Table of Content

16 August 2023, Volume 56 Issue 16

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Function of Maize ZCN7 in Regulating Drought Resistance at Flowering Stage

LI Yan, TAO KeYu, HU Yue, LI YongXiang, ZHANG DengFeng, LI ChunHui, HE GuanHua, SONG YanChun, SHI YunSu, LI Yu, WANG TianYu, ZOU HuaWen, LIU XuYang

Scientia Agricultura Sinica. 2023, 56(16):  3051-3061.  doi:10.3864/j.issn.0578-1752.2023.16.001

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【Objective】The main producing areas of maize is mostly located on the arid or semi-arid region that relying on the rainfed farming in China. The maize production losses caused by drought is a great threaten to food security. As a cross-pollinating crop, maize is mostly sensitive to water stress during flowering time. Drought at flowering stage will lead to asynchronous development between the male and female flower and cause massive grain yield loss. Thus, mining drought resistance related genes at flowering stage is important for maize drought resistance improvement and breeding. 【Method】In the present study, the phylogenic tree of 24 ZCN genes in maize genome, which is homologs of Arabidopsis FT gene, was build. The gene expression patterns of ZCN7 were analysis using qRT-PCR and in vivo GFP fluorescence imaging. A maize natural population consisting of 118 diverse inbred lines were planted in three environments, Beijing in 2021 and 2022 and Urumqi in 2022, to identify the flowering time related traits under different water treatments. The genomic variants around ZCN7 were detected by PCR and Sanger sequencing. The candidate gene association analysis was performed based on mixed linear model and the significant associated variants with drought induced anthesis-silking interval was obtained. The gene expression level of ZCN7 in natural population at flowering time was also measured by qRT-PCR. The differences of drought resistance traits and ZCN7 expression were compared between different haplotypes of significant associated variant. The Ubi1:ZCN7 overexpression transgenic maize were obtained, and the phenotypic performance was identified under different water treatments. 【Result】The 24 ZCN genes in maize genome included 15 FT like genes, 6 TFL1 like genes and 3 MFT like genes. The protein sequence of ZCN genes varied from 111 nn to 193 nn. The ZCN7 showed close relationship with ZCN8 and the protein sequence identity was 83.3% between the two genes. ZCN7 showed highest gene expression in the leaf blade at V12 stage. And the ZCN7-promoter:GFP vector was transformed to Arabidopsis and the GFP showed enriched signal at the blade edge of mature leaf. The candidate gene association analysis revealed a SNP variant at 1001 bp upstream of ZCN7 start codon had highest association signal with drought induced anthesis-silking interval under drought. The A/A and G/G haplotypes of SNP-1001 included 78 and 27 inbred lines, respectively. The anthesis-silking interval of A/A haplotype lines were significantly lower than G/G lines. And the ZCN7 gene expression of A/A haplotype lines were significantly higher than G/G lines. In addition, the ZCN7 overexpression transgenic lines showed significantly decreased anthesis-silking interval than wild type lines. Under drought, the anthesis-silking intervals of OE1 and OE2 were 2.3 and 2.6 days shorter than wild type lines. And the grain yield per plant and kernel number per plant of transgenic lines were significantly higher than wild type lines under drought, while the hundred kernel weight, kernel length and kernel width showed no significant difference. 【Conclusion】The maize ZCN7 played positive role in drought resistance and its overexpression improved grain yield by reducing anthesis-silking interval under drought.

A Comprehensive Assessment of Proso Millet Varieties Tested in the State Multi-Region Trial by GYT Biplot Analysis

MA ZhiXiu, CHAI ShaoHua, GUO Yan, SHI Xing, YANG QingHua, GAO JinFeng, GAO XiaoLi, FENG BaiLi, YANG Pu

Scientia Agricultura Sinica. 2023, 56(16):  3062-3076.  doi:10.3864/j.issn.0578-1752.2023.16.002

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【Objective】GYT biplot analysis was employed to analyze agronomic traits, yields and cultivar-trait interactions of test proso millet varieties in different trial sites and comprehensively asses and classify these test varieties in term of their multiple traits, so as to provide a theoretical basis for reasonable arrangements and zonations of proso millet varieties in China.【Method】GGE biplot, GT biplot and GYT biplot along with the analysis of correlation were employed to comprehensively assess 20 varieties tested in the State Multi-region Trial for Proso Millet in 15 trial sites in 2019-2020 in terms of their growth period, plant heights, node numbers, main spike lengths, grain weights per spike, 1000-kernel weights and yields.【Result】It was showed by the analysis of correlation that in both non-waxy and waxy proso millet varieties, the yields were significantly and positively correlated with the growth period and negatively correlated with the main spike lengths, and the plant heights was significantly and positively correlated with the growth period and main spike lengths. In non-waxy proso millet varieties, the yields were also significantly negatively correlated with the node numbers, and the node numbers and 1000-kernel weights were significantly and positively correlated. In waxy proso millet varieties, the yields were significantly and positively correlated with the 1000-kernel weights, and the growth period were significantly and positively correlated with the plant heights and 1000-kernel weights. The analysis of genotype-trait interactive effects by GT biplot showed that the main components of GT biplot PC1 and PC2 explained 61.81% and 69.96% of genotype-trait interactive effects in no-waxy and waxy proso millets, respectively. The correlations between agronomic traits of the tested varieties displayed by GT biplot were basically consistent with Pearson correlation coefficients. The correlations between the yield-trait combinations of the tested varieties were analyzed by GYT biplot, and all the traits of these combinations were significantly and positively correlated. In terms of calculated ideal indexes, Yi 11-02-92-4, Gu 19-63, 0515-2-2, Yi 11-03-3-2-2 and Zhenglongmi 1 were identified as varieties with a good yield-traits combination performance. In terms of comprehensive performances, Chimi 3, Xinong 2018-N02, Xinong 2018-N10, Y1660, Xinong 18-W02 and Xinong 18-W06 poorly performed. It followed that Yi 11-02-92-4 and 0515-2-2 had wider adaptabilities and better yields than the other varieties in different planting areas, showing an absolute regional yield advantage. 【Conclusion】In assessing multiple traits of millet varieties, GYT biplot analysis was more reliable than GGE and GT biplot analysis, so that it was an effective method to scientifically assess merits of proso millet varieties. Among the varieties tested in the state multi-region trial for proso millet, the no-waxy variety with the best comprehensive performance was Yi 11-02-92-4 and suitable to be planted in spring planting areas of proso millet in Northeast China and spring and summer-planting areas of proso millet on the Loess Plateau. The waxy variety of 0515-2-2 with a better comprehensive performance was suitable to be planted in spring-planting areas of proso millet in northern China and spring- and summer-planting areas of proso millet on the Loess Plateau.

COXⅡ Functional Analysis in Tobacco sua-CMS Line

WANG Jin, LIU YanFang, LIU WenWen, WANG YaQi, SONG ShiYang, ZHANG XingZi, LI FengXia

Scientia Agricultura Sinica. 2023, 56(16):  3077-3087.  doi:10.3864/j.issn.0578-1752.2023.16.003

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【Objective】Cytoplasmic male sterility (CMS) is controlled by the cytoplasmic genome, mainly the mitochondrial genome, which plays an important role in breeding and production of hybrids. Sterile lines and hybrids are the main types of tobacco cultivation in China, accounting for more than 80% of the total tobacco cultivation area. The sterile type of tobacco in China is sua-CMS, which is also the only sterile type of tobacco production, but the control gene and sterility mechanism of sua-CMS are not clear. COXⅡ is the second subunit of cytochrome oxidase (COX), encoded by the mitochondrial genome. Nucleotide base variants occur at the terminal of the COXⅡ in sua-CMS lines, which resulted in the replacement of the C-terminal of COXⅡ Thr²⁵⁷ by Ser. The function analysis of COXⅡ in sua-CMS tobacco provides a foundation for the elucidation of the molecular mechanism of sterility in tobacco.【Method】In this study, four sets of sua-CMS lines and fertile controls were used as materials, and RNA blot hybridization and qRT-PCR methods were used to analyze the transcript and gene expression of COXⅡ between sua-CMS lines and fertile controls, and Western blot and COX enzyme activity experiments analyzed COX protein expression and COX enzyme activity, and ATP content were measured. The COXⅡ of fertile tobacco was overexpressed in sua-CMS line, and the effects of COXⅡ overexpression on COXⅡ expression, COX enzyme activity, ATP content and stamens development of tobacco were studied.【Result】COXⅡ multi-sequence alignment showed that Thr²⁵⁷ at C-terminal was highly conserved between species. Expression analysis showed that the COXⅡ transcript in sua-CMS line was significantly lower than that of the fertile control, and quantitative expression of four sets of sua-CMS lines and fertile controls showed that the expression amount of COXⅡ in sua-CMS lines was only 0.3%-0.4% of the fertile controls, the COX protein abundances and enzyme activity were significantly lower than that of the fertile control, and the ATP content of sua-CMS line was 52% of that of the fertile control. When COXⅡ was overexpressed in sua-CMS line, the expression levels of COXⅡ in transgenic plants increased by up to 171 times, the COX enzyme activity increased by 70%, the ATP content increased by 90%. However, all indicators including COXⅡ expression levels, COX enzyme activity and ATP content in transgenic plants were lower than those of fertile tobacco. We also observed significant changes in anther morphology in transgenic plants, and the differentiation of the anther wall appeared, but there was no pollen grains in the anthers.【Conclusion】This study shows that the mutant COXⅡ of sua-CMS line affects COX function, which is a main reason of anther energy deficiency, and it is also an important factor affecting the development of anthers.

Water Use Characteristics of Increased Plant Density and Reduced Nitrogen Application Maize in Oasis Irrigated Area

JIAO ZhiHui, CHEN GuiPing, FAN Hong, ZHANG JinDan, YIN Wen, LI HanTing, WANG QiMing, HU FaLong, CHAI Qiang

Scientia Agricultura Sinica. 2023, 56(16):  3088-3099.  doi:10.3864/j.issn.0578-1752.2023.16.004

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【Objective】In arid irrigation area, the problem of limited water resources, high nitrogen fertilizer input, and low water use efficiency (WUE) in maize production is serious, it’s necessary to investigate the viability of dense planting to compensate for the negative impact of reduced nitrogen application on the water use efficiency of maize, so as to provide academic foundation for the maize production with reduced nitrogen but high water use efficiency.【Method】From 2019 to 2022, a split-plot experiment was carried out in Wuwei, Gansu Province. Two levels of nitrogen application rate, including reduced nitrogen application (N₁, 270 kg·hm^-2) and traditional nitrogen application (N₂, 360 kg·hm^-2) were set in the main plot. Three planting densities, including traditional density (M₁, 78 000 plants/hm²), medium density (M₂, 103 500 plants/hm²), and high density (M₃, 129 000 plants/hm²) were set in the split-plot, N₂M₁ was set as the control. The effects of nitrogen application, plant density and their interaction on water consumption characteristics and water use efficiency of maize were mainly studied.【Result】In the trial year, the water consumption of reduced nitrogen application was 4.7% lower than that of traditional nitrogen application. The water consumption of the combination of reduced nitrogen application and medium density (N₁M₂) was not significantly different from that of the control. The water consumption of high and medium density treatments increased by 8.4% and 4.2% respectively compared with that of traditional density treatment. The grain yield of maize decreased with the reduction of nitrogen application. Medium density could increase grain yield compared with high density and traditional density. The combination of reduced nitrogen application and medium density increased the grain yield compared with the control. In the test year, the grain yield of the reduced nitrogen application treatment was 4.6% lower than that of traditional nitrogen application treatment, the grain yield of the medium density treatment was increased by 5.6% and 8.2% respectively compared with high density treatment and traditional density, and the grain yield of N₁M₂ was 4.3% higher than that of N₂M₁. Reducing nitrogen application reduced IWUE, but maintained the same WUE as traditional nitrogen application; Medium density was beneficial to improve WUE and IWUE, and could compensate for the WUE decrease caused by reduced nitrogen application. In the four study years, the IWUE decreased by 4.5% due to reduced nitrogen application, and there was no significant difference in WUE between the two nitrogen application levels; Compared with the traditional and high density treatments, the IWUE of the medium density treatment increased by 8.6% and 6.4%, respectively, and the WUE increased by 4.5% and 10.1%, respectively; The compensation effects of medium density on the IWUE and WUE were 4.3% and 5.2%, respectively.【Conclusion】In arid irrigation area, applying nitrogen of 270 kg·hm^-2 and density of 103 500 plants/hm² during the whole growth period of maize can increase the yield and water use efficiency compared with the existing water and nitrogen management measures, which is a production technology for nitrogen saving and water efficient utilization of maize in this area.

Studies on the Suitable Nitrogen Supply Level of Rapeseed Blanket Seedling for Mechanized Transplanting

LI Jing, QIAN Chen, LIN GuoBing, WANG Long, LI YiYang, ZHENG JingDong, YOU JingJing, LENG SuoHu, ZUO QingSong

Scientia Agricultura Sinica. 2023, 56(16):  3100-3109.  doi:10.3864/j.issn.0578-1752.2023.16.005

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【Objective】The density of rapeseed blanket seedling suitable for mechanized transplanting is large and individual plant is small, so the adversity-resistant ability of rapeseed blanket seedling is poor. In order to determine the appropriate nitrogen (N) supply level, the effects of different N levels on the agronomic traits, physiological indexes and survival rate after mechanized transplanting were studied.【Method】In 2020 and 2021, two rapeseed varieties, Qinyou 10 and Ningza 158, were planted. On the basis of spraying 0.25 g N/tray at the cotyledon stage, five nitrogen fertilizer levels (0, 0.5, 1.0, 1.5 and 2.0 g N/tray) were set at one-leaf and one-tip stage. The agronomic traits and physiological indexes were measured 30 days after sowing, and the survival rate was investigated 10 days after mechanized transplanting.【Result】At 30 days after sowing, the seedling numbers per tray ranged from 627 to 669, and the seedling numbers increased first and then decreased with the increase of N supply level. The increase of N supply level increased N contents, and the N content rates in shoots were higher than those in roots. The carbon (C) decreased in general as N level increased, and the C content rates in roots were higher than those in shoots. The variation range of C/N ratios in shoots and roots was 6.98-9.69 and 12.35-16.26, respectively, which showed a downward trend with the increase of N supply level. The plant height, leaf area, fresh weight, dry weight and moisture content increased gradually with the increase of N level, and among them, the increasing extent of the fresh weight in roots was the most. When N level increased from zero to 2.0 g N/tray, the fresh weight in roots per tray in Qinyou 10 and Ningza raised by 106.3% and 95.0%, respectively. The root collar diameter and survival rate after planting showed a trend of first increasing and then decreasing as increasing of N supply level. In the same year and variety experiment, the root collar diameter of 1.5 g N/tray treatment was the highest, and the survival rates ranged from 81.7% to 97.1%. The treatments of 1.0 and 1.5 g N/tray were higher, both above 95%, and there was no significant difference between them.【Conclusion】In the process of rapeseed blanket seedling cultivation, based on 0.25 g N/tray at cotyledon stage, the N supply level from 1.0 to 1.5 g N/tray at one-leaf and one-tip stage is easy to obtain strong seedlings. Under this management, the N content and C/N ratio were appropriate, and seedling number, root collar, survival rate after mechanized transplanting were high.

PLANT PROTECTION

Mechanism of StLAC2 and StLAC6 Differentially Affecting Setosphaeria turcica Based on Non-Targeted Metabonomics Analysis

ZOU JinPeng, YUE HaoFeng, LI HaiXiao, LIU Zheng, LIU Ning, CAO ZhiYan, DONG JinGao

Scientia Agricultura Sinica. 2023, 56(16):  3110-3223.  doi:10.3864/j.issn.0578-1752.2023.16.006

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【Background】As a polyphenol oxidase, laccase plays an important role in fungal growth, development and secondary metabolism. A plurality of laccase genes are encoded in the genome of Setosphaeria turcica, among which StLAC2 and StLAC6 have differential effects on the growth, development, and pathogenicity of S. turcica.【Objective】To clarify the differential mechanisms of StLAC2 and StLAC6 on S. turcica and explore new targets for developing new fungicides and disease control strategies by mining differential metabolites.【Method】StLAC6 was connected with pHZ100-GFP plasmid by seamless cloning, and the complementary expression vector of StLAC6 was constructed. Using PEG-mediated protoplast transformation method, the constructed vector was transferred into the protoplast of StLAC6 gene knockout mutant, and the positive transformants were identified by PCR, RT-qPCR and GFP fluorescence verification, and the StLAC6 revertant strain was successfully constructed. The effects of knocking out and reverting StLAC2 and StLAC6 on melanin synthesis and oxidation resistance in and out of S. turcica were analyzed. Taking wild-type (WT), StLAC2 and StLAC6 gene knockout mutants as experimental materials, the differential metabolites were analyzed by non-targeted metabonomics, and the mechanism of the differential action of StLAC2 and StLAC6 was analyzed by KEGG.【Result】StLAC2 and StLAC6 have differential effects on melanin synthesis in mycelium and secreted into culture medium, and StLAC2 also affects antioxidant activity of S. turcica. Metabolomic analysis found that compared with the WT strain of S. turcica, there were more differential metabolites in the mycelium or secreted into the culture medium after knocking out StLAC2, and KEGG analysis showed that the differential metabolites were mainly lipids, especially phospholipids. Meanwhile, the absence of the StLAC2 caused down-regulation of various flavonoids and polyphenols. The contents of intermediates of the 1, 8-dihydroxynaphthalene melanin biosynthesis pathway, scytalone and vermelone, significantly increased in ΔStLAC2 and decreased in ΔStLAC6.【Conclusion】The StLAC2 participates in melanin polymerization, the StLAC6 negatively regulates melanin biosynthesis in S. turcica, and the differential effects of StLAC2 and StLAC6 affect lipid metabolism and intermediates of the melanin biosynthesis pathway in S. turcica. The absence of StLAC2 caused down-regulation of various flavonoids and polyphenols, leading to decreased antioxidant activity.

Identification of StCYP83 Gene Family in Potato and Analysis of Its Function in Resistance Against Late Blight

KONG LeHui, ZONG DeQian, SHI QingYao, YIN PanPan, WU WenYu, TIAN Peng, SHAN WeiXing, QIANG XiaoYu

Scientia Agricultura Sinica. 2023, 56(16):  3124-3139.  doi:10.3864/j.issn.0578-1752.2023.16.007

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【Objective】The objective of this study is to identify StCYP83 gene family in potato and analyze their expression patterns in response to Phytophthora infestans infection, so as to mine the StCYP83 genes with potential function in resistance to late blight and provide novel resistance gene resources for molecular resistance breeding in potato.【Method】The members of StCYP83 gene family were identified by bidirectional BLAST method. The basic information of StCYP83 protein sequence, subcellular localization and conserved motifs were analyzed by ExPASy Prot Param, Cell-Ploc 2.0 and ESPript, etc. The qRT-PCR was used to analyze the expression pattern of StCYP83 genes in response to P. infestans infection. The immune function of candidate gene StCYP83B1 against P. infestans was analyzed in either Agrobacterium tumefaciens-mediated transient transformation in leaves of Nicotiana benthamiana or stably transformed potato lines with overexpression (OE) of StCYP83B1.【Result】A total of 10 StCYP83 genes were identified in the potato genome, which were named StCYP83B1-StCYP83B10, respectively, with the encoded protein lengths ranging from 387 to 503 aa and molecular weights ranging from 44 to 57 kDa. The subcellular localization of StCYP83 proteins was predicted in the endoplasmic reticulum. The qRT-PCR confirmed that members of StCYP83 could be induced in response to P. infestans infection, suggesting that StCYP83 genes might play a role in the interaction between potato and P. infestans. Accordingly, StCYP83B1 with the highest homology to AtCYP83B1 was selected as a candidate gene for subsequent immune functional analysis. The pathogenicity assay on N. benthamiana leaves showed that overexpression of StCYP83B1 could enhance plant resistance against P. infestans. In accordance with this, overexpression of StCYP83B1 could significantly promote the up-regulation expression of PTI marker genes (NbWRKY7 and NbWRKY8), SA signaling marker genes (NbPR1 and NbPR2), JA signaling marker genes (NbPR3 and NbLOX) and enhance the reactive oxygen species (ROS) burst induced by flg22. In addition, cysteine site in the conserved motif of StCYP83B1 protein was required for its immune function. StCYP83B1 overexpression (StCYP83B1-OE) lines showed enhanced resistance to P. infestans. In accordance with this, StCYP83B1-OE could enhance PTI immune responses, including the increased level of ROS induced by flg22 and the significantly up-regulated expression of PTI marker genes (StWRKY7, StWRKY8 and StACRE31) as well as SA-mediated signaling marker genes (StPR1, StPR2, StPR5 and StPAL2) and JA-mediated signaling marker genes (StLOX, StAOS and StOPR3) in response to P. infestans infection.【Conclusion】A total of 10 members of StCYP83 family were identified, which could be induced by P. infestans infection in different degrees. StCYP83B1 regulates plant resistance to P. infestans by activating PTI, SA and JA signaling pathways. The cysteine site in the heme binding domain of StCYP83B1 is required for its immune function.

SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT

Spatial-Temporal Variation Characteristics of Wheat and Maize Stalk Resources and Chemical Fertilizer Reduction Potential of Returning to Farmland in Recent 30 Years in China

LIU ShuJun, LI DongChu, HUANG Jing, QU XiaoLin, MA ChangBao, WANG HuiYing, YU ZiKun, ZHANG Lu, HAN TianFu, LIU KaiLou, SHEN Zhe, ZHANG HuiMin

Scientia Agricultura Sinica. 2023, 56(16):  3140-3155.  doi:10.3864/j.issn.0578-1752.2023.16.008

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【Objective】China has rich wheat and maize stalk resources. It is necessary to clarify the spatial-temporal characteristics of the wheat and maize stalk resources, the corresponding nutrient resources and chemical fertilizer reduction potential through stalk incorporation, which can provide decision basis for promoting the utilization of stalk resources and reducing chemical fertilizer application. 【Method】Based on the soil long-term monitoring data of the Ministry of Agriculture and Rural Affairs in wheat and maize planting areas of China from 1988 to 2019, this study analyzed the amount of wheat and maize stalk and its nutrient resources and fertilizer reduction potential through stalk incorporation in different years in each region of China. 【Result】The annual average amount of wheat and maize stalk resources reached 1.62×10⁸ t and 4.23×10⁸ t, respectively in 2010s, which were increased by 0.16×10⁸ t and 2.04×10⁸ t compared with 1990s, respectively. The annual average amount of wheat and maize stalk NPK resources reached 278.19×10⁴ t and 901.08×10⁴ t, respectively, which were increased by 27.97×10⁴ t and 434.82×10⁴ t compared with 1990s, respectively. Both of them increased most in North China (NC). The annual average amount of wheat stalk resources and NPK resources decreased first and then increased with planting years, while maize increased. In the first stage (1990s to 2000s) and the second stage (2000s to 2010s), the annual variation rate (AVR) of wheat stalk resources were -42.47×10⁴ t·a^-1 and 205.10×10⁴ t·a^-1, and the AVR of nutrient resources were -0.26×10⁴ t·a^-1 of N, -0.03×10⁴ t·a^-1 of P, -0.44×10⁴ t·a^-1 of K and 1.27×10⁴ t·a^-1 of N, 0.14×10⁴ t·a^-1 of P, and 2.11×10⁴ t·a^-1 of K, respectively. In the first stage and the second stage, the AVR of maize stalk resources were 397.82×10⁴ t·a^-1 and 1 643.60×10⁴ t·a^-1, and the AVR of nutrient resources were 3.46×10⁴ t·a^-1 of N, 0.56×10⁴ t·a^-1 of P, 4.46×10⁴ t·a^-1 of K and 14.30×10⁴ t·a^-1 of N, 2.30×10⁴ t·a^-1 of P, and 18.41×10⁴ t·a^-1 of K, respectively. There were more than 80% of wheat stalk and its nutrient resources distributed in NC and Middle and lower reaches of Yangtze River (MLRYR), with the highest in NC (0.93×10⁸ t, 160.31×10⁴ t of NPK), and the lowest in Southwest China (SW) (0.09×10⁸ t, 16.05×10⁴ t of NPK). About 70% of maize stalk and its nutrient resources were distributed in Northeast China (NE) and NC, with the highest in NE (1.39×10⁸ t, 296.96×10⁴ t of NPK), and the lowest in MLRYR (0.21×10⁸ t, 44.40×10⁴ t of NPK). The annual average nutrient-release amount of wheat stalk incorporation per unit area were 21.1 kg·hm^-2 of N, 3.0 kg·hm^-2 of P and 62.3 kg·hm^-2 of K in China, with the highest in NC, and the lowest in SW. The annual average nutrient-release amount of maize stalk incorporation per unit area were 48.6 kg·hm^-2 of N, 10.6 kg·hm^-2 of P and 97.7 kg·hm^-2 of K in China, with the highest in Northwest China (NW), and the lowest in SW. The annual average nutrient-release amount of wheat and maize stalk incorporation per unit area increased continually during 1988-2019. The proportion of wheat stalk returning nutrients to the annual chemical fertilizer application were 9.13%-10.82%, 4.26%-6.43% and 88.02%-111.86%, respectively, and that of maize stalk were 22.99%-24.37%, 16.04%-28.67% and 150.29%-171.95%, respectively. 【Conclusion】In general, using wheat and maize stalk properly could satisfy the basic potassium requirement for crop production and reduce the application of about 10%-20% nitrogen fertilizer and 5%-20% phosphorus fertilizer. Making full use of stalk resources was an important guarantee for reducing fertilizer application and increasing efficiency.

Effect of Organic Fertilizer Replacing Chemical Fertilizer on Nitrous Oxide Emission from Wheat-Maize Rotation System in Lime Concretion Black Soil

LIU GaoYuan, HE AiLing, DU Jun, LÜ JinLing, NIE ShengWei, PAN XiuYan, XU JiDong, LI Jue, YANG ZhanPing

Scientia Agricultura Sinica. 2023, 56(16):  3156-3167.  doi:10.3864/j.issn.0578-1752.2023.16.009

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【Objective】 Under the condition of organic fertilizer replacing chemical fertilizer, the relationship between nitrous oxide (N₂O) emissions and soil environmental factors in wheat-maize rotation system of lime concretion black soil was researched, which could provide theoretical basis for greenhouse gas emission reduction from farmland. 【Method】 In this study, taking wheat-maize rotation system of lime concretion black soil as research object, the effects of no fertilization (CK), conventional fertilization (CF) and organic fertilizer-N replacing 20% or 40% of chemical fertilizer-N (R2FM and R4FM, organic fertilizer-N were applied in the wheat season) on N₂O emissions and environmental factors driving N₂O emissions were analyzed by the method of static box-gas chromatography and conventional soil parameter analysis. 【Result】 The wheat yields under R2FM and R4FM were significantly increased by 12.2% and 10.2% than those under CF, respectively, but there was no significant difference in the maize yields between fertilization treatments. Average annual N₂O emission fluxes under CK, CF, R2FM, and R4FM were 5.9, 50.3, 43.9 and 39.6 μg·m^-2·h^-1, with 3.1, 23.6, 25.0 and 26.4 μg·m^-2·h^-1 in the wheat season and 8.8, 77.0, 62.8 and 52.9 μg·m^-2·h^-1 in the maize season, respectively. Under fertilization treatments, N₂O emission fluxes were significantly positively correlated with soil NO₃^--N in the whole season, as well as soil temperature in the wheat season and soil water content in the maize season. Cumulative annual N₂O emissions were 2.38, 2.44 and 2.53 kg·hm^-2 for CF, R2FM and R4FM, which were significantly increased by 325%-354% in comparison to CK (0.56 kg·hm^-2), and the range of their emission factors was 0.40%-0.44%. However, there was no significant difference between cumulative annual N₂O emissions or N₂O-N emission factors under CF, R2FM and R4FM. Cumulative seasonal N₂O emissions were obviously different between fertilizer treatments, such as R2FM and R4FM significantly increased cumulative N₂O emissions by 28.3% and 62.6% in the wheat season in comparison to CF, and their N₂O-N emission factors (0.35% and 0.41%) were also significantly increased, but they significantly decreased cumulative N₂O emissions by 15.8% and 33.8% in the maize season, respectively. Cumulative N₂O emissions were significantly positively correlated with soil total nitrogen, alkali-hydrolyzable nitrogen, microbial biomass carbon in the wheat season as well as soil total nitrogen in the maize season, but they were significantly negatively correlated with soil organic carbon in the maize season, respectively. 【Conclusion】 Under the condition of organic fertilizer-N replacing chemical fertilizer-N, optimizing fertilization management in the wheat season is the key to reduce N₂O emissions from wheat-maize rotation system in lime concretion black soil.

HORTICULTURE

Mining Genes Related to Fruit Quality in Sweet Oranges Based on Specific Locus Amplified Fragment Sequencing

LI RenJing, SHEN WanXia, ZHAO WanTong, CHENG Li, LI Pei, JIANG Dong

Scientia Agricultura Sinica. 2023, 56(16):  3168-3182.  doi:10.3864/j.issn.0578-1752.2023.16.010

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【Objective】By using specific locus amplified fragment sequencing (SLAF-seq) sequencing method and combined with fixation indices analysis on different sweet orange populations, the candidate genes related with some excellent traits in sweet orange were mined, so as to provide some candidate genes for the further molecular breeding in sweet orange. 【Method】SLAF-seq technology was used to genotype 240 sweet orange germplasm resources, which represented extensive genetic diversity and from different geographical origins accessions preserved in the National Citrus Germplasm Repository in Beibei of Chongqing. The genomic SNP genotype data were obtained using the sweet orange genome as the reference, and the Fst and XP-CLR values between different subpopulations were calculated. 【Result】A total of 497.82 Mb_-reads were obtained by sequencing 240 sweet oranges accessions with SLAF-seq technology, after the sequencing reads were mapped on to the reference genome by BWA software, and a total of 1 467 968 SNPs were identified by GATK and samtools. The Fst and XP-CLR indices were used to screen out candidate genes related to three traits, including fruit navel occurrence, fruit weight, and titratable acid content. Those genes adjacent to the selected SNPs were called. The result showed that orange1.1G044639m and orange1.1g023641m which annotated auxin efflux vector were related to the formation of navel in sweet orange. SNP in orange1.1G023641m which encoded E3 ubiquitin ligase had the highest Fst score between two different single-fruit weight population, and orange1.1G046891m might affect fruit weight by regulating carbohydrate formation; orange1.1G011684m encoding dihydrolipoamide transacetylase of PDH, and the SNP caused a nonsynonymous mutation in its CDS region, which might determine the citric acid content in fruit; orange1.1G034502m encodes Serine/threonine protein kinase, annotated as phosphorylation dephosphorylation function also related with the citric acid content in fruit. 【Conclusion】In this study, the Fst indices were used to screen six candidate genes associated with three fruit traits in sweet orange by using SNPs from SLAF-seq data. Finally, the relevant genes were screened out for three horticultural traits, including navel occurrence, fruit weight and titratable acid content, and the identified candidate genes should be giving further investigation to explore their regulation mechanism in these traits.

Identification of Callose Synthetase Gene Family and Functional Analysis of PlCalS5 in Paeonia lactiflora

HE Dan, YOU XiaoLong, HE SongLin, ZHANG MingXing, ZHANG JiaoRui, HUA Chao, WANG Zheng, LIU YiPing

Scientia Agricultura Sinica. 2023, 56(16):  3183-3198.  doi:10.3864/j.issn.0578-1752.2023.16.011

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【Objective】 The CalS family plays an important role in regulating callose synthesis in plants. In this study, the members of the CalS family were identified and their bioinformatics and expression patterns were analyzed, which provided an evidence for distant cross incompatibility of Paeonia lactiflora (P. lactiflora). 【Method】The pollen germination and tube growth in self- and cross-pollinated stigma were observed by fluorescence microscope. The callose content, endogenous Abscisic acid (ABA) content and β-1,3-glucanase activity in stigma were measured. Eight members of the CalS family were cloned, and Expasy, MEME, TBtools, MEGA 7.0 and so on were used to predict the basic physicochemical properties and conserved motifs of proteins of the PlCalS family members and to construct a phylogenetic evolutionary tree. The expression of eight PlCalS in stigma at 24 h of self-pollination, 24 h of cross-pollination and 36 h of cross-pollination were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Multiple sequence alignment of CalS5 was performed, a phylogenetic tree was constructed, and the expression characteristics of PlCalS5 in response to ABA treatment at different concentrations were analyzed. 【Result】Fluorescence microscopic observation of pollen tube showed that callose plugging occurred in the stigma of hybrid, which restricted the pollen germination and tube growth. Callose content in cross-pollinated stigma was found to be higher in most periods than that in self-pollinated stigma. Then, the callose activity of β-1,3-glucose and ABA content in stigma with different pollination affinity showed regular differences. The members of the PlCalS family of P. lactiflora were identified and named by integrity analysis of the structural domains and sequence matching, and 15 conserved motifs with stable distribution in the PlCalS family were encoded by every eight genes. Multi-species phylogenetic relationships showed that the CalS family could be divided into three branches, with the PlCalS family distributed in only two branches, of which PlCalS5 was more closely related to CalS5 in Paeonia suffruticosa, Arabidopsis thaliana and Solanum lycopersicum. The bioinformatics analysis showed that the eight family members encoded 1 745-1 951 amino acids, with a total number of 28 583-31 870 atoms and isoelectric points of 7.99-9.13. The analysis of the FPKM values in transcriptome showed that the PlCalS family members were highly expressed in the same period of hybridization and at 36 h of hybridization under the same treatment. qRT-PCR showed that the relative expression levels of 8 genes at 24 h of self-pollination were lower than those at 24 h and 36 h of cross-pollination. In addition, the PlCalS5 gene was found to be more sensitive to high ABA treatment. 【Conclusion】 There were eight gene members in the CalS family of P. lactiflora, which played an important role in regulating the callose formation of P. lactiflora. The expression level of the PlCalS gene in cross-pollinated stigma was higher than that in self-pollinated stigma in most periods, which might be involved in callose abnormal deposition. Heterologous pollen stimulation to the stigma of P. lactiflora could enhance a certain ABA synthesis pathway. ABA induced callose deposition by positively regulating the expression of callose synthesis genes, thus inhibiting pollen germination and tube elongation, and finally affecting pollination compatibility.

FOOD SCIENCE AND ENGINEERING

Effect of Boiling Coconut Water on Flavor Formation of Wenchang Chicken

WU YuCan, ZHANG ZiHan, ZHAO GuiPing, WEI LiMin, HUANG Feng, ZHANG ChunHui

Scientia Agricultura Sinica. 2023, 56(16):  3199-3212.  doi:10.3864/j.issn.0578-1752.2023.16.012

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【Objective】The aim of this study was to clarify the quality characteristics of Wenchang chicken cooked with coconut water, so as to provide an effective basis for the research on the interaction mechanism of Wenchang chicken and coconut water components and the standardized production.【Method】With Wenchang chicken and Hainan green coconut as the main raw materials, the odor substances of Wenchang chicken, Wenchang chicken breast meat, and chicken leg meat cooked with coconut water were analyzed and compared using an electronic nose and gas chromatography-mass spectrometry (GC-MS), combined with odor activity value (OAV) and principal component analysis (PCA). The taste substances of chicken breast meat and chicken leg meat were analyzed using electronic tongue, free amino acid, and nucleotide detection techniques. The physical and chemical indexes of chicken and coconut water, as well as the cooking loss and texture indexes of coconut-boiled chicken and water-boiled chicken, were determined to clarify the changes in the quality characteristics of Wenchang chicken after cooking with coconut water.【Result】The protein content in chicken leg meat was 21.0 g/100 g, and the fat content was 3.08 g/100 g. The protein content in chicken breast meat was 23.6 g/100 g, and the fat content was 1.29 g/100 g. The content of reducing sugar in coconut water was 4.75 g/100 g. The cooking loss of chicken cooked with coconut water was the highest compared with that boiled with water, and there was no significant correlation between the two texture indicators (P>0.05). After cooking with coconut water, five new aldehydes were produced in the chicken, namely 2-heptanal, (E)-2-octenal, (E)-2-nonenal, 2, 5-dimethyl benzaldehyde, and 2-undecenal. The electronic nose was able to distinguish the volatile substances in boiled chicken legs from those in coconut water. After cooking with coconut water, the content of sweet amino acids in chicken legs and chicken soup increased, but the content of savory amino acids in boiled chicken legs, chicken breasts, and broth was higher than that in chicken cooked with coconut water. The contents of 5'-AMP, 5'-IMP, and 5'-GMP in chicken cooked with coconut water were higher than those in boiled chicken.【Conclusion】The fat content in the chicken leg and protein content in the chicken breast differed in various parts of the Wenchang chicken. Boiling Wenchang chicken with coconut water increased the nucleotide content in the muscles and the free amino acid content in the chicken leg meat, significantly enhancing the taste. Boiling Wenchang chicken with coconut water increased the types of aldehydes in the thigh meat, and resulted in better flavor formation than the breast meat.

ANIMAL SCIENCE·VETERINARY SCIENCE

Analysis of Drug Resistance and Epidemic Characteristics of optrA/lsa(E) in Enterococcus faecalis from Pig Farms in Aksu Area of Xinjiang

WANG Dong, CHEN WanZhao, LI HongBo, QIN Lei, XU QiQi, LIU ZePeng, XIA LiNing

Scientia Agricultura Sinica. 2023, 56(16):  3213-3225.  doi:10.3864/j.issn.0578-1752.2023.16.013

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【Objective】 The purpose of this study was to investigate the prevalence of optrA and lsa(E) genes in Enterococcus faecalis isolates from pig farms in Aksu region of Xinjiang and the resistance profiles of these optrA/lsa(E)-carrying isolates, which would provide scientific data for evaluating the impact and hazard of these two important resistance genes. 【Method】PCR screening of optrA and lsa(E) genes were conducted for the collected 322 E. faecalis isolates in laboratory of veterinary pharmacology from Xinjiang Agricultural University. The minimum inhibitory concentrations (MICs) of doxycycline, linezolid, florfenicol, vancomycin, levofloxacin, enrofloxacin, gentamicin and valnemulin were determined by agar dilution method recommended by CLSI documents. Corresponding resistance genes, including ermA, ermB, ermC, fexA, fexB, tet(K), tet(L), tet(M), aac(6')-le- aph(2")-Ia, and cfr, were detected by PCR method. 31 strains with different backgrounds were selected to determine the MLST types and subjected to conjugation transfer experiments. 【Result】A total of 193 E. faecalis isolates were positive for optrA and/or lsa(E), of which 38 strains co-harbored optrA and lsa(E), 9 strains produced only optrA gene, and 146 strains contained only lsa(E) gene. The 193 E. faecalis strains showed 100% resistance to erythromycin, tylosin, tetracycline, doxycycline, gentamicin and valnemulin. Although the detection rate of optrA gene was much lower than that of lsa(E), the bacterial resistance degree in optrA-carrying isolates was significantly higher than that in strains carrying only lsa(E) gene and optrA and lsa(E)-negative strains. The detection rates of resistance genes also showed similar trend. E. faecalis strains carrying both optrA and lsa(E) and strains carrying only optrA showed multiresistance to 8 and 10 antibiotics, accounting for 84.6% and 100.0%, respectively. E. faecalis strains carrying only lsa(E) and optrA and lsa(E)-negative strains were mainly resistant to 6-8 antibitoics, accounting for 84.6% and 100.0%, respectively. Further analysis showed that detection rates of optrA and lsa(E) genes were higher in the pig farms with high resistance degree. Screening of resistance genes showed that ermB, ermC, tet(K), tet(L), tet(M), aac(6')-Ie-aph(2")-Ia, and fex(A) genes were presented in the 193 E. faecalis strains. In general, the detection rates of resistance genes in E. faecalis strains carrying both optrA and lsa(E) and single optrA genes were higher than that in strains carrying single lsa(E) and optrA and lsa(E)-negative strains. The results of MLST and conjugation transfer experiments indicated that optrA/lsa(E)-producing E. faecalis strains were genetically complex and clonal and horizontal transfer co-existed in these pig farms. 【Conclusion】The high carriage rate of optrA and lsa(E) genes and the high level of drug resistance in E. faecalis in pig farms in Aksu region of Xinjiang indicated that antibiotic resistance was serious in this region, which could threaten not only pig industry, but also public health. Preventive and control actions should be taken immediately to prevent further deterioration of antibiotic resistance.

Isolation and Pathogenicity of Fowl Adenovirus Serotype 8a Strain

LI HuiXin, SONG WenPing, HAN ZongXi, LIU ShengWang

Scientia Agricultura Sinica. 2023, 56(16):  3226-3236.  doi:10.3864/j.issn.0578-1752.2023.16.014

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【Objective】 Fowl adenovirus (FAdV) circulates in chicken flocks with multiple serotypes, while there is less information about the pathogenicity of all serotype strains. To understand the capability of causing disease as a primary pathogen to chicken of the FAdV-8a strain, we carry out the experiment for evaluating the pathogenic characteristics of this serotype strain, which will help making the control strategy for breeding. 【Method】 In 2017, liver tissue was collected form the diseased flocks. The liver tissue homogenates were inoculated into the embryo egg for isolating the virus. The isolate was determined as a FAdV strain after PCR detection. To classify the isolate, genomic sequencing and the genetic evolution based on the complete genome sequence and the hexon gene sequence were performed. To clarify the pathogenicity, thirty 10-day-old SPF chicks were randomly divided into 2 groups and exposed to the isolate JL/170408 via nasal inhalation and eye droppings. The clinical syndrome (including morbidity and mortality), viremia, virus shedding, circulating antibody, postmortem examination and histopathological detection at 5 days post infection (d.p.i.), viral distribution and the tropism on tissues were performed to evaluate the pathogenic capability and characteristics of JL/170408 to SPF chicks. 【Result】 The complete genome sequencing showed that there were the highest identity between the isolate and the FAdV-8a TR59 strain. They showed high identity in the genomic structure and the encoding gene. Phylogenetic analysis based on the complete genome sequence, the isolate JL/170408 was in the cluster of FAdV-E, further was grouped into the branch of serotype 8a based on the hexon gene. Consequently, the isolate JL/170408 was determined as FAdV-8a serotype within the species of FAdV-E. The clinical peak was observed from 3 to 13 d.p.i. without death. The virus shedding and viremia was detected as early as 3 d.p.i. and last for a long period at least 51d, the antibody was not positive conversion completely and the mean ELISA titer S/P<1, which didn’t provide enough neutralizing ability to eliminate the virus in the blood and the intestinal tract. At 54 d.p.i., the ELISA titer reached a peak with a mean S/P>2, with the consequence of viremia disappearing and a sudden drop of virus shedding. Postmortem examination and histopathological detection at 5 d.p.i. showed no obvious pathologic change. While the viral load was detected in 15 tissues, suggested that the isolate propagated in multiple tissues and exhibited higher tropism to gizzard. By monitoring the circulating antibody, the infected birds showed later antibody positive conversion until 15 d post infection. Not all birds showed positive seroconversion even at 51 d post infection, and the antibody level was stable. At 54 d post infection, the antibody titer reached peak, suggesting that birds may suffer a second infection. Virus neutralization test based on the antiserum of 63 d.p.i. showed that there was no obvious correlation between the circulating antibody and the neutralization antibody. 【Conclusion】 The FAdV-8a strain JL/170408 causes disease to 10-day-old chicks as single pathogen without leading to death, the isolate is determined as low pathogenic strain. JL/170408 propagates in multiple tissues with higher tropism to gizzard. The infected chicks show a long duration of virus shedding with a repetitive characteristics.

Development and Application of a Mab-Based iELISA for the Detection of Antibodies Against African Horse Fever Virus

GUO Kui, ZHANG ZeNan, WANG YaoXin, LI ShuaiJie, CHU XiaoYu, GUO Wei, HU Zhe, WANG XiaoJun

Scientia Agricultura Sinica. 2023, 56(16):  3237-3246.  doi:10.3864/j.issn.0578-1752.2023.16.015

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【Objective】In order to effectively address the risk of introduction of African horse fever into China, this study aimed to develop an indirect ELISA (iELISA) antibody test specific for African horse fever virus (AHSV) based on monoclonal antibodies, so as to monitor the antibody status of African horse fever in horses in China.【Method】Firstly, Mabs against VP7 antigen were prepared by immunizing mice with the VP7 antigen. Secondly, the ELISA method was developed by optimizing the amount of coating Mab, antigen and secondary antibody concentrations. Negative sera (n=1 000) were used to establish the baseline for a negative population, and then the specificity and sensitivity of it was estimated. The stability of the method was determined by an accelerated test using AHSV-positive reference sera by three independent operators. 10 known positive sera and 400 negative sera for African horse fever were tested using the established method and compared with the results of foreign commercial kits. Finally, the method was tested on 947 clinical samples from 18 provinces or regions in China in 2021 to assess the risk of African horse fever in horses in China.【Result】 Five Mabs were obtained against the VP7 antigen of AHSV, and the 3G9 Mab had the best antigen capture performance. Using the 3G9 Mab as the capture antibody, an ELISA method for the detection of AHSV was established by optimizing different reaction conditions. A cut-off value of 0.25 was selected in AHSV iELISA. The sensitivity of ELISA method was comparable with that of commercial kits and the sensitivity of both methods was consistent. The intra-group coefficients of variation from 3 operators ranged from 3.19% to 7.02%, 0% to 3.11%, and 0.27% to 5.76%, respectively, and the inter-group coefficients of variation for the three operators ranged from 1.17 to 5.03%. The accelerated test at 37 °C demonstrated that the components of the testing kit had good stability over 7 days, thus demonstrating the stability of the method. The results showed that the agreement between the AHSV iELISA and commercial AHSV C ELISA kits were 100%. In this study, a serological survey on AHSV was performed by using 947 serum samples collected from 18 provinces of China in 2021, and the results showed that the positive rates were 0%. 【Conclusion】 A Mab-based AHSV iELISA method was established for the detection of AHSV antibodies, which had the good specificity and sensitivity, and could be a promising candidate tools for use serological surveillance for AHS.