Scientia Agricultura Sinica

Physiological Characteristics in Response to Salt Stress and Allelic Variation and Expression of Salt-Responsive Genes in Seedling Stage of Nangeng Rice Varieties with Salt-Tolerance Ability

DENG LiCheng, LI Cheng, HE Lei, AN HongQiang, WANG CaiLin, ZHANG YaDong, ZHAO ChangJiang, LU Kai

Scientia Agricultura Sinica. 2025, 58(12): 2275-2290. doi:10.3864/j.issn.0578-1752.2025.12.001

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【Objective】Salt stress is one of the main environmental stresses that restrict rice production. Studying the physiological characteristics under salt stress and analysis the allelic variation and expression of salt-tolerance genes provide key gene resources and genetic materials for breeding salt-tolerance rice varieties. 【Method】This study first evaluated the salt-tolerance ability of the Nangeng series high-quality rice varieties/lines during the seedling stage, using survival rate as an indicator for screening salt-tolerance varieties, which physiological changes under salt stress were analyzed, including chlorophyll, Na⁺, K⁺, MDA, H₂O₂ and soluble sugar. The variation types and expression levels of salt-tolerance genes in rice varieties with resistance to high salt concentration were also analyzed to explaining their molecular mechanisms in response to salt stress. 【Result】Under the condition of treating with 140 mmol·L^-1 NaCl for 6 days, the survival rates of NG9108, NG5718, and NGY1 were greater than 60%, with the highest survival rate among the tested varieties. Compared with Nipponbare, the seedlings of NG9108, NG5718, and NGY1 under salt stress had higher chlorophyll content and lower MDA content, indicating that salt stress caused less cell damage to the three varieties. The Na⁺/K⁺ values in the roots of NG9108, NG5718, and NGY1 were significantly higher than those in Nipponbare, while the Na⁺/K⁺ values in the aerial parts were significantly lower than those in Nipponbare, implying that the three varieties absorb or store more Na⁺ in roots, but transport less Na⁺ upwards, which is beneficial for maintaining cell ion balance and causing less ion toxicity and osmotic stress in aerial parts of the seedlings. The three salt-tolerance varieties have 94 SNPs or InDel sites, distributing in exons, introns, 5′UTR, and 3′UTR of the 23 salt-tolerance genes. 24 variation sites of 11 genes occur in the exons, including 7 genes with frameshift mutations or missense mutations which distributed in Os02g0813500 (OsGR2), Os05g0343400 (OsWRKY53), Os06g0685700 (OsRST1), Os07g0685700 (OsEIL2), Os10g0431000 (OsPQT3), Os11g044600 (OsRSS3), Os12g0150200 (P450). Salt stress significantly induces expression of OsSKC1, OsBAG4, OsGPX1, OsCCX2, OsGR3, OsDREB2a, OsRAB21, OsP5CS, OsbZIP23, OsAPX37 and OsLEA3, which help to enhance salt tolerance and reduce the adverse effects of salt damage on rice growth. 【Conclusion】NG9108, NG5718 and NGY1 showed strong salt tolerance phenotype during the seedling growth stage, which is closely related to the balance of sodium and potassium ions under salt stress, allelic variations of multiple salt tolerance genes, and gene expression levels. NG9108, NG5718 and NGY1 have pyramided multiple salt tolerant and high-quality genes, which can be used as backbone parents for genetic improvement and breeding.

Resistance Evaluation and Genetic Stability Analysis of Insect- Resistant and Glyphosate-Tolerant Transgenic Cotton Lines

WEN Jin, NING YanFang, QIN Xin, LIU Yuan, ZHANG XiaoLing, ZHU YongHong, TIAN ShiMin, MA YanBin

Scientia Agricultura Sinica. 2025, 58(12): 2291-2302. doi:10.3864/j.issn.0578-1752.2025.12.002

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【Objective】Cotton bollworm (Helicoverpa armigera) and field weeds are major constraints to high-yield cotton production. Existing varieties with single traits (insect resistance or herbicide tolerance) fail to meet the demands of efficient cultivation. Developing transgenic cotton varieties with combined insect resistance and glyphosate tolerance will provide high-efficiency germplasm resources for stress-resistant cotton breeding.【Method】The insect-resistant fusion gene cry1Ac-vip3Da and glyphosate-tolerant gene g10-epsps were introduced into cotton R15 through Agrobacterium-mediated method, regenerated transgenic plants were screened via PCR, positive lines underwent multi-generation self-pollination to achieve homozygosity, and stable lines with superior resistance were selected. The expression of target genes in different tissues of transgenic lines was analyzed using qRT-PCR and ELISA. Bioactivity assays and glyphosate tolerance tests were conducted to evaluate the genetic stability of insect resistance and herbicide tolerance across generations (T₄-T₆). Agronomic traits of transgenic lines were comprehensively assessed. 【Result】Eight positive transgenic lines with dual resistance were identified through PCR screening, and CA-6, CA-7 and CA-17 lines exhibited higher resistance. qRT-PCR revealed high expression of cry1Ac-vip3Da and g10-epsps in all tissues of these lines, and expression levels varied significantly among tissues. ELISA analysis demonstrated significant differences in Cry1Ac-Vip3Da and G10-EPSPS protein content across tissues of the three transgenic lines, with the highest levels observed in leaves. Protein accumulation gradually decreased during the developmental stages (from the four-leaf stage to boll-opening stage), but remained stable across T₄-T₆ generations. Bioactivity assays and glyphosate tolerance tests demonstrated that three transgenic cotton lines (T₄-T₆ generations) exhibited corrected mortality rates of 65.12%-82.75%, tolerated glyphosate at over four times the recommended dosage, and showed no attenuation of resistance across generations. There were no significant differences in plant height, number of fruit branches, number of bells per plant, bell weight, lint percentage, seed cotton yield, and lint cotton yield between transgenic lines and R15.【Conclusion】The exogenous genes cry1Ac-vip3Da and g10-epsps were stably inherited across generations in transgenic lines CA-6, CA-7, and CA-17, conferring dual insect resistance and glyphosate tolerance without compromising agronomic performance.

Evaluation of 322 Peanut Germplasms for Resistance to Aspergillus flavus Infection

CUI MengJie, SUN ZiQi, QI FeiYan, LIU Hua, XU Jing, DU Pei, HUANG BingYan, DONG WenZhao, HAN SuoYi, ZHANG XinYou

Scientia Agricultura Sinica. 2025, 58(12): 2303-2315. doi:10.3864/j.issn.0578-1752.2025.12.003

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【Objective】Aflatoxin contamination is one of the important factors that hinders sustainable development of the peanut industry. Precise evaluation of germplasm resources from China and abroad for resistance to A. flavus infection and creation of new resistant germplasms will facilitate the development of resistant cultivars. 【Method】The A. flavus infection index of 322 peanut germplasm lines were characterized following in-vitro inoculation of seeds harvested from 3 different “environments” (CA2020, CS2020, CS2021). Aspergillus flavus strain As 3.4408, known for its strong infectivity and high toxin production, was used as the inoculation strain. The botanical type, plant type and nutritional quality of kernels were measured and analyzed. Accessions exhibiting resistance with novel traits were comprehensively evaluated and screened. 【Result】Thirteen accessions with stable resistance were identified, accounting for 4.04% of the total germplasm lines evaluated, most of which belonged to var. hypogaea, including two with stable and high resistance (C203 and C206), while no accession was observed to be immune to Aspergillus flavus infection. The frequency distribution of infection index of 322 accessions exhibited continuous variation, with the broad-sense heritability exceeding 0.8, indicating that the A. flavus-resistance of kernels was significantly influenced by genotypes and “environments”, and the phenotypic variation was primarily controlled by genetic factors. Correlation analysis revealed significant positive correlation of infection index of accessions among the different “environments” (P<0.001), and the phenotype of each accession harvested from various “environments” was relatively consistent. Additionally, no significant correlation was found between nutritional quality and infection index. Comparative analysis of infection index among peanut accessions of different botanical and plant types revealed that var. hypogaea/prostrate-type peanuts were more likely to exhibit resistance to A. flavus infection within the existing peanut germplasm resources. 【Conclusion】The phenotypes of peanut germplasms harvested from different “environments” in response to A. flavus infection were relatively stable. Variation of kernels resistance to A. flavus infection was primarily controlled by genotype. Accessions C203 and C206, exhibiting stable and high resistance, can serve as excellent resistant parents for the mining of aflatoxin resistance genes and for the improvement of peanut varieties resistant to aflatoxin contamination.

Effects of Nitrogen Panicle Fertilizer on Yield, Quality and Aroma of Southern Japonica Rice Nanjing 9108

WANG RuiZhi, LI Tao, QIAN XiangLing, ZHANG Ya, YANG XiaoZhuo, LI GuangYan, WEI HaiYan, ZHANG HongCheng, LIU GuoDong

Scientia Agricultura Sinica. 2025, 58(12): 2316-2332. doi:10.3864/j.issn.0578-1752.2025.12.004

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【Objective】Nitrogen panicle fertilizer is one of the key factors affecting rice yield and quality. Studying its impact on the yield, quality, and aroma of aromatic japonica rice in southern China could provide a theoretical basis for high-yield and high-quality cultivation of southern japonica rice. 【Method】 Conducted from 2022 to 2023, this study used Nanjing 9108, a representative variety of aromatic japonica rice in southern China, as the material, and three nitrogen application modes were set up: no nitrogen fertilizer (N0), no panicle fertilizer (N1), and conventional application of panicle fertilizer (N2, with 70% base and tillering fertilizer + 30% panicle fertilizer). In addition, the experiment of applying ear fertilizer at different leaf age stages, including the top sixth leaf, fifth leaf, fourth leaf, third leaf, second leaf and first leaf just after emerging from the sheath (designated as L6, L5, L4, L3, L2, and L1), was conducted to study the synergistic regulation mechanism of nitrogen panicle fertilizer on yield, quality, and aroma of Nanjing 9108.【Result】Compared with no nitrogen fertilizer application and no panicle fertilizer application, the application of panicle fertilizer could significantly increase the effective panicle number per unit area and grains per panicle of aromatic japonica rice, thereby enhancing its yield. As the period of panicle fertilizer application was delayed, the yield first increased and then decreased, reaching a maximum at the treatment of applying fertilizer at the fourth leaf from the top (counted downwards from the flag leaf). The application of panicle fertilizer improved rice processing quality, appearance quality, and aroma quality. The period of panicle fertilizer application had an impact on these qualities of aromatic japonica rice. With the delay in the period of panicle fertilizer application, the milled rice rate of Nanjing 9108 showed an increasing trend, but the chalkiness degree increased, leading to a deterioration in appearance quality. Simultaneously, the amylose content decreased while the protein content increased, resulting in a decline in taste value and eating quality. The content of 2-acetyl-1-pyrroline (2-AP), as the main component of aroma, also decreased with the delay in the period of panicle fertilizer application. The application of panicle fertilizer significantly increased the proline content and proline dehydrogenase activity in grains. Advancing the period of panicle fertilizer application had a significant promoting effect on proline accumulation during the rice filling stage, and proline dehydrogenase activity also increased, which was conducive to maintaining higher proline content and proline dehydrogenase activity in grains during the maturity stage, thereby promoting the synthesis of 2-AP in rice grains. Based on a comprehensive evaluation of the effects of panicle fertilizer application period using indicators, such as actual yield, milled rice rate, chalkiness degree, taste value, and grain 2-AP content, it was found that the treatment of applying fertilizer at the fourth leaf from the top had the highest comprehensive score. 【Conclusion】Under the experimental conditions of this study, the application of panicle fertilizer contributed to the synergistic improvement of yield and quality. On the basis of ensuring stable yield, the application of panicle fertilizer at the fourth leaf from the top achieved the best overall benefits in terms of yield, taste, and aroma.

Effects of Multiple Cropping Green Manure Combined with Different Nitrogen Fertilizer Levels on Yield and Quality of Spring Wheat

LI Rong, LI ZhengPeng, YAN QingBiao, GUO RanRan, HAN Mei, XU Ke

Scientia Agricultura Sinica. 2025, 58(12): 2333-2345. doi:10.3864/j.issn.0578-1752.2025.12.005

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【Objective】This study aimed to explore the effects of multiple cropping green manure combined with different levels of nitrogen fertilizer on the yield and grain quality of spring wheat, so as to provide the theoretical guidance for the construction of chemical fertilizer reduction and high-quality production mode based on green manure in Qinghai province.【Method】The split plot experiment was carried out in the experimental site of the Academy of Agricultural and Forestry Sciences of Qinghai University from 2023 to 2024. Two planting patterns were set up in the main area: multiple cropping green manure after wheat (W-G) and leisure after wheat (W), and three nitrogen fertilizer levels in the sub-area included: no nitrogen application (N0), nitrogen fertilizer reduction by 30% (N1, 157.5 kg N·hm^-2), and the local custom of nitrogen application (N2, 225 kg N·hm^-2). Wheat yield and grain quality (grain protein content, sedimentation value, formation time, stability time, wet gluten, etc.) were determined.【Result】The grain yield of spring wheat under W-GN1 treatment was 5.5% and 13.4% higher than that under WN1 in 2023 and 2024, and 2.0% and 5.3% higher than that under WN2 treatment, respectively; the biological yield under W-GN1 in 2023 and 2024 was 5.1% and 10.6% higher than that under WN1, and 1.5% and 4.6% higher than that under WN2, respectively. W-G could obtain higher harvest index than W. The compensation effect of multiple cropping green manure was negative under no nitrogen application level, and it was between 2.0%-14.0% under nitrogen application level. The contribution of multiple cropping green manure combined with 30% reduction of nitrogen fertilizer to increasing crop yield was the best. At the same time, W-GN1 could improve grain quality by increasing grain protein content, sedimentation value, formation time and stabilization time. The grain protein content under W-GN1 treatment was 10.62% and 9.48% higher than that under WN1, respectively. The grain sedimentation value increased by 25.05% and 18.13%, respectively. The grain formation time increased by 34.70% and 8.66%, respectively. The grain stability time increased by 41.30% and 13.68%, respectively. Through principal component analysis, it demonstrated that the multiple cropping of green manure after wheat had a more significant promoting effect in grain protein content, sedimentation value, formation time and stability time.【Conclusion】The grain yield and quality of spring wheat were significantly improved by 30% reduction of nitrogen fertilizer (N1,157.5 kg N·hm^-2) combined with green manure after wheat harvest, which could be used as a suitable planting mode and nitrogen application level for improving quality and stable yield of wheat under the condition of reducing chemical fertilizer in Qinghai Province.

The Impact of Sowing Methods on the Seed Germination Environment and Cotton Emergence and Growth

DONG Ming, QI Hong, ZHANG Qian, WANG Yan, WANG ShuLin, FENG GuoYi, LIANG QingLong, GUO BaoSheng

Scientia Agricultura Sinica. 2025, 58(12): 2346-2357. doi:10.3864/j.issn.0578-1752.2025.12.006

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【Objective】The current post-planting film-covering technology in the Yellow River Basin cotton-growing areas relies on manual seedling release and thinning, which hinders the development of full-scale mechanization. This study explored the adaptability of the dry sowing and wet emergence technology (used in northwestern inland cotton regions) to the Yellow River Basin. By comparing the effects of different sowing methods on seed germination environment, cotton emergence rate, and seedling growth, this study aimed to identify key environmental constraints and provide the theoretical support for optimizing mechanized planting techniques. 【Method】From 2023 to 2024, using the cotton cultivar Ji863 as the experimental material, four treatments were implemented: single-seed seeding without mulching (T1), single-seed post-planting film covering (T2), dry sowing and wet emergence with single-seed sowing (T3), and dry sowing and wet emergence with double-seed sowing (T4). The study was conducted in Quzhou County, Hebei Province, and the effects of these treatments on soil environment, cotton emergence, and growth of above-ground and root systems were evaluated. 【Result】Compared with T1, T2 and T3 significantly increased soil temperature at 5 cm, soil moisture content, emergence rate, plant height, above-ground dry matter weight, root length, root surface area, root volume, and root vigor, while significantly reduced soil compaction, emergence time, and root diameter. Compared with T2, in 2023 and 2024, T3 reduced the daily temperature range at 5 cm soil depth by 3.67 and 1.58 ℃ within 30 days after sowing, and reduced soil compaction by 9.36% and 27.06% within 10 days after sowing, respectively, which decreased emergence days in 2024 by 0.6 days, and increased root length, surface area, volume, and root activity in both years. Compared with T4, single-grain sowing in 2023 and 2024 significantly increased emergence time and decreased emergence rate but increased aboveground dry matter weight by 13.98% and 55.00%. The structural equation model showed that different sowing methods affected cotton seedling emergence rate by altering soil temperature at 5 cm depth, daily temperature range, soil moisture content, and soil compaction, while seedling dry matter weight was mainly affected by soil moisture content, plant height, and soil compaction. 【Conclusion】In the Yellow River Basin cotton areas, the dry-sowing and wet-emergence improved emergence speed and rate by reducing the diurnal temperature fluctuation at 5 cm and soil compaction, thereby promoting uniform and robust seedlings, and sowing one seed per hole was the optimal strategy.

Isolation and Identification of Pathogen of Plum Bacterial Shot Hole and Laboratory Test of Bactericide Toxicity

ZHAO YuMeng, LI GuangXu, DAI QiDong, LIU JiaCheng, ZHAO HaiJuan, ZHANG YuJun, YANG Hua, LIU Shuo

Scientia Agricultura Sinica. 2025, 58(12): 2358-2370. doi:10.3864/j.issn.0578-1752.2025.12.007

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【Objective】Plum cultivation is an important industry in Liaoning region, while the large-scale epidemic of bacterial shot hole has seriously affected the sustainable development of plum cultivation. The purpose of this paper is to clarify the pathogenic bacteria species of plum bacterial shot hole in Liaoning, and to explore the toxicity of different agents against the pathogenic bacteria, so as to provide a theoretical basis for the prevention and control of the disease.【Method】From 2023 to 2024, the occurrence of plum bacterial shot hole was investigated in 10 plum production areas of Liaoning Province. By collecting 20 disease samples of leaves, fruits, and twigs with typical symptoms, a total of 30 pathogenic bacteria were isolated, purified and preserved. The characteristics of these colonies including morphology, color, size and edge morphology were observed and recorded. Transmission electron microscopy was employed to further observe their morphology and the Gram staining was performed. The bacterial 16S rDNA universal primer 27F/1492R and multiple gene loci were used for amplification. The sequencing result was compared by BLASTn analysis in the NCBI database and the reference sequences of the corresponding genera were downloaded. Then, a multi-gene joint phylogenetic tree was constructed using the maximum likelihood method on the website of GIPRES Science Gateway. Subsequently, the leaves of ‘Qiu Ji’ plum were inoculated with pathogenic bacteria by the stab inoculation of bacterial suspension, and then cultivated at 28 ℃ under high humidity. The disease occurrence of the leaves was regularly observed and recorded. Furthermore, the pathogenic bacteria were re-isolated from the disease-health junction of the leaves to complete the verification of the whole Koch’s postulates. The sensitivities of pathogenic bacteria to 0.15% tetramycin, 80% ethylicin, 3% benziothiazolinone, 1.8% octylamine, 3% zhongshengmycin and 6% kasugamycin were determined by the inhibition zone method, respectively.【Result】Based on morphological observation and molecular biology identification, the pathogenic bacteria were identified as Xanthomonas arboricola pv. pruni (Xap) (36.67%, 11 strains) and Pantoea vagans (63.33%, 19 strains). The sensitivity results demonstrated that the best inhibitory activities were achieved by 0.15% tetramycin and 80% ethylicin, with the EC₅₀ of 0.15% tetramycin being 0.026 and 0.502 μg·mL^-1 for Xap and P. vagans, respectively, and that of 80% ethylicin being 1.162 and 25.643 μg·mL^-1, respectively. The second was 3% benziothiazolinone, and its EC₅₀ values against Xap and P. vagans were correspondingly 5.200 and 96.075 μg·mL^-1, followed by EC₅₀ of 1.8% octylamine against Xap and P. vagans of 176.008, 273.072 μg·mL^-1, and 3% zhongshengmycin against Xap and P. vagans of 621.697 and 72.270 μg·mL^-1. As for 6% kasugamycin, it had an EC₅₀ of 886.467 μg·mL^-1 for P. vagans and was less effective in inhibiting Xap.【Conclusion】The pathogens causing plum bacterial shot hole in Liaoning were identified as Xap and P. vagans. Moreover, the 0.15% tetramycin and 80% ethylicin were screened and selected as exerting better inhibitory effects on these two pathogens. The results of the study will provide a theoretical basis for the precise prevention and control of plum bacterial shot hole in the field.

Establishment and Application of RT-RAA-CRISPR/Cas12a-Based Visual Detection of Prunus Necrotic Ringspot Virus

ZHANG XiaoQi, SHEN JianGuo, LIAO FuRong, LI WeiMin, JIN YuJie, WUFUER Shayidan, ZHENG LuPing

Scientia Agricultura Sinica. 2025, 58(12): 2371-2381. doi:10.3864/j.issn.0578-1752.2025.12.008

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【Objective】The study aims to establish a novel visual detection technique for prunus necrotic ringspot virus (PNRSV) by combining reverse transcription recombinase-aided amplification (RT-RAA) with CRISPR/Cas12a system (RT-RAA-CRISPR/ Cas12a).【Method】Primers with high amplification efficiency and strong specificity were designed and selected based on the conserved regions of the coat protein (CP) gene of PNRSV. The detection conditions, including primer, probe concentration, temperature, and reaction time were optimized to develop a visual detection method for PNRSV by RT-RAA-CRISPR/Cas12a technology. The specificity of this method was evaluated by detecting PNRSV and common Prunus viruses, including plum pox virus (PPV), apple mosaic virus (ApMV), cucumber mosaic virus (CMV), potato virus X (PVX), and potato virus Y (PVY). The total RNAs from PNRSV-infected fruit were diluted in 10-fold gradients, then RT-PCR, RT-RAA and RT-RAA-CRISPR/Cas12a were performed to compare the sensitivity of the three methods. The RT-RAA-CRISPR/Cas12a and RT-PCR methods were used to detect 31 peach fruit test samples suspected to be infected with the virus collected at the port to verify the practicability of the visual detection method.【Result】The RT-RAA-CRISPR/Cas12a-based visual detection method for PNRSV was successfully established. The optimized working concentrations were as follows: RT-RAA-PNRSV-F2/R2 primers at 0.4 μmol·L^-1, fluorescent reporter (FQ) at 800 nmol·L^-1, CRISPR-Cas12a at 200 nmol·L^-1, and PNRSV-crRNA (CRISPR RNA) at 240 nmol·L^-1, the reaction conditions were performed at 41 ℃ for 45 min. This method showed high specificity for PNRSV and had no cross-reaction with other common Prunus viruses. The limit of detection for PNRSV RNA in peach fruit samples reached 3.06 pg·μL^-1 and 306 fg·μL^-1 using RT-RAA and RT-RAA-CRISPR/Cas12a methods, respectively, showing the sensitivity of RT-RAA-CRISPR/Cas12a was 10 times higher than that of RT-RAA and RT-PCR. Among the 31 tested peach fruit samples at the port, 14 positive samples were identified by RT-PCR, while 15 positive samples were found by RT-RAA-CRISPR/Cas12a, indicating a high level of consistency between the two methods.【Conclusion】The RT-RAA-CRISPR/Cas12a visual detection method for PNRSV has been established. It is characterized by simplicity, rapidity, high sensitivity, high specificity, and visual readability, making it well-suited for rapid on-site detection of PNRSV.

Phosphorus Fractions in Rhizosphere and Bulk Soil of Wheat and Their Availability Under Long-Term Fertilization

LU Peng, FENG Jia, LI Li, YANG Li, YANG Lei, LÜ DeZhi, XUE YanFei

Scientia Agricultura Sinica. 2025, 58(12): 2382-2396. doi:10.3864/j.issn.0578-1752.2025.12.009

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【Objective】 By studying the effects of long-term different fertilization on organic phosphorus and inorganic phosphorus in wheat rhizosphere and bulk soil, the contribution of organic phosphorus and inorganic phosphorus to soil available phosphorus was revealed, which could provide a theoretical basis for rational fertilization of soil and effective utilization of accumulated phosphorus in soil.【Method】Wheat rhizosphere and bulk soil samples were collected from different fertilization treatments (control without fertilizers (CK), nitrogen and potassium (NK), nitrogen, phosphorus and potassium (NPK), and NPK plus organic manure (MNPK)). The content of inorganic phosphorus fractions (dicalcium phosphate (Ca₂-P), octa-calcium phosphate (Ca₈-P), apatite (Ca₁₀-P), aluminum bounded phosphate (Al-P), iron bounded phosphate (Fe-P), and occluded phosphate (O-P)) in the soil were determined by Chang and Jackson's inorganic phosphorus classification method improved by JIANG Bofan and GU Yichu, and organic phosphorus fractions (labile organic phosphorus (L-OP), moderately labile organic phosphorus (ML-OP), moderately resistant organic phosphorus (MR-OP), and high resistant organic phosphorus (HR-OP)) were determined by Bowman-Cole's organic phosphorus classification method.【Result】The long-term balanced fertilization (NPK, MNPK), especially the application of organic fertilizer (MNPK), significantly improved the level of soil organic carbon and decreased the soil pH, and significantly increased the content of L-OP, ML-OP and MR-OP in soil organic phosphorus and Ca₂-P, Ca₈-P, Al-P, Fe-P and O-P in inorganic phosphorus. Organic phosphorus mainly existed in ML-OP, followed by MR-OP, L-OP, and HR-OP, accounting for 73.4%, 15.6%, 5.4%, and 5.6% of the total organic phosphorus in rhizosphere soil, respectively, and 72.1%, 15.8%, 6.0% and 6.1% in bulk soil, respectively. The average content of inorganic phosphorus was Ca₁₀-P>Ca₈-P>Al-P>O-P>Fe-P>Ca₂-P, accounting for 51.2%, 15.7%, 12.3%, 11.4%, 7.2%, and 2.2% of the total inorganic phosphorus in rhizosphere soil, respectively，and accounting for 53.2%, 15.4%, 11.6%, 10.9%, 6.7% and 2.2% of the total inorganic phosphorus in bulk soil, respectively. In the rhizosphere soil, the phosphorus fractions affecting the phosphorus activation coefficient (PAC) were ranked as ML-OP>Al-P>Ca₂-P>L-OP>Ca₈-P>MR-OP>Fe-P>O-P>Ca₁₀-P>HR-OP. In bulk soil, the order of phosphorus fractions affecting PAC was Ca₂-P>L-OP>Ca₈-P>Al-P>ML-OP>Fe-P>MR-OP>Ca₁₀-P>HR-OP>O-P.【Conclusion】Long-term balanced fertilization, especially the application of organic fertilizer, significantly improved the availability of moderately labile organic phosphorus and moderately labile inorganic phosphorus in rhizosphere soil. The availability of labile organic phosphorus and labile inorganic phosphorus in bulk soil was also significantly improved. Available phosphorus in the rhizosphere soil mainly came from ML-OP and Al-P, while available phosphorus in bulk soil mainly came from L-OP and Ca₂-P. The reasonable soil fertilization could effectively improve soil phosphorus availability.

Effects of Straw Returning Methods and Nitrogen Application Rates on Soil Organic Carbon Components and Enzyme Activity

MA YuJie, LI Xu, ZHAI YingFang, LI JingYu, FU Xin, PENG ZhengPing

Scientia Agricultura Sinica. 2025, 58(12): 2397-2410. doi:10.3864/j.issn.0578-1752.2025.12.010

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【Objective】Straw returning is the main conservation tillage measure in the North China Plain, but long-term shallow rotary tillage has caused problems, such as shallow soil layer and nutrient accumulation, which has become an obstacle to the improvement of cultivated land quality in this area. This study was based on a 2-year field experiment to investigate the effects of different straw returning methods and nitrogen application rates on soil organic carbon components and enzyme activity, for providing a theoretical basis for the rational application of straw in North China.【Method】 The experiment set up different straw returning methods (rotary tillage without straw returning (CK), rotary tillage with straw returning (RT), and deep tillage with straw returning (DT)) and nitrogen fertilizer application rates (180 kg N·hm^-2(N1), 240 kg N·hm^-2(N2), and 300 kg N·hm^-2(N3)) as dual factor treatments to analyze the changes in soil organic carbon and labile fractions and enzyme activity under different straw returning methods and nitrogen fertilizer application rates.【Result】 In 2021-2022 and 2022-2023, compared with CK treatment, straw returning treatment significantly increased the soil organic carbon (SOC) content in the 0-20 and 20-40 cm soil layers, with an increase of 5.6%-20.8% and 10.1%-18.8%, respectively, and increased the content of active organic carbon components, such as soil particulate organic carbon (POC), dissolved organic carbon (DOC), and easily oxidizable organic carbon (ROC). Compared with RT treatment, DT treatment could significantly increase the POC content in the 20-40 cm soil layer and the ROC content in the 0-40 cm soil layer. Under different straw returning methods, the effect of nitrogen application rate on SOC did not reach a significant level, but nitrogen application rate increased the content of some activated carbon components. N2 nitrogen application level was more conducive to increasing POC content in the 0-20 cm soil layer, while N3 nitrogen application level was more conducive to increasing microbial biomass carbon (MBC) and DOC content in the 0-20 cm soil layer. In 2022-2023, compared with the CK treatment, the straw returning treatment significantly increased enzyme activities in the 0-20 and 20-40 cm soil layers by 2.3%-30.3% and 5.1%-50.3%, respectively. Compared with RT treatment, the activity of N-acetylglucosamine glucosidase (N-AG) was significantly increased in the 0-20 cm soil layer treated under DT, but the activities of urease (UG) and β - glucosidase (β-G) were significantly decreased. Under RT treatment, the nitrogen application rate of N2 could significantly increase the activity of UG and N-AG compared to other nitrogen application rates; under DT treatment, the nitrogen application rate of N2 could significantly increase the activity of UG and β - G. In the 0-20 cm soil layer, SOC and DOC were positively correlated with catalase (CG) activity, while ROC was positively correlated with CG and N-AG activity. In the 20-40 cm soil layer, ROC and MBC were positively correlated with most enzyme activities. 【Conclusion】In summary, returning straw to the field could enhance the organic carbon, active components, and enzyme activity of surface and sub surface soils. Deep tillage straw returning combined with 240 kg·hm^-2 nitrogen fertilizer was more conducive to increasing the accumulation of soil particulate organic carbon and easily oxidizable organic carbon, as well as enzyme activity, promoting soil fertility and farmland quality improvement.

The Release Characteristics of Medium and Trace Elements and Their Effects on Soil Available Nutrients after the Continuous Return of Green Manure in Peach Orchards

GUO Lei, ZHANG BinBin, SHEN ZhiJun, YAN Juan, XU JianLan, CAI ZhiXiang, YU MingLiang, WANG FaLin, SONG HongFeng

Scientia Agricultura Sinica. 2025, 58(12): 2411-2426. doi:10.3864/j.issn.0578-1752.2025.12.011

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【Objective】The objective of this study is to simulate green manure management in the actual production of grassy peach orchards, investigate the release law of medium and trace elements (MTE) after green manure was continuously incorporated into the soil, and to examine the effects of continuous grass on soil available nutrients in peach orchards, so as to provide reasonable grass planting and scientific formulation of nutrient management measures in peach orchard.【Method】The grass-planting experiment was carried out in a peach orchard where grass was sown in 2016. Two treatments were set up: inter-row planting of ryegrass (RG) and hairy vetch (HV), and the mulching of black ground fabric (BF) was used as the control. In 2021 and 2022, soil available MTE content was measured at four key stages: pre-surface return (before mowing) and pre-burial (before incorporation) of green manure. The nutrient release characteristics of green manure were conducted using the nylon net bag method. The first stubble green manure was subjected to 100 d of surface decomposition followed by 350 d of burying decomposition, and the second stubble green manure was subjected to 100 d of surface decomposition. Element release patterns and residual quantities per unit area of green manure residues were analyzed over two consecutive years.【Result】There was no significant difference in the initial content of Fe between the two types of green manure, while the initial content of Ca, Mg, and Cu in hairy vetch was significantly higher than in ryegrass. The accumulated release ratios of Ca, Mg, Mn, Cu, and Zn in ryegrass ranged from 2.65% to 55.43% during the surface decomposition of the first crop of green manure, all of which were significantly higher than those in hairy vetch. After the burying back to the field, the accumulated release ratios of Mn, Cu, and Zn in the residue increased overall. By the end of the test, the accumulated release ratios of Mn and Zn in hairy vetch reached 82.59% and 90.02%, respectively, which were significantly higher than those in ryegrass. Both species exhibited persistent Fe accumulation, with accumulated release ratios reaching -360.78% (RG) and -371.63% (HV) after 450 d. During the surface decomposition of the second crop green manure, Ca, Mg, and Zn were enriched in hairy vetch only, while Fe, Mn, and Cu were enriched in both of green manures. The differences in the release levels of various elements resulted in the residual amounts of Ca and Mg per unit area of green manure, which reached their highest values after 350 d of returning the first crop of green manure. However, the residual amounts of Fe, Mn, and Cu continued to increase at the end of the test. After continuous grass returning, the increase range of soil exchangeable Ca content in hairy vetch and ryegrass plots exceeded 13.13% and 23.56%, respectively, and the exchangeable Mg content increased by more than 13.85% and 20.51%, respectively. Compared with BF, by the end of the experiment, the available Mn content in the soil of HV increased by 63.16%, and the available Zn content in RG increased by 134.16%. However, the available Cu content of the HV and RG decreased by 37.90% and 36.06%, respectively, and the available Fe content was only 46.87% and 34.61% of the BF, respectively.【Conclusion】In the long run, perennial green manure application enhances soil exchangeable Ca and Mg, but reduces available Fe and Cu in peach orchards. In the short term, MTE fixed by green manure fails to completely release after 450 d of returning, and the enrichment time of Fe is the longest. In the early stage of grass planting in peach orchards, it is advisable to choose the mixed coverage mode of covering ground fabric in rows or under tree trays and planting green manure between rows. During the growth period of green manure, supplemental MTEs fertilization should be applied to effectively mitigate nutrient competition between peach trees and green manure and to promote the improvement of soil fertility in peach orchards.

Screening of Transcription Factors for Key Enzyme Gene CsAlaDC Involved in Ethylamine Synthesis in Yinghong 9 (Camellia sinensis)

RUAN QiaoJun, MAO MiaoMiao, ZHANG YuanYuan, LIN XiaoRong, CHEN ZhongZheng

Scientia Agricultura Sinica. 2025, 58(12): 2427-2438. doi:10.3864/j.issn.0578-1752.2025.12.012

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【Objective】Theanine is synthesized from substrates ethylamine and glutamic acid catalyzed by theanine synthase. Ethylamine, the primary limiting factor in theanine biosynthesis in tea plants (Camellia sinensis), is generated via the decarboxylation of alanine catalyzed by alanine decarboxylase (CsAlaDC). This study aims to identify transcription factors regulating CsAlaDC through yeast one-hybrid screening of a tea plant cDNA library, thereby elucidating the molecular mechanisms underlying ethylamine biosynthesis regulation from a transcriptional perspective. The findings provide a theoretical foundation and technical references for advancing theanine biosynthesis and genetic improvement in tea plants.【Method】Using the Yinghong 9 tea cultivar, the promoter of CsAlaDC was cloned via PCR and employed as bait for yeast one-hybrid screening to identify candidate transcription factors from a cDNA library. Coding sequences (CDS) of candidate transcription factors were cloned and reversed to validate them. Bioinformatics analyses were conducted to characterize protein domains, molecular weights, and solubility of the candidates. Dual-luciferase assays were performed to assess transcriptional regulation of CsAlaDC by the candidate transcription factors, while subcellular localization experiments determined their cellular distribution.【Result】The CsAlaDC promoter cloned from the Yinghong 9 tea cultivar contained core eukaryotic promoter elements such as TATA-box and CAAT-box, along with light-responsive motifs (I-box, G-Box, TCT-motif, TCCC-motif, Box4), a meristem-specific CAT-box cis-element, stress-responsive elements (ABRE, TC-rich repeats), and binding sites for transcription factor families including MYB, MYC, and WRKY. Using the CsAlaDC promoter as bait, yeast one-hybrid screening of the Yinghong 9 cDNA library identified 17 potential binding proteins. After preliminary screening, five candidate transcription factors were selected for CDS cloning and reverse validation, ultimately confirming three transcription factors: CsFBX, CsBBX, and CsASR. Dual-luciferase assays demonstrated that CsBBX and CsASR significantly activated CsAlaDC expression, whereas CsFBX exhibited no regulatory effect. Subcellular localization revealed nuclear localization of CsFBX and CsBBX, while CsASR was localized in both the nucleus and cytoplasm.【Conclusion】Three transcription factors (CsFBX, CsBBX, and CsASR) regulating the ethylamine biosynthesis gene CsAlaDC were identified from the Yinghong 9 cDNA library. All three factors bound to the CsAlaDC promoter, with CsBBX and CsASR demonstrating transcriptional activation capabilities.

Starch Digestion Characteristics and Hypoglycemic Activity of Brown Rice

YU Jing, ZHU DaWei, ZHENG Xin, XU QingYu, DONG HuiYing, CHEN MingXue, SHAO YaFang

Scientia Agricultura Sinica. 2025, 58(12): 2439-2452. doi:10.3864/j.issn.0578-1752.2025.12.013

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【Objective】This study elucidated the starch digestion characteristics of different colored rice grains, and identified the differences in their hypoglycemic activities, such as α-glucosidase and dipeptidyl peptidase-IV inhibition capacity, during cooking and in vitro digestion. It would provide a basis for dietary optimization of whole rice grains diets and the development of functional rice products. 【Method】 An in vitro digestion method was employed to determine the starch digestion rates of polished and brown rice of non-pigmented, red, and black rice grains. Simultaneously, the differences was systematically analyzed and compared among these rice varieties based on their percent of bran part in brown rice, total starch, apparent amylose, total protein, protein fractions (albumin, globulin, prolamin and glutenin), and pasting properties of rice flour. Correlation analysis was conducted to explore the relationships among these indicators. The INFOGEST method was used to simulate the digestion of three colored rice grains cooked in a high-pressure rice cooker. The α-glucosidase and dipeptidyl peptidase-IV inhibitory capacities of bioactive constituents were measured in raw, cooked, and digested rice grains.【Result】 Brown rice exhibited a lower degree of starch digestion, and its starch digestion characteristics were related to its chemical composition. The final degree of starch digestion and the rapid digestion rate of rice starch were not directly correlated, but they showed a negative and positive correlation with the digestion coefficient, respectively (P<0.05). The starch digestion rate constant was significantly positively correlated with prolamin content. The rapid starch digestion rate was significantly positively correlated with percent of bran part in brown rice, total starch, and albumin content, while it was significantly negatively correlated with globulin and apparent amylose content. The final degree of starch digestion was significantly negatively correlated with percent of bran part in brown rice, albumin, and prolamin content (P<0.05). The digestion characteristics of starch also exhibited a certain correlation with its pasting properties. Rice with lower trough and final viscosity and higher breakdown value tended to have a higher rapid starch digestion rate. Colored rice varieties exhibited higher hypoglycemic activities, where red rice had higher α-glucosidase inhibitory capacity and black rice had higher dipeptidyl peptidase-IV inhibitory capacity. During cooking and in vitro digestion, the α-glucosidase inhibitory capacity of non-pigmented and red rice decreased first and then increased, while that of black rice remains basically unchanged. The DPP-IV inhibitory capacity of non-pigmented rice gradually increased during cooking and in vitro digestion, especially after digestion, while that of red and black rice exhibited initial decrease and then increase.【Conclusion】The starch digestion degree of brown rice is lower than that of polished rice. After cooking and digestion, the differences in α-glucosidase or dipeptidyl peptidase-IV inhibitory capacities among different colored rice grains were not significant. However, considering the synergistic effects of these two hypoglycemic activities, black rice grains showed certain advantages in reducing blood glucose levels.

Effects of Cycloastragenol on Cellular Senescence of Pig Donor Fibroblast, Cytoskeletal Dynamic, and Early Developmental Stage of Nuclear Transfer Embryo

ZHOU Qi, ZHANG Liang, PAN Yu, TU Zhi, WANG Zheng, LIU HangHang, XIAN LingJin, XIA YunHong, PAN HongMei, LONG Xi

Scientia Agricultura Sinica. 2025, 58(12): 2453-2474. doi:10.3864/j.issn.0578-1752.2025.12.014

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【Background】 Germplasm resources are the foundation of the revitalization of the swine breeding industry, and their protection and utilization are critical. However, the outbreak of African swine fever and increasing market competition have caused a significant decline in the number of local Chinese pig breeds, therefore threatening the genetic diversity of pig germplasm resources, with many breeds now facing endangerment. Somatic cell nuclear transfer (SCNT) is a promising strategy for restoring endangered pig breeds, but the aging of donor cells severely limits the efficiency of pig SCNT. Cycloastragenol (CAG) has demonstrated anti-aging effects in earlier studies. 【Objective】 This study aimed to evaluate the impact of CAG on aging-related phenotypes of pig ear marginal fibroblasts (PEMFs) and to explore the molecular mechanisms by which CAG regulates cellular senescence using RNA sequencing. Additionally, the potential of CAG as a pre-treatment for SCNT donor cells was further investigated. 【Method】 PEMFs exhibiting aging phenotypes were obtained through continuous passaging. Cell proliferation assays and flow cytometry were performed to detect Senescence-associated β-galactosidase (SA-β-Gal) activity, in order to analyze the optimal concentrations and treatment times of CAG for PEMFs at different stages of aging. The anti-aging effects of CAG were further assessed by measuring p53 protein levels and the expression of Senescence-associated secretory phenotype (SASP) markers, coupled with immunofluorescence staining of the cytoskeleton. By integrating phenotypic analysis with transcriptomic data, the potential mechanisms by which CAG slowed down PEMF senescence were elucidated. Additionally, SCNT embryo cleavage and blastocyst rates were statistically analyzed, followed by staining and quantification of the total cell number and trophectoderm cell number in SCNT blastocysts to investigate the effects of CAG-pretreated PEMFs on SCNT embryo developmental potential. 【Result】 SA-β-Gal assay and CCK-8 cell proliferation assay demonstrated that the anti-aging and proliferation-inhibitory effects of CAG were concentration- and time-dependent. Based on dose-effect balance analysis, the recommended treatment conditions were 100 μmol·L^-1 CAG for 24 hours in P3/P6 PEMFs and 12 hours in P9 PEMFs. Under these conditions, CAG significantly reduced SA-β-Gal activity, p53 protein expression, and the secretion of certain SASP factors in PEMFs. Additionally, CAG treatment led to a significant reduction in cell surface area, focal adhesion number, and stress fiber thickness and quantity, while also reorganizing the cytoskeleton. Transcriptomic analysis and subsequent validation results revealed that CAG modulates pathways such as focal adhesion and extracellular matrix (ECM) signaling, thereby affecting the stability of the cytoskeleton. In embryo culture experiments, PEMFs treated with CAG exhibited higher cleavage and blastocyst formation rates, as well as greater blastocyst total cell numbers and trophoblast cell counts, indicating a significant improvement in the quality and developmental potential of early embryos.【Conclusion】 CAG effectively delayed donor cell senescence, reorganized the cytoskeleton, and significantly enhanced SCNT efficiency and embryo developmental potential. This discovery offered a novel approach to addressing the challenges of pig germplasm conservation and low cloning efficiency.

Effects of Methionine Supplementation on Growth Performance, Carcass Trait, and Plasma Biochemical Indice of Growing Pekin Ducks Fed a Low-Energy and Low-Protein Diet

WU YongBao, TANG Jing, CAO JunTing, WANG QiMeng, XIE Ming, ZHOU ZhengKui, HOU ShuiSheng, WEN ZhiGuo

Scientia Agricultura Sinica. 2025, 58(12): 2475-2486. doi:10.3864/j.issn.0578-1752.2025.12.015

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【Objective】The aim of this experiment was to investigate the effects of methionine supplementation on growth performance, carcass traits, and plasma biochemical indices of growing Pekin ducks fed a low-energy and low-protein diet (maintaining a constant energy-to-protein ratio), and to determine the methionine requirement of Pekin ducks under such dietary conditions, so as to provide the data support for feeding low energy and low protein diet in meat duck industry. 【Method】Using one-factor completely random design, a total of 240 Pekin ducks, with similar weight (560.26 ± 3.82 g) at 15 days of age, were randomly divided into 5 groups, with 6 replicates per group and 8 ducks per replicate. The basal diet was formulated based on China’s Feeding Standard of Meat-Type Ducks, the ratio of metabolizable energy (ME: 12.56 MJ·kg^-1) and crude protein (CP: 17.5%) of growing Pekin ducks (CP/ME: 13.9 g·MJ^-1), a low-energy and low-protein corn-soybean meal diet (ME: 11.90 MJ·kg^-1; CP: 16.6%) with a constant CP/ME was formulated as the experimental basal diet (methionine analyzed value of 0.287%). Ducks were fed diets that the basal diet supplemented 5 methionine levels (0, 0.075%, 0.15%, 0.225%, and 0.30%), respectively. The experiment lasted for 28 days. 【Result】(1) Compared with the non-supplemented group (Control group), the average body weight at 28, 35, and 42 days of age, and average daily weight gain and methionine intake at each period of Pekin ducks were significantly increased (P<0.05) as dietary methionine increased in low-energy and low-protein diet. Average daily feed intake during 15 to 28, 15 to 35, and 15 to 42 days of age exhibited linear increases (P<0.05) with dietary methionine supplementation. (2) The methionine supplementation in low-energy and low-protein diets increased the breast muscle percentage (P<0.05), while abdominal fat percentage linearly decreased (P<0.05) in 42-day-old Pekin ducks. No significant differences were observed on organ indices (P>0.05). (3) There was no significant effect on the biochemical indices in plasma of 42-day-old Pekin ducks (P>0.05), though albumin content showed a quadratic response (P<0.05), peaking at intermediate methionine levels in the low-energy, low-protein diet. (4) Based on the linear-broken line model, the methionine requirement of growing Pekin ducks fed low-energy and low-protein diets ranged from 0.349% to 0.362% for optimal growth performance, and 0.375% for optimal breast meat rate, respectively. 【Conclusion】Taken together, the dietary methionine supplementation could increase the growth performance and improve carcass traits of growing Pekin duck in low-energy and low-protein diets, and the methionine requirement of Pekin ducks in this diet ranged 0.35% to 0.38% for optimal growth performance and carcass traits.

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