Scientia Agricultura Sinica

Identification and Candidate Gene Analysis of the ABNORMAL HULL 1 (ah1) Mutant in Rice (Oryza sativa L.)

ZHANG BiDong, LIN Hong, ZHU SiYing, LI ZhongCheng, ZHUANG Hui, LI YunFeng

Scientia Agricultura Sinica. 2024, 57(3): 429-441. doi:10.3864/j.issn.0578-1752.2024.03.001

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【Objective】Rice is the staple grain crop worldwide, and the morphology of its grains directly influences its ultimate yield, nutritional excellence, and economic significance. Moreover, the intricate interplay between floral development and grain morphology adds further significance to this relationship. Thus, exploring novel rice floral development regulatory genes and molecular regulatory mechanisms lays the foundation for larger and more plump grains rice varieties. 【Method】Ethyl methyl sulfonate (EMS) was used to mutate XD1B (xian-type maintainer line), and a dwarf mutant abnormal hull 1 (ah1) with abnormal formation of glume and lodicule was identified. The agronomic traits of both the wild-type and mutant were observed and recorded. Spikelets from various flowering stage were collected to histological and morphological analysis. The F₂ segregating population was established by ah1 and 56S (xian-type thermo-sensitive sterility line), and utlized for genetic analysis and gene mapping. RNA was isolated from young panicles of both the wild-type and mutant, then reverse transcribed into cDNA. The RT-qPCR analysis was performed to analyze the relative expression levels of the genes regulating floral development and the key genes in the ABA synthesis pathway. 【Result】The observation of agronomic traits revealed that the dwarfed plant was caused by the dramatic shortening of the internodes. At the same time, the mutant is also accompanied by severe spikelet abnormalities and low fruit setting rate. Histological and morphological analysis revealed that the ah1 mutant spikelets exhibited varying degrees of degeneration in floral organs such as palea, lemma, lodicules, and stamens. Some severely affected spikelets displayed altered floral organ characteristics and determinacy of floral meristems, often accompanied by extensive whitening. Based on the extent of degeneration, these spikelets could be classified as slight or severe mutant phenotypes. Genetic analysis showed a segregation ratio of 3﹕1 for the wild-type and mutant within the segregating population, indicating that the mutant traits of ah1 were controlled by a single recessive locus. The AH1 was mapped between the molecular markers RM6716 and RM128 on the chromosome 1, with a physical distance of approximately 8 Mb. Resequencing analysis of the mutant revealed that the LOC_Os01g53450 and LOC_Os01g51860 within this interval showed variation between wild-type and mutant, thus these two genes were provisionally identified as candidate genes. RT-qPCR analysis revealed significant alterations in the relative expression levels of floral organ development regulatory genes during the early developmental stages of mutant panicles; meanwhile, the relative expression levels of OsNCED1/OsNCED2/ OsNCED3/OsNCED4/OsNCED5, the ABA synthesis pathway key genes, were severe inhibited.【Conclusion】AH1 plays a crucial role in the morphological formation of floral organs, such as palea and lemma in rice. LOC_Os01g53450 and LOC_Os01g51860 were provisionally identified as candidate genes in this work.

Function Analysis of the Soybean Transcription Factor NAC1 in Tolerance to Low Phosphorus

XIONG ChuWen, GUO ZhiBin, ZHOU QiangHua, CHENG YanBo, MA QiBin, CAI ZhanDong, NIAN Hai

Scientia Agricultura Sinica. 2024, 57(3): 442-453. doi:10.3864/j.issn.0578-1752.2024.03.002

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【Objective】The productivity of acid soil crops is severely impacted by the limited availability of phosphorus. Soybean (Glycine max) is an important grain and oil crop, known for its preference for phosphorus. Phosphorus deficiency significantly affect both the yield and quality of soybean. While the NAC (NAM, ATAF1/2, CUC2) transcription factor family has been recognized for its involvement in regulating plant responses to various biotic and abiotic stresses, its role in soybean under low phosphorus stress remains largely unexplored. In this study, we focused on the low-phosphorus-tolerant wild soybean variety BW69 as our material, with the objective of cloning and analyzing the expression patterns and functions of the low-phosphorus-tolerant gene GsNAC1. This investigation lays the foundation for a deeper understanding the mechanisms behind the regulation of GsNAC1 response to low phosphorus stress. 【Method】The full-length sequence of GsNAC1 was cloned from BW69, and the characteristics of its encoded amino acid sequence were explored by bioinformatics analysis. In addition, the tissue expression patterns of GsNAC1 were examined through quantitative real-time PCR (qRT-PCR). The subcellular localization of GsNAC1 was observed using laser confocal microscopy. Furthermore, soybean genetic transformation experiments were conducted for further phenotype analysis, and RNA-seq analysis was performed to identify differentially expressed genes (DEGs) related to low phosphorus stress. 【Result】The GsNAC1 gene was successfully cloned, with a full-length coding region of 876 bp. Phylogenetic analysis showed a 62.46% sequence similarity between GsNAC1 and AtATAF1, and no difference was observed with the GmNAC1 sequence from the Williams 82 reference genome. Subcellular localization experiments further revealed that GsNAC1 was localized in the nucleus. Using qRT-PCR, it was discovered that GsNAC1 is expressed in roots, stems, leaves, apes, flowers and pods, with the highest relative expression level found in the roots. Notably, GsNAC1 exhibited significant upregulation in response to low pH and low phosphorus conditions. To assess the phenotypic effects, we performed experiments using both hydroponic and soil cultivation methods under low phosphorus conditions. The transgenic lines showed notable increases in root/shoot ratio, total root length, root surface area, root volume, and phosphorus content compared to the wild type (WT). Transcriptome analysis revealed that GsNAC1 may enhance tolerance to low phosphorus stress by promoting the expression of genes such as GmALMT6, GmALMT27, GmPAP27, and GmWRKY21. 【Conclusion】The expression of GsNAC1 was up-regulated by low pH and low phosphorus, and overexpression of GsNAC1 significantly enhanced the tolerance to low phosphorus stress in soybean, playing a promoting role in the response to low phosphorus stress. Besides, GsNAC1 may enhance the tolerance to low phosphorus stress in soybean by regulating the expression of downstream genes.

Progress on Genetic Transformation of Sorghum

HAN LiJie, CAI HongWei

Scientia Agricultura Sinica. 2024, 57(3): 454-468. doi:10.3864/j.issn.0578-1752.2024.03.003

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Sorghum is the fifth largest grain crop in the world and can be used for food, feed, brewing and bioenergy. Sorghum genetic transformation technology is an essential and important tool in the research of sorghum functional genomics and can also serve as an important complement to traditional breeding methods. In this review, we summarize the research progress of sorghum transformation in recent years, analyze the problems in sorghum genetic transformation and propose strategic solutions to them in order to provide a reference for further improvement of sorghum genetic transformation technology. By summarizing more than 50 literatures on sorghum tissue culture and genetic transformation in recent years, we introduced the current research status of sorghum genotypes, explant sources, and regeneration system construction for genetic transformation, and compared the advantages and disadvantages of four commonly used methods for sorghum genetic transformation: electroporation, pollen-mediated transformation, particle bombardment and Agrobacterium-mediated transformation, summarized the effects of the main components of genetic transformation vectors, including promoters, target genes, selective marker genes and reporter genes, on transformation efficiency, explained the current application status of sorghum genetic transformation, analyzed the main bottleneck problemns in sorghum genetic transformation technology, and studied countermeasures. Sorghum genotypes have a significant influence on tissue culture and P898012 and Tx430 are the most widely used. Gene bombardment and Agrobacterium-mediated transformation are the most commonly used methods for sorghum genetic transformation, and the advantages of Agrobacterium-mediated transformation are gradually emerging. In vector construction, CaMV35S and ubi1 are the most commonly used promoters, and antibiotic resistance genes (nptII, hpt), herbicide resistance genes (bar), and nutrient assimilation genes are the three commonly used selection markers. With the development of sorghum genetic transformation technology and CRISPR/Cas9-mediated gene editing technology, some genes with important agronomic traits have been successfully transferred into sorghum. However, strong genotype dependence, long tissue culture cycle, and poor genetic transformation stability are the main bottlenecks that limit the genetic transformation of sorghum. By introducing morphogenesis regulatory factors, somatic cell generation can be directly performed, which shortens the tissue culture cycle, improves the transformation efficiency, and expands the source of explants. This has become a major breakthrough in sorghum genetic transformation technology. The use of morphogenesis regulatory factors and adoption of cut-dip-budding (CDB) delivery system can further improve the sorghum genetic transformation technology. Combined with the application of CRISPR/Cas9 gene editing technology, they will surely provide an important technical basis for the sorghum molecular breeding and gene function identification.

Differences in Physicochemical Characteristics and Eating Quality Between High Taste Northern Japonica Rice and Southern Semi- Glutinous Japonica Rice Varieties in China

ZHU DaWei, ZHENG Xin, YU Jing, MOU RenXiang, CHEN MingXue, SHAO YaFang, ZHANG LinPing

Scientia Agricultura Sinica. 2024, 57(3): 469-483. doi:10.3864/j.issn.0578-1752.2024.03.004

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【Objective】Clarifying the differences of physicochemical characteristics and sensory taste qualities between northern japonica and southern semi-glutinous high eating quality japonica rice varieties in China is of great significance to the cultivation of high eating quality japonica rice. 【Method】In this study, representative northern japonica varieties and southern semi-glutinous japonica varieties were used as materials, Japanese Koshihikari rice was used as a control to systematically analyze the differences in appearance quality, starch fine structure, protein components, pasting properties of rice flour, cooked-rice texture, and sensory taste qualities of the two categories of japonica rice. 【Result】Although the three northern japonica varieties and the three southern semi-glutinous japonica varieties had high similar sensory taste values, they had significant differences of starch fine structure, protein components, pasting characteristics, and cooked-rice texture. For cooked rice sensory taste, three high taste northern japonica varieties showed better palatability (96-98 points), and three southern semi-glutinous japonica varieties showed better cold rice texture (92-100 points). For physicochemical characteristics, the three northern japonica varieties showed higher apparent amylose content (17.6%-19.6%), lower crude protein, gliadin and glutenin content. The three southern semi-glutinous japonica varieties showed lower apparent amylose content (8.5%-10.5%) and higher gliadin and glutenin content. For starch fine structure, the amylose content and amylose/amylopectin ratio of the three southern semi-glutinous japonica rice varieties were low (54.7% and 55.6% lower on average, respectively), and they had similar amylopectin chain length distribution. In addition, the ratio of amylose short chain to long chain in Nanjing 46 and Ningxiangjing 9 was higher than that in Wuyoudao 4 and Tianlongyou 619 (43.2% higher on average). As a result, the rice flour of southern semi-glutinous japonica varieties had higher pasting temperature, peak viscosity, breakdown value, and lower setback viscosity, and the cooked rice had higher stickiness, less hardness and elasticity. 【Conclusion】Taste quality is a synergy of multiple factors of cooked rice, such as appearance, taste and cold rice texture. Appropriate protein components and amylose ratio are the key factors affecting pasting characteristics of rice flour and sensory taste of rice. In this study, the high eating quality northern japonica varieties had lower pasting temperatures, moderate hardness, and better elasticity, while the high eating quality southern semi-glutinous japonica varieties exhibited higher breakdown value, and the cooked rice had higher viscosity and better appearance.

A VSURF-CA Based Hyperspectral Disease Index Estimation Model of Wheat Stripe Rust

MEI GuangYuan, LI Rong, MEI Xin, CHEN RiQiang, FAN YiGuang, CHENG JinPeng, FENG ZiHeng, TAO Ting, ZHAO Qian, ZHAO PeiQin, YANG XiaoDong

Scientia Agricultura Sinica. 2024, 57(3): 484-499. doi:10.3864/j.issn.0578-1752.2024.03.005

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【Objective】Stripe rust is a serious threat to the growth and yield of wheat. Accurate monitoring and diagnostic assessment are fundamental prerequisites for effective prevention and control of stripe rust. The objective of this study is to construct a wheat stripe rust estimation model using remote sensing technology, enable the rapid and precise estimation of the disease index (DI), and to provide technical support for precise prevention and control.【Method】The hyperspectral data of wheat at different growth stages (heading period, grain-filling period, and maturity period) were acquired through the ASD spectrometer. Initially, the variable selection using random forests (VSURF) method, combined with correlation analysis (CA), was applied to select characteristic bands from the original spectrum (OR) and the first-order differential spectrum (FD). Subsequently, the random forest (RF) algorithm was utilized to compare modeling results of characteristic bands from different datasets, identifying the feature set with the most effective model. Further, models such as partial least squares regression (PLSR), extreme gradient boosting (XGBoost), and back-propagation neural network (BPNN) were employed to compare the modeling effects of different feature sets within various algorithms. This comprehensive analysis aimed to determine the optimal estimation model for wheat stripe rust DI across the entire growth period. Simultaneously, to validate the effectiveness of the feature set across different growth stages, the feature set was used to rebuild models during each of the three distinct growth periods.【Result】The comparative analysis of model effects revealed that the VSURF-CA-FD feature set (537 nm in the green range and 821, 846 nm in the near-infrared range) demonstrated the most effective estimation within the RF model, achieving an R² value of 0.89 and an RMSE of 12.34. These feature bands also exhibited precision in models constructed with other algorithms, including XGBoost (R²: 0.87, RMSE: 13.15), BPNN (R²: 0.84, RMSE: 15.19), and PLSR (R²: 0.69, RMSE: 20.92). For models constructed during different growth stages, the early growth stage (heading period) exhibited an R² value of 0.54, RMSE of 1.29, and NRMSE of 0.21, meeting the requirements for disease estimation. In the middle growth stage (grain-filling period), the model performed well with an R² of 0.66, RMSE of 12.24, and NRMSE of 0.21. In the late growth stage (maturity period), the model’s effectiveness surpassed that of the previous two stages, with an R² of 0.75, RMSE of 10.77, and NRMSE of 0.15.【Conclusion】Utilizing characteristic bands selected through the VSURF-CA method, an RF model with excellent estimation accuracy for wheat stripe rust DI can be established. The research outcomes will provide valuable insights and methodologies for predicting early and mid-stage stripe rust DI.

Stability and Mechanism of Wheat Straw Fermentation Products of Chaetomium globosum Against Phytophthora capsici

LIAO HongJuan, TAN JiaSi, ZHANG ZhiBin, YU JingRong, ZHANG XinYue, JIANG YuMei, ZHU Du

Scientia Agricultura Sinica. 2024, 57(3): 500-513. doi:10.3864/j.issn.0578-1752.2024.03.006

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【Objective】Crop blight caused by Phytophthora capsici has caused huge economic losses to crop industries such as pepper. The aim of this paper is to explore the inhibition stability and mechanism against P. capsici by metabolites of Chaetomium globosum, and to provide references for the research and development of microbial fungistatic agents for the inhibition of P. capsici.【Method】To investigate the thermal, acid-base, light and time stability of the crude extract against P. capsic, the crude extracts were treated at different temperatures (40-121 ℃), pH (1-13), light times (0-12 d) and storage times (0-60 d), and the inhibition rate of crude extracts against P. capsici after different treatments was determined by mycelial growth inhibition method. The effect of the crude extract on the mycelia morphology of P. capsici was observed by optical microscopy. The effects of the crude extract on cell wall, cell membrane, active oxygen metabolism, protein content, reducing sugar content and pathogenicity of P. capsici after being treated with P. capsici for 12-72 h were investigated by various physiological and biochemical experiments.【Result】Within the treatment range set in this experiment, the inhibition rate of the crude extract (1 mg·mL^-1) against P. capsici did not decrease significantly after treatment with different light times and storage times, and the inhibition rate remained at about 93%. The inhibition rate of crude extract against P. capsici did not decrease significantly in the heat treatment range of 40-70 ℃, while the heat treatment above 70 ℃ significantly reduced the inhibition rate, but the inhibition rate was not less than 70%. The inhibition rate of crude extract against P. capsici was significantly reduced in the acid-base treatment range of pH 1-5 and pH 9-13, but the inhibition rate was also not less than 70%. The treatment of crude extract affected the morphology of P. capsici mycelia, caused severe distortion and shrinkage of mycelia, and also affected the metabolism of active oxygen species, resulting in large accumulation of active oxygen species in mycelia. The alkaline phosphatase activity, β-glucosidase activity, nucleic acid and protein contents in culture medium of P. capsici were significantly increased after being treated with crude extract for 12-72 h, and the contents of malondialdehyde and hydrogen peroxide in P. capsici mycelia were significantly increased. Catalase activity and the contents of soluble protein and reducing sugar in the mycelia of P. capsici were significantly decreased within 12 to 72 h after the crude extract treatment, but the activities of superoxide dismutase, peroxidase, polygalacturonase and β-glucosidase were significantly decreased only for a certain time.【Conclusion】In the treatment range set in this experiment, the inhibition effect of the crude extract of C. globosum wheat straw fermentation is not affected by the light and storage time, but the heat treatment of more than 70 ℃ and the pH treatment of 1-5 and 9-13 significantly reduce the inhibition effect of the crude extract against P. capsici. In addition, the crude extract inhibits P. capsici by changing the morphology of mycelia, damaging cell walls and membranes, causing intracellular material leakage, reducing the contents of protein and reducing sugar in mycelia, inhibiting the activity of antioxidant enzymes, interfering the metabolism of reactive oxygen species and causing a large accumulation of reactive oxygen species.

Population Colonization, Re-Establishment and Development of the Tomato Leafminer (Tuta absoluta)

ZHANG GuiFen, WAN Kun, PAN MengNi, WANG Long, HUANG Cong, WANG YuSheng, ZHANG YiBo, XIAN XiaoQing, YANG NianWan, GUI FuRong, LIU WanXue, WAN FangHao

Scientia Agricultura Sinica. 2024, 57(3): 514-524. doi:10.3864/j.issn.0578-1752.2024.03.007

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【Background】The tomato leafminer (Tuta absoluta), one of the most destructive insects of Solanaceae crops, is a worldwide quarantine pest. T. absoluta was first recorded in open field in northwestern China, Ili, Xinjiang in 2017, and detected in greenhouses in southwestern China, Lincang, Yunnan in 2018. At present, T. absoluta has rapidly spread to more than 20 provincial-level regions in China.【Objective】The colonization, re-establishment and development ability of invading populations of T. absoluta were evaluated under indoor caged conditions using adults as the research object.【Method】The female fecundity and percentage of larval hatching of each propagule were tested by introducing 5 different numbers of propagules (2, 4, 6, 8, and 10 individuals) of T. absoluta adults (sex ratio = 1﹕1) into different cages. Subsequently, the effect of propagule size on the ability of population colonization of T. absoluta was evaluated. During the population re-establishment and development starting from one pair of adults (2 individuals, sex ratio = 1﹕1), the number of individuals (including adults, eggs, and larvae), the percentage of individuals (including different developmental stages and larval instars) and the richness (totally 6 developmental stages and larval instars) of T. absoluta were investigated. Subsequently, the population re-establishment and development starting from the minimum number (one pair of adults) of adult individuals of T. absoluta were evaluated.【Result】Introducing adults with 5 propagule sizes (i.e., 2, 4, 6, 8, and 10 individuals; sex ratio=1﹕1) into each cage, the numbers of eggs laid by all females were 93.1, 194.9, 271.3, 311.5, and 400.2 eggs/cage, respectively, significant differences were detected between the 5 propagule sizes. The average fecundity was 93.1, 97.4, 90.4, 77.9, and 80.0 eggs/female, respectively; significant differences were detected between them with that the average fecundity (per female) detected in propagule sizes 1♀﹕1♂, 2♀﹕2♂, and 3♀﹕3♂ was significantly higher than that of propagule sizes 4♀﹕4♂ and 5♀﹕5♂. The percentage of larval hatching in all the 5 propagule sizes was 91.3%-92.7%, all of which were relatively high. Comprehensive evaluation, based on above three indexes, showed that the population colonization ability of the initial number of propagules 5♀﹕5♂ was the highest, and then followed by the 3♀﹕3♂, the next was the 4♀﹕4♂, and then the 2♀﹕2♂, that of 1♀﹕1♂ was the lowest. One pair of T. absoluta adults (2 individuals, sex ratio=1﹕1) could re-establish a population; after ~90 d (3 generations), the population could be in a stable state. Three distinct population peaks were observed in the initial 0-91 d of population re-establishment and development (~3 generations). The numbers of total individuals (pupa not included) of T. absoluta at the three peak days increased by 77.8-, 60.5-, and 60.1-fold compared to the initial number of propagules (2 individuals), and adults increased by 6.4-, 4.1-, and 7.7-fold, respectively. Afterwards, all developmental stages, including eggs, 1-4 instar larvae, and adults, occurred simultaneously showing that the phenomenon of overlapping generations was obvious. The population tended to stabilize and in fluctuating growth with the total population size of 96.6-140.6 individuals and the ratios of different developmental stages (egg and adult) and larval instars (1-4 larval instars) around 16.67%, and the richness index R was stable at 1.011-1.094.【Conclusion】One pair of T. absoluta adults had high reproductive and survival abilities. The larger the initial number of invaded individuals, the greater the likelihood of successful colonization of T. absoluta. One female and one male adults of T. absoluta could successfully re-establish a population and maintain long-term stability of the population. The introduction of one pair of T. absoluta adults could likely cause significant risk of invasion and might generate enough progenies to meet the needs of population development and expansion. In the work of prevention and control, the monitoring and warning of T. absoluta should be strengthened and it should be eliminated as early as possible.

Characteristics of Acidity and Nutrient Changes in Red Soil After Conversion of Paddy Field to Dry Land and Vegetable Field

QIU HaiHua, KUAI LeiXin, ZHANG Lu, LIU LiSheng, WEN ShiLin, CAI ZeJiang

Scientia Agricultura Sinica. 2024, 57(3): 525-538. doi:10.3864/j.issn.0578-1752.2024.03.008

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【Objective】 The aim of this study was to analyze the characteristics of changes in soil acidity and nutrient content after the conversion of paddy fields with different parent material development into dryland and vegetable fields in Qiyang, a typical county in the red soil area, so as to provide a scientific basis for the rational use of land to prevent acidification in the area. 【Method】18 sites were selected and collected from paddy fields and adjacent dryland and vegetable fields to analyze the changes of soil pH, exchangeable acid, exchangeable salt-based ions, organic matter, cation exchange, and nutrient content and their interrelationships. 【Result】 The pH of all the soils developed with alkaline parent material was significantly higher than that of the soils developed with acidic parent material. Soil pH decreased by 0.48 units after the conversion of acidic parent material developed paddy fields to vegetable fields; soil pH decreased by 0.74 and 0.53 units after the conversion of alkaline parent material developed paddy fields to drylands and vegetable fields, respectively. The bilinear model fit analysis showed that the soil exchangeable aluminum content increased rapidly when the soil pH was below 5.88, 5.78 and 5.63 in the paddy field, dryland and vegetable field, respectively, and the increment of soil exchangeable aluminum content increased by 1.09, 2.33 and 2.93 cmol₍₊₎·kg^-1 by one pH unit, respectively. Soil organic matter and total nitrogen content decreased by 11.06 and 0.42 g·kg^-1, respectively, in the acidic matrices developed paddy fields converted to drylands, while no significant changes were observed in vegetable fields; soil organic matter and total nitrogen content decreased significantly in the alkaline matrices developed paddy fields converted to drylands and vegetable fields, by 13.88-17.28 and 0.57-0.71 g·kg^-1, respectively. The total and effective phosphorus content of the soil increased significantly from 0.41-0.48 g·kg^-1and 26.79-28.69 mg·kg^-1 after the conversion of the paddy field with alkaline parent material to dryland and vegetable field, respectively. Correlation analysis showed that soil pH was significantly and positively correlated with soil exchangeable calcium and magnesium, cation exchange, organic matter content and total nitrogen content (P<0.01); soil exchangeable acid and aluminum were significantly correlated with effective phosphorus content (P<0.05) and negative correlation (P<0.01) with soil exchangeable calcium and magnesium, cation exchange, organic matter and total nitrogen. 【Conclusion】Soil organic matter and total nitrogen content decreased significantly after the conversion of acidic and alkaline parent material developed paddy fields to drylands, while soil total phosphorus and effective phosphorus content tended to increase. Soil acidification was observed after the conversion of paddy fields to vegetable fields for acidic parent materials or to drylands and vegetable fields for alkaline parent materials; the increased nitrification and increased leaching of salt-based ions from paddy fields to drylands and vegetable fields might be one of the main reasons for soil acidification.

Effect of Continuous Reduction of Fertilizer Application on Yield Stability of Spring Wheat in Yellow River Irrigation Area of Ningxia

WANG YueMei, TIAN HaiMei, WANG XiNa, HAO WenYue, LÜ ZheMing, YU JinMing, TAN JunLi, WANG ZhaoHui

Scientia Agricultura Sinica. 2024, 57(3): 539-554. doi:10.3864/j.issn.0578-1752.2024.03.009

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【Objective】The objective of this study was to explore the effects of continuous reduction of chemical fertilizer on supply of soil nitrogen, phosphorus and potassium, and grain yield of spring wheat in Yellow River Irrigation Area of Ningxia (NYRIA), and to analyze the factors affecting yield stability, and then to provide a theoretical basis for rational fertilizer reduction and high yield and stable yield of spring wheat. 【Method】The field positioning experiment of chemical fertilizer reduction application was carried out for four consecutive years from 2019 to 2022, with Ningchun 4 of spring wheat cultivar as the test crop. The field treatments included conventional fertilization (CF with N 270 kg·hm^-2, P₂O₅150 kg·hm^-2, K₂O 75 kg·hm^-2), the lower limit of reduced fertilization (RF1) with N180 kg·hm^-2, P₂O₅45 kg·hm^-2, and K₂O 30 kg·hm^-2 (which reduced N, P₂O₅, and K₂O by 33.3%, 70.0%, and 60.0%, respectively, compared with conventional fertilization), the upper limit of fertilization reduction (RF2) with N 225 kg·hm^-2, P₂O₅75 kg·hm^-2, K₂O 45 kg·hm^-2 (which reduced N, P₂O₅, K₂O by 17.0%, 50.0%, 40.0%, respectively, compared with traditional fertilization), and no fertilization (CK). The climatic factors during the growth period of spring wheat, soil moisture content before sowing and harvest of spring wheat, soil mineral nitrogen, available phosphorus and available potassium content, dry matter accumulation in shoot, grain yield and yield components of spring wheat at harvest were analyzed, and correlations among them were also discussed. 【Result】The soil moisture content before sowing were inter-annual differences in 2019-2022, among which the average was only 19.5% in 2022; the soil moisture content before sowing was significantly affected by fertilization treatment in 2020, there was no significant difference of soil moisture content before sowing between fertilization treatments in other years. The content of mineral nitrogen, available potassium and available phosphorus in the soil before sowing and harvesting were higher under conventional fertilization CF treatment, followed by RF2 treatment; there was no significant difference between them, while which of the RF1 treatment tended to decrease. In 2019, the CF treatment had the highest accumulation of dry matter in shoot and grain yield, which was 23 261.7 kg·hm^-2 and 9 449.0 kg·hm^-2, respectively, and had an increase of 2.8%-4.5% and 3.2%-16.0% compared with the RF2 treatment. However, from 2020 to 2022, the RF2 treatment had the highest accumulation of dry matter in shoot and grain yield, and there was no significant difference between the RF2 treatment and the CF treatment, but the yield of RF2 treatment performed the most stable at 4 years. From the perspective of inter-annual changes, the number of ears, 1000 grain weights and yields of hectares for all treatments showed a downward trend year by year, so the fertilization rate was not the main reason for the inter-annual difference in grain yield, but which was closely related to soil moisture before sowing, precipitation, temperature, humidity and wind speed. The decrease in yield in 2022 was accompanied by a lower soil moisture before sowing, hot dry air phenomenon during the filling period and a change in the nitrogen fertilizer base ratio. 【Conclusion】 In NYRIA, continuous and appropriate reduced application of chemical fertilizer with N 225 kg·hm^-2, P₂O₅75 kg·hm^-2, K₂O 45 kg·hm^-2 would not significantly reduce the supply capacity of soil nitrogen, phosphorus and potassium, and increase the number of ears, ear grains and 1000 grain weights in the hectares of spring wheat, and promote the transfer and accumulation of dry matter in shoot to grains to a certain extent, then tend to increase the grain yield of spring wheat. However, there were interannual differences in spring wheat yield due to climate factors, such as rainfall, wind speed, and humidity, as well as soil moisture, continuous cropping obstacles, and nitrogen fertilizer application ratios. Among them, temperature, relative humidity, and wind speed were the main factors affecting interannual variation of wheat yield, and their impact on fertilization effects needed further research.

Effects of Wheat and Common Vetch Cover Crops on Chinese Cabbage Seedling Growth and Soil Microbial Community Structure

WANG QingHui, LI NaiHui, ZHANG YiPing, DI ChengQian, WU FengZhi

Scientia Agricultura Sinica. 2024, 57(3): 555-569. doi:10.3864/j.issn.0578-1752.2024.03.010

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【Objective】 This study aimed to investigate the impact of cover crops (wheat and common vetch) on the growth of Chinese cabbage seedlings and the structure of the soil microbial community. The findings could provide the theoretical and technical support for using wheat and common vetch cover crops to alleviate continuous cropping obstacles in Chinese cabbage production. 【Method】In this experiment, there were four treatments: wheat cover crop treatment (W), common vetch cover crop treatment (P), mixture of wheat and common vetch treatment (WP), and no cover crop treatment (CK). The effects of different cover crop treatments on the growth of Chinese cabbage seedlings were studied. Moreover, the effects of wheat and common vetch cover crops on the microbial community of Chinese cabbage rhizosphere were also investigated through qPCR and Illumina MiSeq techniques. In addition, Spearman correlation analysis was conducted to identify the key soil microbial taxa related to Chinese cabbage growth. Then, the changes in soil chemical properties on soil microbial community structure were explored by environmental factor correlation analysis. 【Result】Compared with CK, the cover crop treatments had positive effects on Chinese cabbage growth and decreased soil electrical conductivity (EC) value. The mixed cover crop treatment significantly decreased soil available potassium content, whereas wheat cover crop treatment increased soil pH. The qPCR results showed that the abundance of soil bacterial community was not significantly affected by the cover crop treatment, but increased the abundance of soil fungal community. Both common vetch cover crop treatment and mixed wheat and common vetch cover crop treatment significantly reduced the abundances of Bacillus spp. and Pseudomonas spp. communities. The Illumina MiSeq analysis showed that the relative abundance of genus TM7a was significantly increased by cover treatments, while the relative abundances of Leptolyngbya_EcFYyyy-00, Lophotrichus, Acaulium, and Sodiomyces were decreased. The mixed cover crop treatment significantly increased the relative abundance of Sphingomonas and Massilia and significantly decreased the relative abundance of Fusarium. Spearman correlation analysis showed that Sphingomonas, TM7a, Massilia, and Gemmatimonas were positively correlated with growth. Leptolyngbya_EcFYyyy-00, Acaulium, Lophotrichus, Sodiomyces, and Fusarium were significantly negatively correlated with the growth of Chinese cabbage. Moreover, these cover crop treatments influenced bacterial and fungal diversity indices. The Shannon index and inverse Simpson index for soil bacterial community and Shannon index of soil fungal community significantly decreased in cover common vetch treatment. In contrast, the inverse Simpson index of soil fungal community was increased. The mixed cover crop treatment increased the fungal Shannon index, while the inverse Simpson index of soil fungal community decreased. Principal Coordinates Analysis (PCoA) showed significant differences in soil microbial community structure, with soil EC value as a major environmental factor affecting the structure.【Conclusion】The cover crop treatments exhibited growth-promoting effects on Chinese cabbage seedlings, and the best effect was found in the mixed cover crop treatment. The relative abundances of some Sphingomonas, TM7a, Massilia, other potential growth-promoting bacteria were increased in the cover crop treatments. The relative abundances of some potential plant pathogens Leptolyngbya_EcFYyyy-00 and Fusarium were decreased and the relative abundance of potential biocontrol agent Chaetomium was increased in the mixed cover crop treatment.

Physiological Response of Potted Tomatoes to NaCl and Na₂SO₄ Brackish Water Irrigation

PEI ShuYao, CAO HongXia, ZHANG ZeYu, ZHAO FangYang, LI ZhiJun

Scientia Agricultura Sinica. 2024, 57(3): 570-583. doi:10.3864/j.issn.0578-1752.2024.03.011

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【Objective】Brackish water irrigation is one of the important means to increase irrigation water sources and to alleviate the shortage of agricultural water in arid areas. However, unreasonable irrigation water quality can severely limit the physiological activity and growth of plants. Carrying out research on the effects of different salt types of brackish water irrigation on the physiological changes of tomato leaves is conducive to reveal the mechanisms of salt tolerance to different types of salt in the salt-sensitive crop tomato at the physiological level, which is of great significance to agricultural production as well as to the use of brackish water for water conservation and salt control. 【Method】In this study, tomato was used as an object of study in a brackish water irrigation pot experiment, and the two factors of irrigation water salt type (NaCl (T1) and Na₂SO₄ (T2)) and salinity (0, 1.5 (S1), 3.0(S2), 4.5 (S3) and 6.0 (S4) dS·m^-1) were set to analyze the changes of physiological indexes, such as leaf gas exchange parameters, osmotic and antioxidant physiological regulation, and ionic balance, in tomato plants subjected to different types and degrees of stress at different reproductive periods. The reasons for the differences in the degree of decline in photosynthetic capacity of tomato under NaCl and Na₂SO₄ stress were explored too. 【Result】Brackish water irrigation caused significant decreases in leaf net photosynthetic rate (P_n), stomatal conductance (G_s), and transpiration rate (T_r) in the late stage of fertility (mature picking stage) and high salinity (S4) treatments compared with CK, and the contents of proline (pro), soluble sugar (SS), malondialdehyde (MDA), and leaf Na⁺ increased continuously during the salt stress and the progression of the fertility period (P<0.05). The superoxide dismutase activity (SOD) showed a trend of increasing and then decreasing with increasing salinity in the late reproductive stage. The highest decreases in P_n, G_s, and T_r could be up to 44.13%, 64.53%, and 33.75%, respectively, whereas the increases in pro, SS, MDA, and Na⁺ could be up to 2.31, 0.77, 0.55, and 5.81 times higher than that of CK, respectively, all of which achieved under T1 stress. The correlations of SS, SOD and K⁺/Na⁺ with P_n were significantly changed under the two salt stresses, in which the slopes of the regression lines of SS and P_n were significantly higher under T1 treatment than T2 (P<0.05), the slopes of the regression lines of SOD and P_n were significantly lower under T1 treatment than T2 (P<0.05), and the regression curves of K⁺/Na⁺ and P_n showed that the T1 curves were relatively leftward. The results of principal component analysis showed that, under T1 treatment, SOD activity value was higher, which had an important role in P_n stabilization, but it was suppressed in the late reproductive stage, and could only enhance the water use efficiency to a certain extent; under T2 treatment, the physiological indexes were less stressed, in which the SS accumulation was related to the photosynthetic products, which could promote the biomass accumulation. 【Conclusion】 Salt stress led to excessive Na⁺ absorption by leaves, causing a decrease in net photosynthetic rate, stomatal conductance and transpiration rate of tomato, and the accumulation of malondialdehyde in leaves, while leaves increased superoxide dismutase activity as well as proline and soluble sugar content to cope with the stress. Under the same irrigation salinity, the net photosynthetic rate and stomatal conductance of tomato leaves were more affected by NaCl stress, soluble sugars maintained the stability of net photosynthetic rate under Na₂SO₄ stress, superoxide dismutase was able to protect the photosynthetic system under NaCl stress, and the NaCl treatment was required to maintain a higher leaf K⁺/Na⁺ level when the net photosynthetic rate was the same. Tomato leaves under Na₂SO₄ stress were less affected by stress, whereas tomato under NaCl stress had higher water use efficiency at the same salinity. The recommended salinity for irrigation of brackish water containing mainly NaCl was less than 3 dS·m^-1, and the salinity for irrigation of brackish water containing mainly Na₂SO₄ was not more than 4.5 dS·m^-1.

Modeling and Optimization of 3D Printing Process of Pleurotus Eryngii Powder Using Neural Network-Genetic Algorithm

SU AnXiang, HE AnQi, MA GaoXing, ZHAO LiYan, YANG WenJian, HU QiuHui

Scientia Agricultura Sinica. 2024, 57(3): 584-596. doi:10.3864/j.issn.0578-1752.2024.03.012

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【Objective】Food 3D printing technology, a promising technology in the field of food, can be affected by multiple factors and thus has problems, such as difficulty in determining printing parameters and poor ability of predicting printing accuracy. This paper aimed to seek out an effective modeling method to optimize 3D printing parameters of Pleurotus eryngii powder and to determine the optimal conditions for 3D printing.【Method】Pleurotus eryngii powder and locust bean gum were adopted as 3D printing ink. Then, based on single-factor experiments, the central composite experimental design was performed to study the influence of four key process parameters - nozzle diameter, printing height, nozzle movement speed and fill density - on the accuracy of 3D printing. In order to optimize 3D printing parameters of Pleurotus eryngii powder, response surface methodology (RSM) and artificial neural network and genetic algorithm (ANN-GA) were employed to achieve different effects.【Result】The determination coefficient (R²), root mean square error (RMSE), relative error (RE), and optimal value of prediction (VOP) of RSM model were 0.8817, 0.2314, 72.73%, and 0.148, respectively; the R², RMSE, RE, and optimal VOP of ANN-GA model were 0.9389, 0.2269, 33.85%, and 0.215, respectively. The ANN-GA model obtained higher R², lower RMSE and RE, and was better fitting ability, and higher optimal VOP than RSM model, so ANN-GA model possessed better prediction ability. Compared with RSM, ANN-GA was more suitable for optimization of 3D printing parameters of Pleurotus eryngii powder. The optimal process parameters of 3D printing obtained by ANN-GA, with Pleurotus eryngii as printing ink, included nozzle diameter 1.2 mm, printing height 1.1 mm, nozzle movement speed 24 mm·s^-1, and fill density 84%. Experimental verification suggested that the deviation of printed samples by ANN-GA was 0.325, which was superior to the actual printing deviation 0.550 by RSM.【Conclusion】ANN-GA was effective in determining the optimal process parameters of 3D printing and accurate in predicting the accuracy of food 3D printing products. Therefore, ANN-GA could serve as an effective and convenient method for optimizing personalized 3D printing parameters of agricultural products and food.

Estrogen Mediates CircZNF423 as a Sponge for oar-miR-541-3p to Target CALM3 for Regulating Myoblast Proliferation in Sheep

CHI RunQing, HAN HaiYin, WANG Peng, LI KaiYang, CHU MingXing, LIU YuFang

Scientia Agricultura Sinica. 2024, 57(3): 597-612. doi:10.3864/j.issn.0578-1752.2024.03.013

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【Background】Estrogen is the main hormone secreted by female mammalian ovarian tissue, and it plays an important regulatory role in muscle growth and development. The circRNA has been found to be involved in a variety of signaling pathways related to muscle growth and development. 【Objective】According to the whole transcriptome integration analysis of ovariectomized and not ovariectomized Sunite sheep in our previous study, the circZNF423 was found to regulate the expression of oar-miR-541-3p/CALM3 as ceRNA. To further investigate the molecular mechanism of estrogen in sheep muscle growth, the effect of exogenous addition of estrogen-mediated regulation of oar-miR-541-3p/CALM3 by circZNF423 on myoblast proliferation was examined by sheep primary myoblast in vitro culture. This study could provide a theoretical basis for further study of the role of estrogen and circRNA in sheep growth and development trait, and also provide new research ideas for sheep molecular design breeding. 【Method】In this study, the sheep longissimus dorsi tissue was collected, and the sheep primary myoblast were isolated and cultured in vitro. The location of circZNF423 expression in myoblast was determined by immunofluorescence staining, RNA in situ hybridization (FISH) and nucleoplasmic separation experiments. RNA hybrid online software was used to predict the existence of binding relationship between circZNF423, oar-miR-541-3p and CALM3, and the binding of circZNF423 to oar-miR-541-3p, oar-miR-541-3p and CALM3 was verified by dual luciferase activity assay and biotin-labeled miRNA pull-down assay. The synthetic circZNF423 overexpression or interference plasmids, oar-miR-541-3p mimics (mimics) or inhibitors (inhibitors), and CALM3 overexpression or interference vectors were constructed in vitro and transfected in primary sheep myoblast, and the expression of proliferation markers and myoblast proliferation rate were detected using RT-qPCR, Western blot, EdU and CCK-8. To further clarify the role of estrogen in sheep muscle growth and development, the exogenous estradiol (E₂) was added at different concentrations in vitro, and the changes in the expression of sheep myoblast proliferation were detected using RT-qPCR, Western blot, CCK-8, and EdU. 【Result】Immunofluorescence staining showed that the isolated primary myoblast were sheep myoblast and could be used for subsequent functional validation experiments. RNA in situ hybridization and nucleoplasmic isolation experiments showed that circZNF423 was mainly expressed in the cytoplasm of sheep myoblast. The results of RNAhybrid, dual luciferase activity assay and biotin-labeled miRNA pull-down assay showed a significant binding relationship between both circZNF423 and oar-miR-541-3p, oar-miR-541-3p and CALM3 3’UTR. The expression of proliferation markers PCNA, CDK2 and Pax7 in sheep myoblast was significant increased after circZNF423/CALM3 inhibition or oar-miR-541-3p overexpression (P<0.05 or P<0.01). The EdU and CCK8 results indicated that the proliferation rate of sheep myoblast was significantly increased after circZNF423/ CALM3 inhibition or oar-miR-541-3p overexpression (P<0.05), while the opposite was true after circZNF423/CALM3 overexpression or oar-miR-541-3p inhibition. The expression of sheep myoblast proliferation markers was significantly higher at 10 nmol·L^-1 than that at 1 nmol·L^-1 and 100 nmol·L^-1 after the addition of different concentrations of exogenous estradiol (E₂) (P<0.05 or P<0.01). The mRNA and protein expression levels of the proliferation markers PCNA, CDK2 and Pax7 significantly increased in sheep myoblast, while the expression of circZNF423 and CALM3 were significantly decreased and the expression of oar-miR-541-3p was significantly increased (P<0.05 or P<0.01). The results of EdU and CCK8 showed that the proliferation rate of sheep myoblast was significantly increased after the addition of estradiol in vitro (P<0.05). 【Conclusion】CircZNF423 as ceRNA regulated the binding and expression of oar-miR-541-3p and CALM3 in sheep myoblast, and the addition of exogenous estrogen promoted the proliferation of sheep myoblast by inhibiting the circZNF423/oar-miR-541-3p/CALM3 pathway. These results provided a theoretical basis for revealing the molecular mechanisms of estrogen and circZNF423 in the developmental traits of sheep skeletal muscle.

Molecular Epidemic Characteristics and Genetic Variation Analysis of Porcine Circovirus 3 in Some Pig Farms in Central China from 2020 to 2022

LI BOTIAN, LI CHUNQI, LIU GUOPING, XIE JUN, ZENG PAN, ZHAO RUNZE, LI TONG, PEI JIE, GUO LIWEI, WU RUI, TAN LEI

Scientia Agricultura Sinica. 2024, 57(3): 613-626. doi:10.3864/j.issn.0578-1752.2024.03.014

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【Objective】 The epidemiological and genetic variation characteristics of porcine circovirus 3 in some parts of central China were investigated through a systematic experimental protocol, which provided the data of basis for the research of PCV3 vaccine。【Methods】 A total of 3 500 clinical samples collected from 15 large-scale pig farms in central China (Hubei, Hunan and Henan) were detected by real-time PCR, and the relationship between different pig herds, different tissues and organs, different amount of detoxification and the characteristics of corresponding epidemiological symptoms was analyzed. The whole genome of PCV3 for some positive samples was amplified, sequenced and analyzed.【Result】 50.86% (1 780/3 500) of the tested samples were positive for PCV3 nucleic acid, pigs at different ages were susceptible to PCV3, and the positive rates of PCV3 were higher in nursing pigs, suckling piglets and growing finishing, which were 70.44% (498/707), 67.19% (596/887) and 41.75% (177/424), respectively. The positive rates of PCV3 in lymph nodes, lung, and postpartum placenta samples were 67.05% (59/88), 63.79% (74/116), and 49.11% (55/112), while the positive rates of PCV3 in blood and colostrum samples were 56.09% (502/895) and 44.20% (278/629), respectively, and PCV3 was detected in nasal swabs and saliva. The positive rates of PCV3 was high in the pig with symptoms of reproductive disorder, which were 44.43% (399/898), 36.43% (431/1183), and 28.04% (233/831), respectively. The length of the whole genome sequence of the 24 PCV3 was 2 000 nt, and the homology of the whole genome nucleotide sequence among the 24 sample strains was 98.4%-100%, and the homology of the whole genome with the reference strain PCV3 at home and abroad was 97.4%-99.5%. Genetic evolution analysis showed that 23 strains of PCV3 belonged to the PCV3b subtype and 1 strain belonged to the PCV3a subtype. The results of Rep amino acid sequence alignment showed that PCV3-L2, L23 and L14 strains had unique variation sites in (N¹²⁴I), (A¹⁸³E) and (V²⁴⁴I), respectively. The results of Cap protein amino acid sequence alignment showed that PCV3-L15, L21, L3 and L19 strains had unique variation sites in (T⁴⁵P), (R²K), (R¹⁴K) and (F⁷L), respectively.【Conclusion】 PCV3 can infect pigs at different ages and is distributed in different tissues and organs. PCV3 can infect pigs through vertical transmission (such as colostrum and semen) and horizontal transmission (such as oral and nasal secretions and saliva). PCV3 infection may be closely related to reproductive disorders, respiratory disorders, multiple organ inflammation and arthritis in pigs. In addition, both PCV3a and PCV3b subtypes were prevalent in large-scale pig farms in the surveyed area, and the PCV3b subtype was the dominant strain. In this study, genome-wide amino acid sequence analysis revealed that some unique variation sites were located in Cap protein, which may lead to changes in the immunogenicity conferred by Cap protein. In this study, the genetic evolution of the whole gene sequence of PCV3 is elaborated, aming to lay a foundation for future research on PCV3 vaccines.

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