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    01 October 2026, Volume 59 Issue 19
    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    Plant Epitranscriptomics: Molecular Mechanisms Regulating Stress Responses and Prospects for Stress-Resistant Breeding
    ZHANG Ce, ZHANG YanYan, LIU XuanDong, LU DuXian, LIN JinXing, CUI YaNing
    Scientia Agricultura Sinica. 2026, 59(19):  4195-4207.  doi:10.3864/j.issn.0578-1752.2026.19.001
    Abstract ( 12 )   HTML ( 1 )   PDF (1204KB) ( 5 )   Save
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    As a critical interface bridging environmental cues and gene expression, plant RNA modifications (the epitranscriptome) empower sessile plants to adapt rapidly to fluctuating environments. This review provides a comprehensive overview of the regulatory mechanisms underlying key modifications, such as N6-methyladenosine (m6A), 5-methylcytosine (m5C), and organellar C-to-U editing, under abiotic (salt, drought, extreme temperatures, heavy metals) and biotic stress conditions. We elucidate how these modifications orchestrate hormone signaling, reactive oxygen species (ROS) homeostasis, and energy metabolism by fine-tuning transcript stability (e.g., SbABA2 in sorghum), translation efficiency (e.g., CBFs in Arabidopsis), and protein structure (e.g., mitochondrial NAD9 in wheat). The emerging significance of modifications on long non-coding RNAs (lncRNAs) in stress resilience is also explored. Technologically, we critically evaluate MeRIP-seq, miCLIP, and Nanopore Direct RNA Sequencing (DRS), highlighting the superiority of Nanopore DRS in resolving full-length transcripts within complex plant genomes, particularly in forestry species. Moreover, advances in functional verification tools, including DART-FISH and CRISPR-dCas13-mediated targeted editing, are presented. Concluding with a forward-looking perspective, we propose an epitranscriptome engineering strategy based on "target mining-precision editing-germplasm creation" to facilitate the breeding of high-yielding, stress-tolerant crops.

    Identification of QTLs and Exploration of Candidate Genes Conferring Anaerobic Germination Tolerance in Rice
    YANG HuiJuan, LIU YiFan, YU XiaoMan, HUANG DanLin, CHEN DanDan, SHU YaZhou, WEI Hua, HE HuiYing, SHANG LianGuang, YUAN QiaoLing
    Scientia Agricultura Sinica. 2026, 59(19):  4208-4220.  doi:10.3864/j.issn.0578-1752.2026.19.002
    Abstract ( 10 )   HTML ( 1 )   PDF (2382KB) ( 4 )   Save
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    【Objective】In direct-seeded rice production, low germination rates and uneven emergence under anaerobic conditions are major bottlenecks limiting yield stability and cultivation area. This study aims to elucidate the genetic regulation of anaerobic germination in rice and identify key genes controlling coleoptile elongation, thereby providing genetic resources for breeding submergence-tolerant direct-seeded rice varieties.【Method】Sixty-nine of 237 accessions were selected to measure anaerobic coleoptile length and seedling establishment rate, and the correlation between the two traits was evaluated. After anaerobic germination treatment, coleoptile length data was collected from all 237-rice pan-genome accessions. A genome-wide association study (GWAS) was performed using these phenotypic data together with high-density SNP markers to identify genetic loci underlying tolerance to anaerobic germination. Candidate genes were further screened through functional annotation, transcriptome analysis, and haplotype analysis.【Result】Coleoptile length was highly significantly correlated with seedling establishment rate (r2=0.4533), confirming that coleoptile elongation is a reliable indicator for anaerobic germination tolerance. GWAS identified two novel significant association loci on chromosomes 8 and 12, named qAG8 and qAG12, neither of which has been reported previously. Within these intervals, 46 and 48 genes were detected, respectively; after functional annotation, 29 genes from qAG8 and 14 from qAG12 were retained. Integration of transcriptome and haplotype analyses revealed four candidate genes potentially involved in anaerobic germination: LOC_Os08g39210 (encoding a cell wall-associated kinase OsWAK74, localized to the cell wall and negatively regulating cell proliferation), LOC_Os08g39290 (containing an EF-hand domain, possibly involved in calcium signaling), LOC_Os08g39550 (encoding a polygalacturonase inhibitor protein, localized to the cell wall), and LOC_Os12g39830 (a rice cyclin CycD5;2, involved in cell cycle regulation).【Conclusion】Two novel loci (qAG8 and qAG12) conferring tolerance to anaerobic germination were identified on chromosomes 8 and 12, and four candidate genes were uncovered through transcriptome and haplotype analyses. These findings provide new insights into the genetic basis of anaerobic germination and offer potential molecular targets for marker-assisted breeding of direct-seeded rice.

    Evaluation of Smut Resistance and Screening of Resistant Germplasm in 291 Sugarcane Accessions
    PU XinYuan, MA YunFei, ZHAO Jun, ZHAO LiPing, ZHAO XingDong, HU Xin, ZAN FengGang, LIU JiaYong, YANG LiHua, XIA HongMing, LU Xin, RAO XiBing, TANG SiQi, BAI YaDong, ZHANG Jing, REN ShengLin, ZHAO PeiFang
    Scientia Agricultura Sinica. 2026, 59(19):  4221-4234.  doi:10.3864/j.issn.0578-1752.2026.19.003
    Abstract ( 5 )   HTML ( 1 )   PDF (776KB) ( 4 )   Save
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    【Objective】Smut is a devastating disease affecting global sugarcane production. Screening for resistant germplasm is crucial for sugarcane disease-resistant breeding. This study aimed to systematically evaluate the resistance levels of sugarcane germplasm from different origins to smut, identify highly resistant germplasm, provide research materials for elucidating resistance mechanisms, and supply excellent parental resources for disease-resistant breeding.【Method】From 2020 to 2021, 291 sugarcane germplasm accessions from domestic and international sources were used. The trials included a plant crop and a ratoon crop (“crop type”) were conducted in Yuanjiang Hani, Yi and Dai Autonomous County, where the smut incidence is the highest in Yunnan province. A mixture of smut teliospores collected from the field (concentration: 5×106 spores/mL) served as the inoculum. Treatments consisted of artificial inoculation and a natural infection area (“treatment”). Disease occurrence in each germplasm was investigated and statistically analyzed. Resistance was evaluated and graded based on incidence, and highly resistant and resistant germplasm were screened, and compared the resistance level among the various sources.【Result】A total of 20 smut-resistant germplasms were screened from 291 germplasms, accounting for 6.87% of the evaluated germplasm, including 4 high-resistant germplasms (1.37%). CP72-3709 showed immunity, and CP96-1252, FR93-910 and Yunzhe02-2332 showed high resistance. Among the remaining germplasms, 14 (4.81%) showed moderately resistant (MR), 74 (25.43%) showed moderately susceptible (MS), 134 (46.05%) showed susceptible (S), and 49 (16.84%) showed highly susceptible (HS). Statistical analysis indicated that disease incidence differed highly significantly (P<0.001) among “treatments”, “crop types”, genotypes, and the interaction between “treatment” and genotype. demonstrating that sugarcane smut incidence is significantly influenced by treatment method, crop type, and genotype. Statistical analysis revealed extremely significant differences (P<0.001) in disease incidence among treatments, crop types, genotypes, and the treatment × genotype interaction. The disease incidence in the artificial inoculation zone (32.16%) was extremely significantly higher than that in the natural infection zone (18.29%), while the incidence in ratoon crops (35.29%) was extremely significantly higher than that in plant cane (15.24%), indicating that sugarcane smut incidence is significantly influenced by inoculation method, crop type, and genotype. The broad-sense heritability was 0.812, suggesting that the variation in disease incidence is primarily governed by genetic factors. Differences in disease incidence were also observed among germplasms from different sources, with germplasms originating from France exhibiting the lowest incidence.【Conclusion】This research obtained the smut resistance of 291 sugarcane germplasms in the highest smut incidence area Yuanjiang county. The 20 resistant germplasms screened in this study collectively constitute a valuable resistance gene pool, providing elite parental resources for resistance breeding. The immune germplasm CP72-3709 can serve as an excellent resistance source for investigating the molecular mechanisms underlying smut resistance, mining resistance genes, and improving smut-resistant sugarcane cultivars. The highly resistant and resistant genotypes, such as CP96-1252, FR93-910, YZ02-2332, CP89-2143, and POJ2878, can be utilized as core parents in resistance breeding programs.

    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY
    Evolution of Source-Sink Traits and End-Use Quality in Winter Wheat During Cultivar Replacement in the Haihe Plain
    DU RuiRui, HUANG XueJue, YU Yang, SUN MengMeng, LI ZhenLei, JIANG WenNa, LIU XiaoLi, ZHEN WenChao, WANG YanDong
    Scientia Agricultura Sinica. 2026, 59(19):  4235-4249.  doi:10.3864/j.issn.0578-1752.2026.19.004
    Abstract ( 14 )   HTML ( 1 )   PDF (4741KB) ( 6 )   Save
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    【Objective】This study aimed to investigate the coordinated evolution of source-sink traits and quality traits in winter wheat during cultivar replacement in the Haihe Plain, thereby providing a theoretical basis for breeding and agronomic management aimed at simultaneously achieving high yield and high quality.【Method】Thirty major winter wheat cultivars approved from the 1950s to the present were evaluated in field experiments conducted over three wheat-growing seasons (2022-2025) at the Xinji Experimental Station, Agricultural University of Hebei. Source traits (leaf area index and net photosynthetic rate), sink traits (grain number per unit area, grain-filling rate, and yield), and quality traits (protein fractions, starch properties, and processing quality) were measured. Carbon-to-nitrogen (C/N) ratios and the activities of enzymes associated with carbon and nitrogen metabolism were also determined. Linear mixed-effects models and Mantel tests were used to characterize the temporal trends in source-sink traits and quality traits and to assess their relationships.【Result】Cultivar replacement improved source-sink traits. Source activity (net photosynthetic rate) increased by 0.66 μmol·m-2·s-1 per decade, whereas sink size (grain number per unit area) and sink activity (average grain-filling rate) increased by 626.56 grains/m2 and 0.03 mg/(grain·d) per decade, respectively, resulting in an increase in sink strength (grain yield) of 551.77 kg·hm-2 per decade. However, this process induced a pronounced “carbon dilution effect”, disrupting the balance between yield and quality: amylopectin content increased significantly by 0.31% per decade, whereas total protein content decreased by 0.23% per decade. Processing-quality parameters, including sedimentation value and wet gluten content, decreased significantly by 0.96 mL and 0.71%, respectively, per decade. Moreover, protein-related traits were significantly negatively correlated with source activity and sink strength. The activities of enzymes involved in carbon and nitrogen metabolism showed that cultivar replacement significantly increased the activities of key enzymes, including sucrose synthase and glutamine synthetase, leading to a 1.44 per-decade increase in the grain C/N ratio, whereas the C/N ratios of stems and leaves decreased significantly. This further indicated that cultivar replacement altered patterns of carbon and nitrogen metabolic partitioning.【Conclusion】Modern high-yielding cultivars achieved substantial yield gains through coordinated source-sink improvement by enhancing source activity and expanding sink capacity; however, relatively insufficient nitrogen accumulation in grains made it difficult to improve yield and quality simultaneously. Agronomic practices, such as shifting nitrogen application to the flowering stage and regulating water supply during grain filling, combined with the breeding of cultivars with high nitrogen assimilation or remobilization efficiency, were recommended to alleviate the carbon dilution effect and achieve coordinated improvements in yield and quality. These findings provid a theoretical basis for breeding and agronomic management strategies that coordinate high yield with high quality and promote high-yield, high-efficiency wheat production.

    Effects of Diversified Crop Rotation on Soil Microbial Community Characteristics and Co-Occurrence Network
    WANG LuYing, YANG Jie, LU HaiTao, WANG Xin, ZHAO YingLong, YANG Jie, WANG GuiYan
    Scientia Agricultura Sinica. 2026, 59(19):  4250-4262.  doi:10.3864/j.issn.0578-1752.2026.19.005
    Abstract ( 11 )   HTML ( 1 )   PDF (2218KB) ( 3 )   Save
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    【Objective】This study investigated the effects of different rotation patterns on soil microbial community characteristics and co-occurrence networks, providing a theoretical basis for improving the soil microenvironment and enhancing the stability and stress resistance of soil ecosystems.【Method】In the North China Plain, winter wheat-summer maize double-cropping continuous cropping (WMWM) was used as the control, and three rotation patterns were established: spring sweet potato-winter wheat-summer maize rotation (SPWM), spring peanut-winter wheat-summer maize rotation (PWM), and spring millet-winter wheat-summer maize rotation (MWM). Based on a 6-year field positioning experiment, amplicon sequencing, random forest, and other methods were used to systematically analyze the effects of different rotation systems on soil microbial diversity, community composition, and co-occurrence networks, as well as their relationships with soil properties and yield.【Result】Among them, the bacterial ACE index under SPWM was significantly increased by 22.8% compared with the control; the fungal ACE index under MWM was significantly increased by 52.2% compared with the control; and the bacterial and fungal Simpson and Shannon indices under PWM and MWM were all significantly increased compared with WMWM. Principal component analysis (PCA) showed that diversified crop rotation changed soil microbial community structure and formed relatively unique microbial communities. Rotation increased the relative abundances of dominant bacteria such as Proteobacteria, Acidobacteria, and Actinobacteria, and dominant fungi such as Ascomycota and Glomeromycota. The relative abundance of Proteobacteria under PWM and MWM increased by 12.8% and 14.0%, respectively, compared with the control; the relative abundances of Ascomycota and Glomeromycota under SPWM, PWM, and MWM were significantly increased by 34.9%, 42.7%, and 46.2%, respectively, compared with the control. Meanwhile, rotation increased the number of nodes in the microbial co-occurrence network, increased the average clustering coefficient and modularity, and elevated the proportion of positive interactions. Correlation analysis showed that bacterial diversity, community structure, and co-occurrence networks were significantly correlated with soil total nitrogen, ammonium nitrogen, nitrate nitrogen, and organic carbon, while fungal diversity, community structure, and co-occurrence networks were significantly correlated with soil total nitrogen, available potassium, available phosphorus, ammonium nitrogen, and microbial biomass nitrogen. The random forest prediction model indicated that ammonium nitrogen, nitrate nitrogen, fungal community abundance, fungal diversity, and fungal co-occurrence network were key factors affecting yield.【Conclusion】Diversified crop rotations with winter wheat-summer maize optimize soil microbial community structure and improve microbial co-occurrence network structure, thereby effectively enhancing the stability and complexity of the soil ecosystem, and can provide a theoretical basis for improving the stability and stress resistance of arable soil ecosystems.

    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    Effects of Long-term Biochar Application on Carbon Sequestration, Emission Reduction and Crop Production in Barren Rainfed Farmland
    DUAN XuKun, WEI HeYa, LI JianZheng, TIAN YuPing, WANG LiGang
    Scientia Agricultura Sinica. 2026, 59(19):  4263-4276.  doi:10.3864/j.issn.0578-1752.2026.19.006
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    【Objective】Effects of long-term biochar application in barren rainfed farmland remain unclear. This study aimed to quantitatively analyze the impacts of biochar application under reduced nitrogen fertilization on carbon sequestration, emission reduction, and crop production in barren farmland within the Huang-Huai-Hai region through a long-term field experiment. The goal was to provide optimized strategies and scientific evidence for biochar application technology in similar low- to medium-yield fields in the region.【Method】Based on a 10-year long-term field experiment at the Ministry of Agriculture and Rural Affairs Key Field Scientific Observation Station for Eco-Environment in Yanshan, four treatments were established in barren farmland: conventional farmer nitrogen application (N), reduced nitrogen fertilization by 40% (RN), reduced nitrogen + annual low-rate biochar application (RNC1), and reduced nitrogen + high-rate biochar application every five years (RNC5). Soil organic carbon content and N2O emission flux were measured, and these indicators were combined with soil physicochemical properties and crop yield to comprehensively evaluate the effects of different biochar application treatments on carbon sequestration, emission reduction, and crop production in barren rainfed farmland.【Result】Compared with the RN treatment, the RNC1 treatment significantly increased soil available nutrients, with available phosphorus and available potassium increasing by 28.5% and 7.9%, respectively. In contrast, the RNC5 treatment focused more on soil nitrogen retention, significantly increasing total nitrogen in surface and deep soil layers by 7.4% and 13.5%, respectively, and significantly increasing alkaline hydrolysis nitrogen in deep soil by 10.3%. Biochar application significantly enhanced soil organic carbon storage and improved soil carbon fractions. Compared with the N treatment, soil organic carbon storage under RNC1 and RNC5 increased by 34.8% and 38.6%, respectively, and particulate organic carbon content increased by 19.5% and 20.5%, while mineral-associated organic carbon content increased by 78.3% and 88.9%, respectively. Combining reduced nitrogen with biochar application also reduced N2O emissions. The cumulative N2O emissions under RNC5 were 13.0% lower than those under RNC1, indicating that applying biochar every few years could effectively leverage its soil amendment properties, which was more beneficial for carbon sequestration and emission reduction in agricultural ecosystems.【Conclusion】Biochar application could substantially enhance soil carbon storage, reduce greenhouse gas emissions, and improve the soil nutrient environment. Applying 3.85 t of biochar every five years could balance carbon sequestration and crop yield stability in barren farmland. This approach could serve as an effective strategy for green and low-carbon agricultural development in barren farmland within the Huang-Huai-Hai region.

    Effects of Continuous Cultivation and Cropland Afforestation on Nitrogen and Phosphorus Nutrients in Typical Steppe
    BAO YuFan, ZHU Na, ZHANG JingMin, ZHAO Man, LIU JiaHui, LUO YuHong, LI Le, XU Nuo, YAN YuChun
    Scientia Agricultura Sinica. 2026, 59(19):  4277-4288.  doi:10.3864/j.issn.0578-1752.2026.19.007
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    【Objective】This study aimed to investigate the effects of long-term cultivation and cropland afforestation on soil nitrogen and phosphorus nutrients in typical grasslands of Inner Mongolia, so as to provide the scientific basis for rational utilization and sustainable development of grassland.【Method】Soil samples were collected in 0 to 50 cm soil layers from natural grassland, cropland (continuously cultivated for 60 years) and shrubland (afforestation for 17-18 years) of typical grassland in XilinGol, Inner Mongolia in August 2020. The content of soil total nitrogen (TN), total phosphorus (TP), ammonium nitrogen (NH4+-N), nitrate nitrogen (NO3--N) and available phosphorus (AP) were compared and analyzed under different land-use types.【Result】Grassland cultivation and the conversion of cropland to shrubland significantly affected soil nitrogen and phosphorus nutrients: (1) Reclamation and long-term cultivation of natural grassland significantly reduced the content of total nitrogen, total phosphorus, and ammonium nitrogen in the 0-10 cm soil layer, while significantly increasing the available phosphorus content in the 0-30 cm soil layer. (2) Following shrubland establishment through cropland conversion, no significant changes were observed in soil total nitrogen or ammonium nitrogen content; however, nitrate nitrogen content throughout the 0-50 cm profile, total phosphorus in the 20-50 cm soil layer, and available phosphorus in the 0-30 cm soil layer all decreased significantly. (3) In response to land-use change, soil nitrate nitrogen and available phosphorus-representing readily available nutrient pools-exhibited more pronounced changes compared to total nitrogen and total phosphorus.【Conclusion】Grassland cultivation significantly decreased the content of soil total nitrogen and total phosphorus, however, the current management model of planting leguminous shrubs combined with understory herb harvesting struggled to achieve effective soil nutrient recovery in the short term and might even exacerbate phosphorus deficiency.

    Inventory and Mitigation Potential of Nitric Oxide Emissions from Agricultural Soils in China Based on Localized Emission Factors
    ZENG HuiMing, LU KeXin, ZHOU WenJing, CHENG Kun
    Scientia Agricultura Sinica. 2026, 59(19):  4289-4300.  doi:10.3864/j.issn.0578-1752.2026.19.008
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    【Objective】Nitric oxide (NO) is not only a crucial precursor for tropospheric ozone formation but also participates in acid rain formation and nitrogen deposition processes, posing a severe threat to ecological security. Agriculture is a key anthropogenic source of NO. Given China’s substantial nitrogen fertilizer consumption, NO emissions from its agricultural soils warrant attention. Addressing the high uncertainty associated with current estimations that rely on global default emission factors (EFs) or a single national EF, this study aims to construct a localized EF dataset reflecting the characteristics of China's geographical regions, crops, and fertilizer types. Furthermore, it seeks to calculate the 2021 NO emission inventory for Chinese cropland soils and assess mitigation potentials, thereby providing a scientific basis for green agricultural development.【Method】This study systematically retrieved 54 field in-situ observation papers from the CNKI and Web of Science databases, extracting 440 pairs of full-growth period data. Three EF calculation methods—Maximum Likelihood Estimation (M1), Linear Regression With Intercept (M2), and Linear Regression Without Intercept (M3)—were compared. Adopting a “70% calculation+30% independent validation” mode, the optimal algorithm was screened to calculate localized EFs for China. By combining statistical data on crop planting areas and nitrogen application rates across provinces in 2021, the NO emission inventory for Chinese croplands was calculated, and uncertainty was quantified using the error propagation method. Finally, optimized management scenarios, such as fertilizer reduction and organic substitution, were established to predict NO mitigation potential.【Result】Independent validation results indicated that the M1 method yielded the highest prediction accuracy (R2=0.56). The calculated localized EFs demonstrated significant differences across crops and fertilization practices. Under sole synthetic fertilizer application, tea plantations exhibited the highest EF (1.91%), while rice paddies had the lowest (0.27%). Combined application of organic and inorganic fertilizers generally resulted in lower EFs compared to sole synthetic fertilizer application. Spatially, EFs presented a pattern of being higher in the southeast and lower in the northwest. It was estimated that the total NO emissions induced by nitrogen fertilizer application in Chinese croplands in 2021 were 150 kt NO-N, with an uncertainty of 9.1%. Regarding crop contribution, grain crops were the primary emission source, accounting for 49.9% of the total emissions. Among them, maize contributed the most (31.8 kt NO-N) due to its large planting area and high nitrogen application rates. Despite their smaller planting areas, tea plantations and sugarcane exhibited high emission intensities per unit area due to intensive nitrogen inputs, reaching 7.02 kg NO-N·hm-2 and 2.55 kg NO-N·hm-2, respectively. Regionally, East China contributed 40.9% of the total NO emissions from cereal crops nationwide (30.6 kt NO-N). Scenario analysis indicated significant mitigation potential for NO emissions from Chinese agricultural soils. Through the implementation of scientific nitrogen reduction and the promotion of organic fertilizer substitution technologies, the national NO mitigation potential could reach 24%, equivalent to an annual reduction of 36 kt NO-N. East, North, and Northeast China together accounted for 78.9% of the national mitigation potential from cereal crops.【Conclusion】By constructing a localized emission factor (EF) dataset based on field measurements, this study controlled the uncertainty of NO emission estimates from Chinese cropland soils to within 9.1%. However, due to the scarcity of field measurement data in regions such as Northeast and Northwest China, there remains some uncertainty in regional estimates. This study clarified the significant spatial heterogeneity and crop specificity of NO emissions from Chinese agricultural soils, identifying East China and high-nitrogen-input cash crops as priority areas for future NO mitigation and control. The findings confirm that optimizing chemical fertilizer application rates and implementing organic fertilizer substitution are effective pathways for reducing agricultural soil NO emissions. These results provide scientific support for formulating differentiated, region- and crop-specific agricultural nitrogen management policies and air pollution control measures.

    Nature-Nurturing Agriculture: Investigation on a New Agricultural Paradigm Based on Cross-Kingdom Synergy
    CHEN KeYe, JIANG ShuoYun, WANG XianZhe, LIANG XinQiang, TANG XiaoTian, JIANG PeiKun, YUE ChunLei, ZHANG ZhiJian
    Scientia Agricultura Sinica. 2026, 59(19):  4301-4317.  doi:10.3864/j.issn.0578-1752.2026.19.009
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    In response to the global challenges of ecological degradation, resource constraints and rising quality demands in agriculture, alongside the inherent limitations of organic farming, this paper proposes a new agricultural development paradigm termed ‘nature-nurturing agriculture (NNA)’. This advanced model prioritized the cultivation of ecological carriers, including soils, biotic communities and material cycling, and leveraged “Biomass Mining” as core resources. Building on the prohibition of synthetic inputs that characterized organic farming, this approach aimed to achieve increased yields of high-quality produce, enhanced health of ecological carriers and greater efficacy of ecological assets. This study combined theoretical analysis and case study research. First, it reviewed and discussed the connections and distinctions between existing sustainable agricultural models and NNA, and then constructed an analytical framework for NNA by integrating ecological holism, circular economy theory, and ecological asset theory. Second, three representative case studies from the kingdoms Plantae, Animalia, and Fungi were examined. Through systematic compilation of ecological indicators, the mechanisms related to resource cycling, resilience enhancement, and ecological asset appreciation facilitated by NNA were analyzed, and the viability of the sequential 'Nurture-Produce-Appreciate' framework was assessed. The study found that: (1) In the plant-domain case, through measures, such as the application of bio-organic fertilizers, the return of green manure to the field, and the construction of ecological water bodies and buffer zones, more than two years of nurturing significantly increased soil organic matter content, markedly increased earthworm density in vegetable fields, substantially reduced the use of chemical fertilizers and pesticides, significantly decreased the incidence of major soil-borne diseases, and generated a certain level of carbon-sequestration benefits and additional agro-tourism income; (2) In the animal-domain case, following ecological pond transformation and more than two years of nurturing, biodiversity in aquaculture water bodies was significantly restored, the food chain in fish ponds became more complete; with the support of Chinese herbal medicine, fungal enzyme preparations, and insect-protein substitution technologies, the year-round elimination of antibiotics and conventional fishery drugs was achieved, while both the yield and quality of largemouth bass (Micropterus salmoides) improved and additional income from by-catch and aquatic plants increased; (3) In the fungal-domain case, rice-straw cultivation of Stropharia rugosoannulata was carried out during the fallow period, and frass of black soldier fly larvae was applied to promote straw decomposition and soil cultivation, thereby achieving the high-value utilization of straw, enhanced soil carbon sequestration, improved fungal community diversity, and the secondary nurturing of fungal residues returned to the field, reflecting the coupling of production processes with ecological nurturing. NNA was defined as an agricultural development pathway that, building upon organic farming, further emphasizes the cultivation of ecological carriers, intra-system cycling, and the appreciation of ecological assets. Its essence lies in the support of stable production and the realization of multiple values through ecological health. Future research should be directed toward region-specific adaptation, the construction of technical frameworks, long-term field monitoring, ecological asset accounting, and standard certification, so as to advance NNA from conceptual exploration toward standardized application.

    SPECIAL FOCUS: NATURE-BASED SOLUTIONS: RESEARCH PARADIGMS AND CHINESE PRACTICES FOR GREEN AGRICULTURAL TRANSITION
    Nature-Based Solutions: Research Paradigms and Chinese Practices for Green Agricultural Transition
    JIA XiangPing, CHENG Gong
    Scientia Agricultura Sinica. 2026, 59(19):  4318-4322.  doi:10.3864/j.issn.0578-1752.2026.19.010
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    Evolutionary Trajectory, Driving Mechanisms, and China’s Pathway in Global Nature-Based Solutions for Agricultural Research
    LIANG XiaoHe, WANG Qi, LEI Jie, ZHOU JiaJia, WU Yu, ZHOU AiLian
    Scientia Agricultura Sinica. 2026, 59(19):  4323-4334.  doi:10.3864/j.issn.0578-1752.2026.19.011
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    Against the backdrop of intensifying global climate changes and multi-faceted challenges to food security, nature-based solutions (NbS) are emerging as a key paradigm driving the green transformation of agriculture. This paper systematically traced the evolutionary logic of international and domestic research on agricultural NbS, identified the driving factors of high-impact outputs, and analyzed the localization dilemmas and institutional bottlenecks faced by China. It aimed to provide a decision-making basis for constructing an agricultural NbS theoretical system with Chinese characteristics and exploring global collaborative pathways. Based on data from the Web of Science and CNKI databases, this study adopted a quantitative analytical framework combining bibliometrics and multiple regression analysis. Scientific knowledge mapping was utilized to extract the core topics and evolutionary patterns of domestic and international research. Meanwhile, a multiple regression model was constructed to quantitatively measure the key mechanisms influencing the academic impact of global agricultural NbS research from four dimensions: collaboration networks, regional geopolitics, resource allocation, and interdisciplinary integration. The findings indicated that international research on agricultural NbS has progressed through three successive stages: Theoretical foundation, application and expansion, and mechanism deepening and model-driven. These stages have coalesced into four core research clusters: synergistic enhancement mechanism of agro-ecosystems, pathways for the ecological transformation of urban agriculture, R&D of intelligent and eco-agricultural facilities, and assessment of benefits in agricultural NbS applications, exhibiting highly empirical and granular characteristics. Regression analysis revealed that funding inputs, transnational collaboration, and interdisciplinary integration significantly elevated academic impact. Europe dominated global rule-making by virtue of its institutional advantages, and the academic spillover effect of Sino-European collaboration was significant, establishing China as a critical hub connecting Western theories with large-scale practical verification. Core funding agencies in the US and Europe effectively translated resources into academic discourse power, whereas the driving effect of China's national-level funding on frontier basic theoretical innovation remains to be strengthened. Furthermore, research within the Chinese context presented a dual-driven feature of macro-level coordination and local-level collaboration, with the research focus gradually shifting from macro-concept discussions to micro-engineering practices. However, the overall research network exhibited fragmentation, and the disciplinary distribution was highly concentrated in environmental science and traditional agricultural and forestry disciplines, lacking a comprehensive modeling paradigm for cross-system coupling. Although China currently ranks among the global leaders in terms of publication volume in agricultural NbS, it still faces challenges, such as a mismatch between publication scale and international impact, an overemphasis on localized technical validation at the expense of systematic integration, a relatively marginalized strategic status of agriculture within the broader NbS agenda, and an incomplete autonomous theoretical system. Moving forward, future research should deepen international cooperation to co-build standardized evaluation metrics, introduce social-ecological-technical system (SETS) analytical frameworks to dissolve disciplinary barriers, and strengthen top-down design to elevate the strategic weight of agricultural NbS. Furthermore, efforts should be dedicated to promoting the deep integration of the international NbS framework with China’s philosophy of a “community of life for mountains, rivers, forests, farmlands, lakes, grasslands, and deserts” thereby constructing an autonomous knowledge system for agricultural NbS that encompasses both Chinese characteristics and a global vision.

    Nature-Based Solutions for Sustainable Transition in High-Yield Cropping Systems
    LUO Ning, LI ZhaoLei, XU CaiLong, NIU Na, WANG Pu, MENG QingFeng
    Scientia Agricultura Sinica. 2026, 59(19):  4335-4348.  doi:10.3864/j.issn.0578-1752.2026.19.012
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    Global agriculture is facing the dual challenge of meeting the rising demand for food production while coping with increasing constraints on resources and the environment. Identifying new concepts and feasible pathways to support the sustainable transformation of crop production has therefore become a key scientific priority. This study focused on the roles and potential of nature-based solutions (NbS) in enhancing both crop productivity and sustainability. It established a theoretical framework for NbS-driven nutrient management, analyzed representative technical pathways and their performance, and summarized recent progress in relevant standards and policies to provide scientific guidance for improving the ecological productivity of agricultural systems. The major approaches currently applied in sustainable farmland management and their limitations were reviewed. Taking nutrient management as an example, a triple-pathway framework for the sustainable transformation of high-yield cropping systems was proposed based on NbS, and the applicability of the Global Standard for NbS developed by the International Union for Conservation of Nature (IUCN) in farmland management was assessed. NbS-based nutrient management could enhance nutrient use efficiency and ecosystem services by harnessing natural ecological processes, optimizing system structures, and improving coordination among multiple factors, thereby supporting high crop productivity, efficient resource use, and environmental sustainability. The three pathways included: (1) Optimization of input use within cropping systems, including adjustments in nutrient sources and application methods; (2) Redesign of cropping systems by introducing legumes into major cereals to promote biological nitrogen fixation, improve nutrient balance, and reduce carbon emissions; (3) Integrated regulation of cropping systems and inputs to facilitate system transformation and strengthen resilience. Evidence from representative regional case studies showed that NbS-driven practices could improve nutrient use efficiency, reduce environmental pollution, and enhance the resilience of high-yield systems, demonstrating both technical feasibility and strong potential for scaling up. NbS thus provides a systematic approach to achieving sustainable transformation of crop production. By integrating natural ecological processes with modern agricultural management, NbS could simultaneously improve nutrient efficiency, restore ecosystem functions, and reduce environmental risk, guiding agricultural systems toward green, low-carbon, and resilient development. Strengthening policy support systems and implementation mechanisms for NbS nutrient management would be essential to achieving coordinated progress in green agricultural development, carbon peaking, carbon neutrality, and food security.

    Key Technologies and Models of Straw Comprehensive Utilization: Grain-Mushroom Rotation in Designated Zones as a Nature-Based Solution
    KONG WeiLi, WANG LiJie, ZHENG JingKang, GAO Fang, LI Tao, ZHANG KeKe, ZHANG YuTing
    Scientia Agricultura Sinica. 2026, 59(19):  4349-4357.  doi:10.3864/j.issn.0578-1752.2026.19.013
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    With the continuous growth of the global population and the expanding demand for food, agricultural production has expanded substantially in scale, and straw, as the output of its by-product, has also risen accordingly. However, the underutilization of straw resources has led to serious problems, such as air pollution and the unsustainability of farmland ecosystems. To address these challenges, the grain-mushroom designated zones rotation technology provides an eco-friendly, economically efficient, and sustainable new model for the comprehensive utilization of straw, which is based on the concept of nature-based solutions (NbS). This study firstly defined the grain-mushroom designated zones rotation technology as the rotational cultivation of grains and Stropharia rugosoannulata within a designated unit, alternating between different plots over the years. Then, a comparative analysis was conducted between this model and other major straw utilization pathways, including straw fertilization, energy conversion, feed processing, substrate production, and raw material utilization. Furthermore, the study examined how this technology embodied key NbS principles from the perspectives of natural system integrity, multi-scale matching, economic feasibility, and responding to social challenges, such as food security and agricultural pollution, it also systematically evaluated the multiple co-benefits like ecological and economic performance based on empirical data obtained from practical applications in parts of China and relevant literature. The grain-mushroom designated zones rotation technology effectively integrated grain production and mushroom cultivation across both temporal and spatial dimensions, thereby overcoming the limitations of other straw utilization models. It achieved the synergistic development of ecological, economic, and social benefits, aligning well with the fundamental principles of NbS. At present, the grain-mushroom designated zones rotation technology still faced gaps in satisfying NbS standards, especially in terms of governance frameworks, monitoring and evaluation, and large-scale implementation. To close the existing gaps, this paper proposes the following practical recommendations: (1) The government should introduce targeted policies to support the promotion and application of this technology; (2) At the county, township, and village levels, a multi-stakeholder co-governance platform should be established, comprising “government + enterprises + cooperatives + farmers + research institutions”, to ensure farmers’ rights to information, participation, and decision-making in model selection and benefit distribution; (3) In collaboration with research institutes, a quantifiable and operational evaluation system should be developed around the three dimensions of ecological, economic, and social benefits, supported by scientific, reliable, and continuous data. This would enable the grain-mushroom designated zones rotation technology to evolve into a mature, replicable, and scalable paradigm consistent with international NbS standards.

    Strengthening the Codex Alimentarius System from a Nature-Based Solution Perspective: Governance Models and Practical Approaches
    NING MingHao, LU XinDan, GAO Fang, XU Shang, JIA XiangPing
    Scientia Agricultura Sinica. 2026, 59(19):  4358-4373.  doi:10.3864/j.issn.0578-1752.2026.19.014
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    Against the backdrop of dual challenges, high-yield demands and ecological constraints, in global sustainable agricultural development, reconciling the structural discrepancy between the rigid governance logic of the Codex Alimentarius (Codex), centered on risk exclusion, and the flexible practice paradigm of nature-based solutions (NbS), focused on multi-objective system optimization, has become a critical issue for enhancing comprehensive agricultural governance effectiveness. This study systematically reviewed the development trajectory, core characteristics, and governance challenges of both Codex and NbS, innovatively proposing the core concept of “deepening the resilience transformation of Codex from an NbS perspective”. Based on the international institutional spectrum model, it revealed Codex as a member-state government-dominated “principal-agent” mechanism and identified the “orchestrator-intermediary” governance framework of NbS formed by multiple stakeholders. To overcome governance fragmentation, three static fusion models, Codex-centric, NbS-centric, and Dual-centered synergy, were proposed, alongside a dynamic cyclic mechanism driven by both government and market forces. This mechanism utilized intelligent switching of critical thresholds to achieve periodic and dynamic role transitions between “command” and “coordination” for governance actors. Incorporating China’s national conditions, the study proposed a hierarchical governance strategy under a simplified quadripartite governance paradigm to achieve a closed-loop cycle of “policy guidance→technological conversion→regional implementation→feedback optimization”. Dynamic cyclic pilots were conducted within the multi-stakeholder co-governance zone, which served as the core platform for systemic integration and dynamic regulation to harmonize Codex’s uniformity requirements with NbS’s local adaptability. Simultaneously, to address challenges, such as high-frequency role transitions and the formalization of community participation, a progressive risk prevention and control mechanism was established. This mechanism ensured that when ecological performance and social benefits fell below warning thresholds for a sustained period, the system automatically switched from a flexible coordination mode to mandatory government intervention, ensuring compliance with rigid baselines. This study provides a theoretical paradigm for reconciling the structural differences between Codex and NbS, achieving an organic integration of NbS flexible goals and Codex rigid benchmarks through localized institutional reconstruction. This integration pathway offered an operational solution for enhancing the resilience and sustainability of agricultural governance and paved a practical path for China’s strategic participation in the reform of the Codex Alimentarius Commission and the global agricultural governance system.

    Exploring Research Paradigms for Agricultural Heritage from the Perspective of Nature-Based Solutions
    XIAO YunZe, ZHANG Yu, CHENG Gong, LIAN ZiRou, ZHOU HouXi
    Scientia Agricultura Sinica. 2026, 59(19):  4374-4390.  doi:10.3864/j.issn.0578-1752.2026.19.015
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    【Background】Agricultural heritage systems are agroecosystems and knowledge systems co-evolved by humans and nature. At a time when global food systems are being challenged by climate change, land degradation, and declining ecosystem service functions, their multifunctional values have attracted growing attention as a key resource for safeguarding food security and advancing sustainable agricultural development. Since 2023, China’s No. 1 Central Documents have incorporated the “conservation and utilization of agricultural cultural heritage” into the national strategy, laying a policy foundation for its institutionalized development. Meanwhile, the International Union for Conservation of Nature (IUCN) has rapidly promoted the concept of nature-based solutions (NbS) in the fields of agriculture and ecological governance, emphasizing the use of ecosystem processes to address societal challenges.【Objective】Using NbS as an analytical framework could enable a deeper, more systematic, and more comprehensive examination of agricultural heritage systems, help establish an interdisciplinary research paradigm, and provide an internationally applicable indicator system for the conservation, monitoring, management, evaluation, utilization, and research of agricultural heritage systems.【Method】Employing conceptual comparative analysis, this paper proposes that agricultural heritage systems constitute a prototypical form of NbS and could effectively address challenges, such as food security, ecosystem degradation, and rural social vulnerability. Drawing on the eight criteria and 28 indicators of the IUCN Global Standard for NbS, an indicator-based evaluation method was applied to conduct a systematic empirical analysis of representative cases selected from Globally Important Agricultural Heritage Systems (GIAHS) and from China’s list of Important Agricultural Cultural Heritage.【Result】Within each category of the eight NbS criteria, three GIAHS cases and three Chinese cases were selected, aiming to cover a wider range of regions, more diverse ecosystems, and more heterogeneous livelihood types, for a total of 16 GIAHS cases and 18 Chinese cases. These cases substantiated all relevant components of the 28 indicators, demonstrating that agricultural heritage systems align closely with the NbS Standard in terms of ecosystem services, biodiversity conservation, farmland governance, climate resilience, community participation, and related dimensions. The NbS indicator system also proved feasible for the evaluation and policy planning of agricultural heritage systems.【Conclusion】In terms of conceptual structure, value objectives, and system attributes, agricultural heritage systems exhibited a high degree of congruence with NbS; agricultural heritage systems could be regarded as a subset of NbS. Accordingly, NbS principles, criteria, and indicators could be adopted to analyze and discuss agricultural heritage systems, providing a globally applicable analytical framework for their conservation, monitoring, management, utilization, and evaluation. This study offers a research paradigm and an empirical pathway for integrating agricultural heritage systems into the global NbS policy architecture and for promoting green transformation in agriculture.

    FOOD SCIENCE AND ENGINEERING
    Cereal Science and Processing Technology from the Perspective of Future Food
    WEI YiMin, LU ShuWen, LIU JingSheng, ZHANG Hao, ZHANG Bo, SHEN Qun, SHE YongXin, HU XinZhong, GUAN ErQi, ZHANG YingQuan, LI Ming, GUO BoLi
    Scientia Agricultura Sinica. 2026, 59(19):  4391-4399.  doi:10.3864/j.issn.0578-1752.2026.19.016
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    Future foods aim to systematically address major issues, such as food supply, quality and safety, nutrition and health, environmental sustainability, and intelligent production, bringing unprecedented challenges and opportunities to traditional cereal science and processing technologies. Currently, the field of cereal science and processing technology is challenged by several key issues, including the high cost and low adoption rates for intelligent online detection and blockchain traceability systems, coupled with their limited accuracy across diverse crop varieties and production scenarios; the absence of a comprehensive theoretical framework for understanding the synergistic interactions among cereal components, which hinders the optimization of personalized nutrition and processing routes; the slow progress, insufficient integration, and limited cost-effectiveness of stand-alone green production technologies; the inability of interdisciplinary technologies to overcome scalable production barriers, and a persistent lack of independent innovation and key equipment in traditional industries; and rapid food safety testing technologies struggle with regional variations and model instability. From the strategic perspective of future food science and engineering, this paper consolidated and refined key research directions and core technologies in the fields of cereal science and processing technology, as follows: (1) Digital and intelligence-driven cereal processing technologies, including online quality detection and grading of cereal raw materials, AI-based process parameter optimization and real-time control, and blockchain traceability systems; (2) Precision nutrition-oriented cereal component and healthy food development, including quantitative health effects of cereal components, targeted delivery systems for functional factors, and personalized food solutions; (3) Sustainable and green cereal processing technologies, including non-thermal processing, full-component utilization and high-value conversion of by-products, and optimized clean production methods; (4) Exploration of novel cereal resources and innovation in traditional food products, including novel physical and bioprocessing techniques, as well as the upgrading and reinvention of traditional foods; (5) Intelligent food quality and safety assurance systems, including the integration of rapid detection technologies with the development and deployment of intelligent risk prediction platforms. Looking ahead, research on future food science will drive the deep integration of digitalization, intelligence, precision nutrition, and sustainable development. These efforts would provide the crucial support for ensuring national food security, revitalizing rural industries, and enhancing public health, thereby contributing to the full implementation of the “Healthy China” strategy.

    ANIMAL SCIENCE·VETERINARY SCIENCE
    Differential Analysis of Hypoxia-Related Factors in the Small Intestines of Yaks and Yellow Cattle
    ZHANG YeQiong, WANG Jun, XU MingWei, HU JiaQi, LIU ShaSha, LAN WenLin, TAN Xiao, YANG Kun
    Scientia Agricultura Sinica. 2026, 59(19):  4400-4416.  doi:10.3864/j.issn.0578-1752.2026.19.017
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    【Objective】This study aimed to investigate the differential expression of hypoxia-related factors—Pyruvate dehydrogenase kinase 1 (PDK1), Hypoxia-inducible factor-1α (HIF-1α), Transforming growth factor-β (TGF-β), Vascular endothelial growth factor (VEGF), Mothers against decapentaplegic homolog 2 (Smad2), and Mothers against decapentaplegic homolog 3 (Smad3)—in the small intestinal tissues of yaks, to elucidate the intestinal adaptation mechanisms of yaks to hypoxic environments.【Method】Small intestinal tissues (duodenum, jejunum, and ileum) from plateau yaks and plain yellow cattle were selected as research subjects. Hematoxylin-eosin staining (H.E), periodic acid-Schiff staining (PAS), Masson’s trichrome staining, and immunohistochemical staining were performed. Tissue sections after staining were photographed under a light microscope. Image analysis software was used to measure villus height (V) and crypt depth (C) in H.E-stained sections, as well as the average optical density (AOD) of PAS, Masson, and immunohistochemical staining. The average optical density value was employed to represent positive expression results, thereby investigating the distribution characteristics and expression differences of hypoxia-related factors in different species and intestinal segments.【Result】H.E staining results indicated that the V/C ratio (villus height/crypt depth), an indicator of intestinal digestion, in both yaks and yellow cattle was significantly higher in the duodenum compared to the jejunum and ileum (P<0.05). Furthermore, the duodenal V/C ratio of yaks was significantly higher than that of yellow cattle (P<0.05). PAS staining showed that PAS-positive substances were expressed with significantly higher intensity in the duodenum than in the jejunum and ileum (P<0.05), with strong positive expression observed in the striated border, goblet cells, and Paneth cells. The expression in all intestinal segments of yaks was significantly higher than in yellow cattle (P<0.05). Masson's trichrome staining results revealed significant interspecies and segmental differences in collagen fiber content within the small intestinal tissues of both yaks and yellow cattle (P<0.05). Specifically, the proportion of collagen fibers in the duodenum and jejunum of yellow cattle was significantly higher than in the corresponding segments of yaks (P<0.05). Immunohistochemical analysis detected distinct positive brown-yellow expression of PDK1, HIF-1α, TGF-β, and VEGF in the small intestinal tissues, primarily localized to the striated border, Paneth cells, and goblet cells. Differences in intestinal gland expression were observed between species: weak positive expression was seen in the duodenal and jejunal glands of yellow cattle, while strong positive expression was observed in the corresponding locations in yaks. Tissue segment analysis showed that the expression levels of these factors were significantly higher in the duodenum than in the jejunum (P<0.05). Regarding species differences, the overall expression levels in yak small intestinal tissues were significantly higher than in yellow cattle (P<0.05). However, the expression patterns of Smad2 and Smad3 differed from those of the aforementioned factors. The expressions of Smad2 and Smad3 in the duodenum were significantly lower than those in the ileum and jejunum, respectively (P<0.05). Furthermore, the expression of Smad2/3 in the duodenum and ileum of yaks was significantly lower than in yellow cattle (P<0.05), while in the jejunum of yaks, it was significantly higher than in yellow cattle (P<0.05).【Conclusion】Results from H&E staining, special staining, and immunohistochemical staining revealed significant differences between yaks and yellow cattle in the morphological structure, carbohydrate distribution, and expression of hypoxia-related factors in the small intestine. These findings suggest that yak intestines possess unique structural and molecular adaptive features to hypoxia, and that factors such as PDK1, HIF-1α, and VEGF may play important roles in the intestinal hypoxic adaptation mechanisms of yaks. This study provides fundamental data for research on intestinal adaptation in high-altitude animals and offers a reference for exploring the mechanisms and prevention of hypoxia-related intestinal diseases.

    Development and Application of a Multiplex qPCR Detection Kit for Pathogens Causing Bovine Respiratory Disease Complex
    LIU XinZhuo, WANG XinYu, ZHANG YueWei, WU Hao, GU Yue, WANG Shuang, WU WenXue
    Scientia Agricultura Sinica. 2026, 59(19):  4417-4432.  doi:10.3864/j.issn.0578-1752.2026.19.018
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    【Background】In recent years, with the continuous growth of China's demand for beef and milk consumption, coupled with the expansion of breeding scale, the need for cattle health monitoring has become increasingly urgent. Bovine respiratory disease complex (BRDC), as one of the most devastating diseases, has shown a significant upward trend in incidence, causing sustained economic losses to the cattle industry. The etiology of BRDC in calves is complex, with frequent co-infections among pathogens, significantly increasing diagnostic difficulty. Current commercial detection kits suffer from limitations such as limited variety and low throughput, failing to meet the demand for early and accurate diagnosis in large-scale farms.【Objective】This study aims to develop a multiplex quantitative real-time PCR (qPCR) kit with high specificity, sensitivity, and reproducibility for the simultaneous detection of four major pathogenic bacteria associated with BRDC, thereby overcoming the long detection cycles and low sensitivity of traditional methods and providing reliable technical support for rapid diagnosis and scientific prevention and control of BRDC.【Method】Nasal swabs were collected from healthy calves rigorously confirmed negative for the four target pathogens. High-throughput sequencing was employed to construct a nasal cavity microecological background nucleic acid database, and total nucleic acids were extracted to prepare "background nucleic acid samples" for simulating the complex interference matrix of clinical specimens. Specific primers and probes were designed targeting conserved gene regions of Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida, and Klebsiella pneumoniae. A multi-step bioinformatics screening strategy was employed to rigorously evaluate primer secondary structures and their homology with non-target background nucleic acids, thereby eliminating the risk of cross-reactivity with non-target flora. Positive and negative controls containing authentic sample interference components were prepared by mixing background nucleic acid samples with buffer solution and pathogen nucleic acid samples, respectively. The annealing temperature was optimized using gradient settings, primer and probe concentrations were optimized using a matrix method, and the kit's sensitivity, specificity, and reproducibility were systematically evaluated. Validation was performed using 117 clinical samples.【Result】The detection sensitivity of the developed kit reached 1 copy/μL for Mp. bovis and M. bovis, and 10 copies/μL for P. multocida and K. pneumoniae, demonstrating high amplification efficiency. Specificity validation showed no cross-reactivity with common bovine respiratory commensal bacteria or other pathogens, and no non-specific amplification signals were observed. In reproducibility tests, both intra-batch and inter-batch coefficients of variation were both below 10%, indicating good stability and repeatability. The detection rates in clinical samples were 17.09%, 14.53%, 6.84%, and 45.30%, respectively, and all positive samples were confirmed by sequencing.【Conclusion】This study successfully developed a Multiplex quantitative real-time PCR detection kit capable of simultaneously, rapidly, sensitively, and specifically detecting the four major BRDC pathogens: Mp.bovis, Mb.bovis, P.multocida, and K.pneumoniae. By incorporating background nucleic acid simulation and a multi-step bioinformatics screening strategy, this kit effectively addresses the issue of non-specific interference in complex clinical samples, ensuring the accuracy and reliability of the detection system. With its high sensitivity, strong specificity, and good reproducibility, it shortens the detection cycle from several days with traditional methods to within 2 hours, significantly improving clinical diagnostic efficiency. This kit provides key technical support for early rapid diagnosis, epidemic surveillance and warning, and precision medication-based prevention and control, demonstrating broad prospects for application and popularization.