manure (T20, T40, T60, T80) and sole ‑ ‑ organic manure (OM) mostly exerted significant influences on total amino acid content and individual amino acid 370 ‑ ‑ concentrations of _D. officinale_ . With increasing proportion of chemical fertilizer substitution, total amino acid 371 ‑ ‑ ‑ content and each amino acid component generally showed an upward then downward trend, with optimal 372 performance observed in T40 and T60 treatments. In respect of total content and ratios of amino acids, compared 373 with CF, essential amino‑acid content increased significantly by 23.2%, 44.0% and 53.3% in T20, T40 and T60, 374 ‑ ‑ while decreasing significantly by 18.2% and 45.4% in T80 and OM. Non essential amino acid content increased 375 significantly by 39.6%, 99.1%, 96.7% and 10.5% in T20, T40, T60 and T80, and decreased significantly by 15.7% 376 ‑ under OM. Total amino acid content increased significantly by 32.5%, 75.0% and 77.7% in T20, T40 and T60, and 377 decreased significantly by 28.7% under OM. Moreover, compared with CF, treatments of reduced chemical 378 fertilizer combined with organic manure (T20, T40, T60, T80) and sole organic manure (OM) significantly reduced 379 EAA/TAA and EAA/NEAA by 6.9%‑23.4% and 11.7%‑35.2%, and significantly elevated NEAA/TAA by 380 ‑ 5.4% 18.2% ( _P_ <0.05). 381 ‑ In terms of amino acid composition, T20, T40 and T60 increased the contents of all 17 amino acids. By contrast, T80 and OM reduced 14 amino‑acid components except aspartic acid, glycine and cystine. Compared 383 with CF, the T20 treatment significantly increased valine, isoleucine, leucine, aspartic acid, glutamic acid, glycine, 384 alanine, cystine, histidine, arginine and proline by 21.7%, 69.9%, 40.8%, 74.8%, 27.5%, 677.1%, 21.6%, 11.3%, 385 113.5%, 26.2% and 9.7%, respectively. T40 and T60 significantly enhanced all amino‑acid components, with 386 increments ranging from 20.6% to 1151.7% and from 20.1% to 1536.9%, respectively. Under T80, threonine, 387 phenylalanine, valine, methionine, leucine, aspartic acid, glycine, cystine, histidine, arginine and proline increased 388 significantly by 33.4%, 24.3%, 20.7%, 43.3%, 18.1%, 18.2%, 25.4%, 907.5%, 11.9%, 42.4%, 12.6%, 29.5% and 389 10.5%, while tyrosine decreased significantly by 91.7%. For the OM treatment, threonine, phenylalanine, lysine, 390 valine, methionine, isoleucine, leucine, tyrosine, serine, glutamic acid, alanine, histidine, arginine and proline 391 decreased significantly by 44.8%, 58.7%, 46.2%, 52.3%, 64.5%, 36.3%, 35.8%, 56.3%, 21.1%, 29.8%, 19.1%, 392 69.6%, 18.7% and 44.8%, respectively; glycine content increased significantly by 594.5%. No significant 393 differences were detected among other comparisons. 394 ‑ ‑ It can be concluded that although reduced chemical fertilizer combined with organic manure application ‑ lowered the yield of _D. officinale_, amino acid contents were markedly improved under appropriate substitution 396 ratios (T20, T40 and T60). From the perspective of amino‑acid profiles, the rank order of amino‑acid types and 397 concentrations was largely consistent across fertilization treatments. Aspartic acid possessed the highest content, 398 followed by glutamic acid, leucine, arginine and lysine. Aspartic acid and glutamic acid are characteristic 399 ‑ ‑ umami taste amino acids, jointly accounting for more than 25% of total amino acids and contributing to flavour 400 regulation. 401 ‑ Table 2 Effects of Different Fertilization Treatments on Amino Acid Composition of _D. officinale_ 402 **Amino acid** **CK** **CF** **T20** **T40** **T60** **T80** **OM** Threonine 0.107±0.01d 0.18±0.006c 0.199±0.023c 0.26±0.016b 0.308±0.013a 0.12±0.01d 0.099±0.02d Phenylala 0.137±0.015e 0.222±0.008c 0.238±0.015c 0.308±0.027b 0.341±0.014a 0.168±0.01d 0.092±0.011f nine Essential amino acid (%) Nonessent ial amino Lysine 0.180±0.006c 0.345±0.015b 0.349±0.055b 0.418±0.015a 0.437±0.025a 0.32±0.024b 0.186±0.018c Valine 0.093±0.005f 0.194±0.004d 0.236±0.016c 0.276±0.015b 0.314±0.018a 0.154±0.017e 0.092±0.017f Methionin 0.015±0.004e 0.044±0.007c 0.049±0.002bc 0.056±0.002a 0.053±0.004ab 0.025±0.002d 0.016±0.003e e Isoleucine 0.103±0.008e 0.152±0.009d 0.258±0.007c 0.299±0.018b 0.322±0.007a 0.144±0.008d 0.097±0.008e Leucine 0.165±0.017e 0.418±0.01b 0.588±0.035a 0.622±0.02a 0.607±0.053a 0.342±0.022c 0.268±0.022d Aspartic 0.167±0.016d 0.334±0.022c 0.584±0.046b 1.003±0.026a 1.08±0.073a 0.641±0.081b 0.411±0.028c acid acid (%) [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.762708; this version posted November 3, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.762708) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) Tyrosine 0.06±0.01e 0.159±0.01c 0.165±0.013bc 0.192±0.012b 0.22±0.02a 0.119±0.028d 0.07±0.004e Serine 0.133±0.01bc 0.137±0.005bc 0.149±0.011b 0.243±0.008a 0.256±0.004a 0.121±0.012cd 0.108±0.017d Glutamic 0.176±0.011f 0.506±0.028d 0.645±0.08c 0.983±0.071a 0.863±0.093b 0.425±0.095de 0.355±0.05e acid Glycine 0.145±0.012e 0.023±0.001f 0.175±0.017d 0.282±0.006b 0.369±0.01a 0.227±0.015c 0.157±0.009de Alanine 0.142±0.01e 0.188±0.01d 0.229±0.025c 0.301±0.019a 0.267±0.012b 0.162±0.02de 0.152±0.009e Cystine 0.043±0.001ab 0.031±0.001d 0.034±0.004bc 0.042±0.002ab 0.045±0.002a 0.034±0.012bc 0.031±0.004d Histidine 0.031±0.003f 0.076±0.007d 0.161±0.01b 0.181±0.002a 0.086±0.007c 0.044±0.002e 0.023±0.003f Arginine 0.206±0.018e 0.296±0.007c 0.373±0.01b 0.437±0.005a 0.424±0.023a 0.259±0.008d 0.24±0.017d Proline 0.108±0.013f 0.249±0.016c 0.273±0.01b 0.315±0.008a 0.32±0.018a 0.176±0.011d 0.138±0.007e Essential amino acid, EAA (%) Nonessential amino acid, NEAA (%) Total amino acid, TAA (%) 0.801±0.031f 1.555±0.011d 1.916±0.02c 2.239±0.085b 2.383±0.012a 1.273±0.061e 0.85±0.064f 1.211±0.018f 1.998±0.03d 2.79±0.086b 3.978±0.056a 3.931±0.06a 2.207±0.004c 1.685±0.064e 2.012±0.048e 3.553±0.038c 4.706±0.105b 6.217±0.03a 6.314±0.048a 3.48±0.057c 2.534±0.113d Eaa/TAA 0.398±0.006b 0.438±0.003a 0.407±0.005b 0.36±0.012d 0.377±0.005c 0.366±0.011cd 0.335±0.014f Neaa/TAA 0.602±0.006d 0.562±0.003e 0.593±0.005d 0.64±0.012b 0.623±0.005c 0.634±0.011bc 0.665±0.014a ‑ Among the 17 amino acids detected in _D. officinale_ under all fertilization treatments, four umami taste amino acids DAA (Asp, Glu, Gly, Ala), eight bitter‑taste amino acids BAA (Val, Met, Ile, Leu, Tyr, Phe, His, Arg), and nine 404 medicinal amino acids MAA (Asp, Glu, Gly, Arg, Met, Phe, Leu, Lys, Tyr) were identified. The contents of 405 ‑ ‑ bitter taste amino acids, umami taste amino acids and medicinal amino acids ranged from 63.3% to 258.0%, 406 81.1% to 237.1%, and 129.9% to 448.2%, respectively. Different fertilization treatments significantly affected the 407 contents of these three categories of amino acids. Compared with the non‑fertilization control (CK), the contents of 408 ‑ ‑ bitter taste amino acids, umami taste amino acids and medicinal amino acids in fertilized _D. officinale_ increased 409 significantly by 66.8%‑309.5%, 10.7%‑192.3% and 40.4%‑245.2%, respectively. Relative to the CF treatment, 410 bitter‑taste amino acids increased significantly by 55.4%, 144.5%, 145.5% and 38.5% under T20, T40, T60 and 411 T80; umami‑taste amino acids increased by 32.6%, 52.0% and 51.7% under T20, T40 and T60, but decreased 412 significantly by 19.7% and 42.5% under T80 and OM; medicinal amino acids increased significantly by 35.2%, 413 84.5%, 89.3% and 6.8% under T20, T40, T60 and T80, and decreased significantly by 23.0% under OM (Figure 2). 414 Notably, medicinal amino acids exhibited relatively high content and proportion. The ratio of medicinal amino acids to total amino acids ranged from 64.5% to 72.6% across treatments, with an average value of 69.3%. The 416 ranking of medicinal‑amino‑acid content from high to low was as follows: T60 = T40 > T20 > T80 > CF > OM > CK 417 (Figure 2). These results indicated that _Dendrobium_ plants are rich in medicinal amino acids, and rational 418 combined application of reduced chemical fertilizer with organic manure can further improve the accumulation of 419 ‑ medicinal amino acid components. 420 421 **Figure 2** Contents of different categories of amino acids under various fertilization treatments 422 **3.4. Correlation Analysis of Various Indicators of** _**D. officinale**_ 423 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.762708; this version posted November 3, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.762708) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) Correlation analysis was performed on multiple indicators of _D. officinale_ (Figure 3), and significant ‑ correlations were observed among these parameters. Pearson correlation analysis revealed that growth related 425 traits including stem length, stem diameter, internode length, leaf number, chlorophyll content, number of fresh 426 stems, yield and dry‑matter conversion rate showed highly significant positive correlations with one another. 427 Except for protein and crude fiber, quality indicators such as dendrobine, polysaccharides, polyphenols, flavonoids 428 ‑ and alcohol soluble extracts also exhibited highly significant positive correlations. Highly significant positive 429 correlations were also detected among essential amino acids, non‑essential amino acids, total amino acids, 430 ‑ ‑ umami taste amino acids, bitter taste amino acids and medicinal amino acids. Furthermore, quality indicators 431 (dendrobine, polysaccharides, polyphenols, flavonoids, alcohol‑soluble extracts) were significantly or highly 432 significantly positively correlated with essential amino acids, non‑essential amino acids, total amino acids, 433 ‑ ‑ umami taste amino acids, bitter taste amino acids and medicinal amino acids. In addition, protein showed highly 434 significant positive correlations with all growth indicators of _D. officinale_ . Crude fiber was significantly positively 435 correlated with most growth indicators but negatively correlated with quality indicators. Leaf number and yield were 436 both highly significantly positively correlated with all categories of amino acids (Figure 3). 437 438 Figure 3 Correlation coefficients among various indicators of _D. officinale_ 439 **3.5. Comprehensive Evaluation of the Effects of Different Fertilization Treatments on Growth Performance** 440 **and Quality of** _**D. officinale**_ 441 ‑ The membership function method was adopted to comprehensively evaluate the effects of various fertilization treatments on the growth performance and quality of _D. officinale_ (Table 3). The results showed that the 443 comprehensive evaluation values under different fertilization treatments were ranked in the order: 444 T40 > T60 > CF > T20 > T80 > OM > CK. The comprehensive evaluation values of sole chemical fertilizer 445 application, combined application of reduced chemical fertilizer with organic manure, and sole organic manure 446 application were all higher than that of the non‑fertilization control. 447 Among all treatments, the T40 treatment (40% organic manure substitution for chemical fertilizer) obtained the highest comprehensive evaluation value of 0.748 for growth performance and quality, followed by the T60 449 treatment (60% organic manure substitution for chemical fertilizer) with a value of 0.682. The sole organic‑manure 450 treatment (OM) had the lowest comprehensive evaluation value of 0.420. 451 These findings indicated that fertilization generally improved the comprehensive performance of _D. officinale_ in terms of growth and quality. Chemical fertilizer mainly promoted plant growth traits and yield, whereas organic 453 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.762708; this version posted November 3, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.762708) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) manure predominantly improved quality attributes. Reasonable allocation of chemical and organic fertilizer 454 dosages could achieve an optimal balance between yield and quality. 455 ‑ ‑ Table 3 Membership function based comprehensive evaluation of growth, yield and nutraceutical quality of _D._ 456 _officinale_ under different fertilization treatments 457 Index CK CF T20 T40 T60 T80 OM Vj Wj Stem length 0.004 0.051 0.047 0.039 0.032 0.028 0.016 0.514 0.055 Stem diameter 0.002 0.047 0.034 0.032 0.031 0.029 0.026 0.451 0.048 Internode length 0.003 0.061 0.052 0.038 0.028 0.018 0.016 0.653 0.070 Number of leaves 0.001 0.047 0.044 0.042 0.038 0.030 0.028 0.456 0.049 Chlorophyll 0.004 0.066 0.048 0.044 0.029 0.023 0.014 0.633 0.067 Number of fresh shoots 0.003 0.058 0.051 0.038 0.028 0.027 0.013 0.600 0.064 Dry weight ratio 0.009 0.053 0.030 0.024 0.032 0.016 0.027 0.552 0.059 Yield 0.001 0.049 0.045 0.044 0.039 0.032 0.023 0.475 0.051 Dendrobine 0.004 0.016 0.033 0.036 0.050 0.041 0.041 0.493 0.053 Polysaccharide 0.004 0.015 0.027 0.034 0.054 0.036 0.028 0.554 0.059 Polyphenols 0.001 0.021 0.022 0.059 0.047 0.047 0.034 0.589 0.063 Flavonoids 0.004 0.007 0.026 0.064 0.060 0.051 0.045 0.633 0.067 Alcohol extract 0.005 0.020 0.025 0.042 0.030 0.027 0.022 0.480 0.051 Protein 0.025 0.062 0.051 0.047 0.021 0.012 0.021 0.630 0.067 Crude fiber 0.037 0.008 0.017 0.045 0.045 0.036 0.032 0.445 0.047 Total amino acids 0.001 0.029 0.050 0.077 0.079 0.027 0.010 0.750 0.080 Comprehensive evaluation value 0.111 0.657 0.648 0.748 0.682 0.514 0.420 Ranking 7 3 4 1 2 5 6 **4. Discussion** 458 _**4.1 Effects of Different Fertilization Treatments on Growth Traits and Yield of D. officinale**_ 459 The present study demonstrated that fertilization could promote the growth and improve quality of _D._ ‑ _officinale_, achieving high yield and good quality simultaneously[35 36]. In this experiment, fertilization significantly 461 improved growth indices including stem length, stem diameter, internode length, leaf number, chlorophyll content 462 and number of fresh stems, and increased yield, which was consistent with the findings reported by Wu et al. Their 463 research confirmed that stem length, stem diameter, fresh weight per clump, water content, crude‑fiber content, 464 alkaloid content, polysaccharide content, total flavonoid content, alcohol‑soluble extract content and protein 465 ‑ content of fertilized _D. officinale_ were all significantly higher than those under non fertilization treatment. 466 In addition, with the increasing substitution ratio of organic manure for chemical fertilizer, growth traits and yield of _D. officinale_ gradually declined. This result agreed with the conclusion of Liu et al.[37]. Chemical fertilizer 468 exhibited prominent advantages in promoting vegetative growth and biomass production of _D. officinale_, while 469 organic manure showed weaker promoting effects on plant vegetative growth. Chemical fertilizer can rapidly 470 release mineral nutrients such as nitrogen, phosphorus and potassium to satisfy the nutritional demands for rapid 471 tillering and stem elongation of _D. officinale_ . It raises leaf chlorophyll concentration, enhances photosynthetic 472 efficiency, and consequently increases yield, which is in accordance with previous studies[24]. Zhang et al. applied 473 special controlled‑release fertilizer for orchids to _Dendrobium denneanum_, and observed significant promotion in 474 stem growth rather than leaf growth; stem length, stem diameter and internode number showed consistent 475 increasing trends[38]. 476 Under the experimental conditions of this study, the yield of T20 and T40 treatments only slightly decreased by 4.2%‑6.0% compared with CF and remained at a relatively high level. Nevertheless, when the organic‑manure 478 substitution ratio reached 80% (T80) or under sole organic‑manure treatment (OM), yield decreased significantly 479 by 20.8% and 32.1% relative to CF, respectively. This experiment adopted an equal‑nitrogen substitution design. 480 Nutrients from organic manure are released slowly. Even with identical total nitrogen input, high‑proportion 481 organic‑manure treatments provide insufficient available mineral nitrogen during the vigorous‑growth stage, failing 482 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.762708; this version posted November 3, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.762708) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) to meet the demand for rapid vegetative growth of _D. officinale_ . This further inhibits tillering, stem elongation and 483 ‑ fresh stem yield formation. These observations are similar to those reported by Kong et al.[39] in fertilizer trials on 484 ‑ _D. officinale_ . Although high rate organic manure can improve substrate properties, insufficient supply of readily 485 ‑ available nutrients restricts above ground biomass accumulation[40]. 486 ‑ All combined fertilization treatments in this study produced significantly higher yield than the non fertilization ‑ control, indicating that reduced chemical fertilizer combined with organic manure was still superior to non fertilized 488 cultivation. In medicinal plant cultivation, sole chemical fertilizer application pursuing high yield usually leads to 489 poor quality, whereas sole organic‑manure application causes yield loss. This represents a practical contradiction 490 ‑ in commercial production of _D. officinale_ . Therefore, an appropriate organic manure substitution ratio should be 491 determined within an acceptable yield‑loss range to realize coordinated improvement of yield and quality. The yield 492 of T40 treatment in this trial was only 5.7% lower than that of CF, showing a minor yield reduction and providing a 493 feasible option for balancing yield performance. It should be noted that this experiment lasted for one annual 494 growth cycle. Substrate physicochemical properties will continuously change along with long‑term decomposition 495 of organic manure. Further long‑term field trials are required to clarify the persistent effects of different fertilizer 496 ‑ combinations on growth and yield of _D. officinale_ under multi year continuous cultivation. 497 _**4.2 Effects of Different Fertilization Treatments on Quality of D. officinale**_ 498 The medicinal value of _D. officinale_ depends on the contents of bioactive components including dendrobine, polysaccharides, polyphenols, flavonoids and alcohol‑soluble extracts, which serve as core indices for evaluating 500 medicinal material quality[2‑4]. The present study showed that the contents of dendrobine, polysaccharides, 501 polyphenols, flavonoids, alcohol‑soluble extracts and protein under all fertilization treatments were higher than 502 those under CK. With the increase in the organic‑manure substitution ratio for chemical fertilizer, the medicinal 503 quality indices of _D. officinale_ generally exhibited a trend of first increasing and then decreasing. The T40 and T60 504 treatments produced the most prominent improvement in polysaccharide, dendrobine, flavonoid and polyphenol 505 contents. Compared with sole chemical‑fertilizer treatment (CF), T40 significantly increased dendrobine, 506 polysaccharides and flavonoids by 47.4%, 32.8% and 45.8%, respectively. These findings support previous 507 ‑ reports that organic manure facilitates the accumulation of secondary metabolite bioactive substances in _D._ 508 _officinale_ and can markedly elevate the contents of polysaccharides, alkaloids and flavonoids relative to sole 509 chemical fertilizer application[24,40]. 510 In addition to macronutrients such as nitrogen, phosphorus and potassium, organic manure contains humic acids, small‑molecule organic acids, trace elements and microbial metabolites. On the one hand, it ameliorates the 512 ‑ micro environment of the cultivation substrate; on the other hand, it induces the expression of 513 ‑ secondary metabolic pathways in _D. officinale_, promoting the synthesis and accumulation of defensive and 514 medicinal bioactive substances such as polyphenols, flavonoids and alkaloids[41]. Nevertheless, quality indices 515 declined under excessive organic‑manure substitution (T80, OM). It is speculated that excessive organic nutrient 516 supply disturbs the carbon‑nitrogen metabolic balance of plants, and more carbon skeletons are consumed by 517 substrate microorganisms, thereby inhibiting the biosynthesis of secondary metabolites. The polysaccharide and 518 ‑ alcohol soluble extract contents in all treatments of this study complied with the quality criteria for _D. officinale_ 519 specified in the _Pharmacopoeia of the People’s Republic of China_ (2020 Edition). Combined fertilization 520 treatments exhibited obvious advantages in quality improvement. Although CF achieved the highest yield, its 521 contents of medicinal bioactive components including polysaccharides and dendrobine remained at relatively low 522 levels, indicating that cultivation relying solely on chemical fertilizer carries risks of insufficient medicinal quality for 523 _D. officinale_ . 524 In the present study, the variation trends of protein and crude fiber were not fully consistent with those of polysaccharides and dendrobine. Protein content decreased with the reduction in chemical‑fertilizer proportion, 526 with the maximum value observed in CF. Higher crude‑fiber contents were also recorded in CF and T20, whereas 527 crude fiber decreased under treatments with high organic‑manure proportion. Correlation analysis revealed that 528 protein was highly significantly positively correlated with growth indices but negatively correlated with most 529 bioactive quality indices. This suggests that protein and crude fiber are more closely associated with the vigour of 530 vegetative growth, whereas dendrobine, polysaccharides and flavonoids represent medicinal secondary quality. 531 These two groups of parameters exhibit distinct response patterns to fertilizer nutrients. This result is similar to the 532 conclusion reported by Wang et al.[42] regarding the differentiated responses of different nutritional components 533 ‑ to nutrient conditions in mycorrhizal inoculated _D. officinale_ . In research on fertilization techniques for _D. officinale_, 534 quality assessment should not rely on a single indicator; instead, yield and multiple bioactive components should 535 be comprehensively taken into consideration. 536 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.762708; this version posted November 3, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.762708) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) _**‑**_ _**4.3 Effects of Different Fertilization Treatments on Amino**_ _**Acid Composition of D. officinale**_ 537 ‑ ‑ Amino acids are important nutritional components of _D. officinale_ . Among them, umami taste, bitter taste and medicinal amino acids are closely associated with its dietary and health‑promoting values[8‑9]. All 17 amino acids 539 were detected across the seven treatments in this experiment. Fertilization significantly increased the contents of 540 ‑ ‑ total amino acids, essential amino acids, medicinal amino acids, umami taste amino acids and bitter taste amino 541 acids. With the increase of organic‑manure substitution ratio, total amino acids, essential amino acids, medicinal 542 ‑ amino acids and umami taste amino acids exhibited a trend of first increasing and then decreasing. The T40 and 543 T60 treatments achieved the best comprehensive performance in amino‑acid profiles. Compared with CF, T40 544 increased total amino acids by 75.0 % and medicinal amino acids by 84.5 %. Nevertheless, further elevation of 545 ‑ ‑ organic manure substitution ratio led to a remarkable decline in various amino acid contents under T80 and OM; 546 ‑ the total amino acid content under OM was even lower than that under CF. 547 ‑ Organic manure contains abundant organic nitrogen substrates such as amino acids and polypeptides, which can be directly absorbed and utilized by the root system of orchid plants. Meanwhile, functional microorganisms 549 including _Bacillus subtilis_ in the substrate can accelerate the mineralization of organic matters, release 550 ‑ ‑ small molecular amino acid substances, facilitate the synthesis and accumulation of amino acids in plants, and 551 consequently raise the contents of essential and medicinal amino acids. The organic manure adopted in this study 552 was fermented from mugwort straw, earthworm cast and _Pleurotus eryngii_ residue, compounded with extracts of 553 _D. officinale_ and mugwort, and inoculated with _Bacillus subtilis_ . It can supply abundant organic nitrogen sources for 554 ‑ _D. officinale_, which forms the material basis for the substantial improvement of amino acid components in T40 and 555 T60 treatments. However, under high‑substitution treatment T80 and sole organic‑manure treatment OM, the 556 supply of readily‑available mineral nitrogen was insufficient, restricting total nitrogen uptake by plants and resulting 557 ‑ ‑ in shortage of precursors for amino acid synthesis, thereby decreasing total amino acid content. 558 ‑ Aspartic acid and glutamic acid occupied prominent proportions in the amino acid profiles of _D. officinale_ in the present study. As umami‑taste amino acids, they jointly accounted for more than 25 % of total amino acids and 560 ‑ ‑ determine the edible flavour of _D. officinale_ . Medicinal amino acid components accounted for 64.5 % 72.6 % of 561 total amino acids, with an average value as high as 69.3 %, which constitutes the material foundation for the 562 ‑ health beneficial effects of _D. officinale_ . T40 and T60 treatments exerted significant effects on enhancing 563 ‑ ‑ medicinal and umami taste amino acids. It indicates that appropriate reduction of chemical fertilizer combined with 564 organic manure can not only improve medicinal bioactive components, but also elevate the dietary nutritional 565 value of _D. officinale_ . Correlation analysis demonstrated that medicinal quality indices such as dendrobine, 566 polysaccharides and flavonoids were significantly or highly significantly positively correlated with total amino acids 567 and medicinal amino acids. This suggests synergistic regulation between the accumulation of secondary bioactive 568 ‑ substances and amino acid metabolism in _D. officinale_ . This finding is consistent with the report by Wang et al.[43] 569 ‑ on mycorrhizal inoculated _D. officinale_, namely that cultivation conditions favourable for secondary metabolism 570 ‑ can simultaneously promote amino acid accumulation. 571 _**4.4 Insights from Comprehensive Evaluation on Yield**_ _**‑**_ _**Quality Synergy of D. officinale**_ 572 ‑ As a medicinal economic crop, _D. officinale_ requires both acceptable fresh stem yield and guaranteed intrinsic medicinal quality in practical production. Neither single yield indicator nor individual bioactive‑component index 574 can objectively judge the merits of fertilization regimes. The membership‑function comprehensive evaluation 575 ‑ method normalizes multi dimensional parameters and enables integrated assessment covering growth, yield, 576 quality and amino‑acid profiles, which has been widely applied in screening cultivation measures for medicinal 577 plants. In the present study, the ranking of comprehensive evaluation values was: 578 T40 > T60 > CF > T20 > T80 > OM > CK. 579 The T40 treatment obtained the highest comprehensive evaluation value (0.748). Its yield decreased only slightly by 5.7% compared with CF which achieved the maximum yield, whereas dendrobine, polysaccharides, 581 flavonoids, total amino acids and medicinal amino acids were all significantly enhanced. It realized maximum 582 quality improvement with minimal yield loss. Although T60 also exhibited high quality indices, its yield decreased 583 by 12.4% relative to CF, resulting in inferior comprehensive performance compared with T40. Sole 584 ‑ chemical fertilizer treatment (CF) ranked first in yield, yet its comprehensive evaluation value was lower than T40 585 ‑ and T60, owing to its insufficient medicinal bioactive components and total amino acid content. Treatments with 586 high organic‑manure proportion (T80 and OM) showed partial improvement in quality indices but suffered 587 considerable yield reduction, leading to unsatisfactory overall performance. 588 ‑ It can be concluded that sole chemical fertilizer application focuses on yield promotion while organic manure prioritizes quality improvement, and a certain trade‑off exists between them. Only an appropriate mixing ratio can 590 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.762708; this version posted November 3, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.762708) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) achieve the balance of both targets. Under the experimental conditions of this study, 40% equal‑nitrogen 591 ‑ ‑ organic manure substitution for chemical fertilizer (T40) was the optimal fertilization scheme for bed cultivated _D._ 592 _officinale_ in greenhouse production to coordinate yield and quality. This experiment was carried out under specific 593 ‑ greenhouse facilities, cultivation substrates and special organic manure in Ruili, Dehong. Multi site verification is 594 still required to confirm whether this ratio is fully applicable under alternative climatic conditions, substrate types 595 and organic‑manure raw‑material sources. For practical popularization in production, further fine‑tuning should be 596 performed considering fertilizer cost, substrate fertility and cultivation duration. 597 **5. Conclusions** 598 ‑ In this study, a greenhouse bed cultivation experiment was conducted to investigate the regulatory effects of ‑ reduced chemical fertilizer combined with organic manure on yield, quality and amino acid composition of _D._ 600 _officinale_ . The results showed that fertilization significantly improved growth traits and enhanced yield and quality 601 ‑ ‑ of _D. officinale_ . Sole chemical fertilizer treatment achieved the best vegetative growth and fresh stem yield, yet the 602 ‑ accumulation of medicinal bioactive components was limited. With the increase in organic manure substitution 603 ratio, stem length, stem diameter and yield of _D. officinale_ exhibited a declining trend, whereas medicinal quality 604 indices including dendrobine, polysaccharides and flavonoids followed a pattern of first increasing and then 605 decreasing. Appropriate organic‑manure substitution significantly increased the contents of total amino acids, 606 essential amino acids, medicinal amino acids and umami‑taste amino acids, with T40 and T60 showing the most 607 ‑ ‑ ‑ pronounced improvement; high rate organic manure substitution led to a reduction in amino acid contents. The 608 average proportion of medicinal amino acids in total amino acids of _D. officinale_ reached 69.3%. Most medicinal 609 bioactive components were significantly or highly significantly positively correlated with amino‑acid components, 610 indicating a synergistic accumulation effect. 611 ‑ ‑ The 40% equal nitrogen organic manure substitution for chemical fertilizer (T40) obtained the highest comprehensive evaluation value. Under this treatment, yield only slightly decreased by 5.7% compared with sole 613 ‑ chemical fertilizer application, while the contents of dendrobine, polysaccharides, flavonoids and amino acids 614 were significantly increased. Therefore, T40 was identified as the optimal fertilization mode for balancing yield and 615 medicinal quality of _D. officinale_ under the present experimental conditions. 616 **Author contributions** 619 **Conceptualization:** Long Zhou, Hongbin Yang. 620 **Data curation:** Long Zhou. 621 **Formal analysis:** Long Zhou, Xudong Zhao. 622 **Investigation:** Long Zhou, Wenna Zhou, Hongbin Yang. 623 **Methodology:** Long Zhou. 624 **Validation:** Long Zhou, Huarong Liu. 625 **Writing – original draft:** Long Zhou. 626 **Writing – review & editing:** Long Zhou, Hongbin Yang. 627 628 **Data availability statement:** All relevant data are within the paper and its Supporting Information files. 629 **Funding:** This research was funded by the Science and Technology Talents and Platform Program of Yunnan 630 Provincial (202405AI350229), the Innovation Guidance and Sci‑tech Enterprise Cultivation Program of Yunnan 631 Province (202604BK090910), and the Scientific Research Fund Project of Education Department of Yunnan 632 Province (2026J0837). 633 **Competing interests:** The authors have declared that no competing interests exist. 634 **References** 635 1. 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