Table of Content

16 April 2022, Volume 55 Issue 8

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Research Progress of Nitrogen Efficiency Related Genes in Rice

SANG ShiFei, CAO MengYu, WANG YaNan, WANG JunYi, SUN XiaoHan, ZHANG WenLing, JI ShengDong

Scientia Agricultura Sinica. 2022, 55(8):  1479-1491.  doi:10.3864/j.issn.0578-1752.2022.08.001

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Over the last few years, China government has put forward a strategy to achieve the goal of “zero growth of chemical fertilizer”. It is particularly important to reduce the input of nitrogen fertilizer in agricultural production and enhance the nitrogen use efficiency in crops. Nitrogen is mostly absorbed from the soil by plant roots in the form of nitrate nitrogen (NO₃^-) and ammonium nitrogen (NH₄^-). It is transported from roots in plants to synthesize essential life substances, such as amino acids and nucleotides. Nitrogen is used as a basic element for crop growth and yield formation. However, excessive application of nitrogen fertilizer destroys the physical and chemical properties of the soil, causes undesirable changes to soil salinization, and pollutes the ecological environment, and pollutes the ecological environment. By reducing the quantity of nitrogen fertilizer, will destabilize the yield potential of field crops including rice and wheat which is being used as a staple food in China. It can threaten food security of the country. To improve the nitrogen use efficiency (NUE) and stabilize the food security, mining nitrogen-efficient genes, such as NRT1.1B, OsGRF4 etc., genetic improvement of current existing varieties through molecular design breeding will help to cultivate new nitrogen efficiently rice lines. Tapping the productive potential of current rice varieties will improve the level of sustainable agricultural development in our country. In this article, based on the nitrogen-efficient genes excavated in the current rice research, this article reviews the PTR (polypeptide transporter) family, NRT (nitrate transporter) family, AMT family (ammonium transporter family), NLP family and other types of rice nitrogen-efficient genes. The future prospects of gene utilization have been prospected. Based on the nitrogen-efficient genes excavated in the current rice research, they are divided into four categories: NRT/PTR, AMT (ammonium transporter), NLP and other types, and summarize their functions and characteristics, and analysis the utilization prospect and existing problems of nitrogen-efficient genes with potential breeding value.

Unconditional and Conditional QTL Analysis of Wheat Spike Length in Common Wheat Based on 55K SNP Array

TANG HuaPing, CHEN HuangXin, LI Cong, GOU LuLu, TAN Cui, MU Yang, TANG LiWei, LAN XiuJin, WEI YuMing, MA Jian

Scientia Agricultura Sinica. 2022, 55(8):  1492-1502.  doi:10.3864/j.issn.0578-1752.2022.08.002

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【Objective】This study is to excavate spike length (SL)-related quantitative trait loci (QTL) with potential breeding value, explore the genetic relationship between SL and other important agronomic traits in wheat, and aim at laying a foundation for fine mapping and molecular-assisted selection breeding. 【Method】A total of 126 F₇ recombinant inbred lines (RIL) constructed by crossing 20828 and SY95-71 were used in this study. The RIL population including their parents were planted in seven different environments for phenotypic evaluation: Wenjiang, Chongzhou, Ya'an of Sichuan Province in China, and Khulna in Bangladesh during 2016-2017 and 2017-2018 growing seasons. Unconditional QTL mapping was performed using a genetic linkage map constructed using the wheat 55K SNP array, and QTLs’ effects were further analyzed. Conditional QTL analysis was performed to analyze the relationship between SL and other agronomic traits including plant height (PH), spike extension length (SEL), spikelet number per spike (SNS) and thousand-kernel weight (TKW). 【Result】Thirteen QTLs controlling SL were identified using unconditional QTL mapping, and they were located on chromosomes 1A, 1D, 2B, 2D, 4B, 6D, and 7A. The LOD values ranged from 2.79 to 6.19, and the phenotypic variation rate ranged from 5.35% to 12.77%. Three stable and major QTLs (QSl-sau-2SY-2B, QSl-sau-2SY-2D.5 and QSl-sau-2SY-4B) were identified, and they explained 6.54% to 11.72%, 10.16% to 12.57%, and 5.35% to 10.92% of phenotypic variation rate, respectively. Furthermore, these three major QTLs could be also detected in multi-environment analysis. Moreover, aggregation analysis suggested that the SL of lines polymerizing the positive allels at these three major QTLs was significantly longer than that of those with any two ones or those carrying only one. Meanwhile, it was found that QSl-sau-2SY-2B had no significant effect on PH, SEL, SNS and TKW. QSl-sau-2SY-2D.5 had a significant effect on improving TKW (3.98%), but no significant effect on PH, SEL and SNS. QSl-sau-2SY-4B had a significant effect on decreasing PH (-12.28%) and SEL (-22.26%), but no significant effect on SNS and TKW. The conditional QTL analysis showed that QSl-sau-2SY-2B was independent of PH and SEL, whereas, affected by SNS and TKW. QSl-sau-2SY-2D.5 was independent of SEL, SNS and TKW, but affected by PH. QSl-sau-2SY-4B was independent of SEL and TKW, but affected by PH and SNS. 【Conclusion】In this study, three stable and major QTLs were identified for SL: QSl-sau-2SY-2B, QSl-sau-2SY-2D.5, and QSl-sau-2SY-4B, among which QSl-sau-2SY-2B may be a novel QTL independent of PH and SEL.

Characteristics and Cold Tolerance of Upland Cotton Genetic Standard Line TM-1

WANG JunJuan, LU XuKe, WANG YanQin, WANG Shuai, YIN ZuJun, FU XiaoQiong, WANG DeLong, CHEN XiuGui, GUO LiXue, CHEN Chao, ZHAO LanJie, HAN YingChun, SUN LiangQing, HAN MingGe, ZHANG YueXin, FAN YaPeng, YE WuWei

Scientia Agricultura Sinica. 2022, 55(8):  1503-1517.  doi:10.3864/j.issn.0578-1752.2022.08.003

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【Objective】We systematically investigated the major agronomic traits and cold tolerance of accession TM-1 at the bud and seedling stages. The relative expressions of cold tolerance-related genes were analyzed by the qRT-PCR method. The cold tolerance mechanism of TM-1 was further discussed, which provides the theoretical basis for the breeding utilization of TM-1.【Method】The major agronomic traits of TM-1 were manually investigated in the field using variety CRI35 as the control. The fiber quality was assessed by an international calibrated cotton standard (HVICC), and the insect resistance (Bt) was detected by kanamycin screening and molecular detection technologies. For the cold tolerance testing, two contrasting accessions, cold-resistant accession Yu 2067 and cold-sensitive variety Hengmian 3 were set as controls, respectively. The cold resistance of TM-1 at bud stage and cotyledon stage was identified, treated at 4℃ and then recovered under normal conditions for 7 days, and the relative cotyledon spreading rate and the cold injury levels of plants were investigated, and cold injury indexes and cold resistance indexes were calculated. The portable chlorophyll meter was used for in vivo testing the leaf relative chlorophyll content (represented by SPAD value). The expressions of cold tolerance-related genes in leaves were measured by qRT-PCR method. 【Result】 The leaves of TM-1 were large and dark green. The pre-frost seed cotton yield was 2 791.50 kg·hm^-2, and the plant height was 94.60 cm. The growth period was about 135 days, and the yield, plant height, fruit branch number per plant, boll number per plant were higher than CRI35, while other agronomic traits were similar to CRI35. TM-1 had medium fiber quality. The test results of kanamycin and test paper showed that TM-1 did not contain the Bt like CRI35. Identification results of cold tolerance at bud stage showed that compared with the control treatment, the relative chlorophyll content and plant height of TM-1 decreased significantly. Low-temperature stress significantly inhibited hypocotyl elongation and chlorophyll synthesis in cotton leaves. Under low-temperature treatment, the taproots of TM-1 were damaged, but the lateral roots were more developed than those of the control. The cold tolerance level of TM-1 reached high cold resistance at the bud stage. Identification of cold tolerance at the cotyledon stage showed that the relative chlorophyll content and plant height of TM-1 decreased significantly compared with the control. The cold tolerance index of TM-1 at the cotyledon stage was 85.32%, which was significantly higher than Yu 2067, and the tolerance level of TM-1 reached cold resistance at the cotyledon stage. After the treatment of low-temperature stress for 24 h at the trefoil stage, nine genes were up-regulated in the TM-1 leaves, and their up-regulated expression folds were significantly higher than those of cold-sensitive accession. Dehydrin gene was up-regulated in TM-1 leaves, and the expression fold was similar to that in the leaves of Yu 2067, which was 4.69 times that in the leaves of Hengmian 3. The expression fold of the LEA3 gene in TM-1 leaves was significantly higher than that of Yu 2067 and Hengmian 3. 【Conclusion】 Accession TM-1 has stable agronomic characters and the medium fiber quality. It can be used as an ideal receptor for transferring exotic genes because without Bt. TM-1 can also be used as an important parent for cotton breeding and a gene source for cloning genes because of its good cold tolerance.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Research Advances of Bradyrhizobia and Its Symbiotic Mechanisms with Peanut

WU Yue, SUI XinHua, DAI LiangXiang, ZHENG YongMei, ZHANG ZhiMeng, TIAN YunYun, YU TianYi, SUN XueWu, SUN QiQi, MA DengChao, WU ZhengFeng

Scientia Agricultura Sinica. 2022, 55(8):  1518-1528.  doi:10.3864/j.issn.0578-1752.2022.08.004

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Nitrogen is one of the essential elements for plant growth, which is obtained by legumes through symbiotic nitrogen fixation with rhizobia. The establishment of symbiotic relationship includes nodulation and nitrogen fixation, involving complex regulatory mechanisms, which is also significantly affected by environmental factors. Symbiosis between peanut and bradyrhizobia is essential for peanut growth and production, but contains many specific and unknown symbiotic mechanisms. In this review, symbiosis between peanut bradyrhizobia and peanut was reviewed, including: (1) Diversity and genomic functions of peanut bradyrhizobia; (2) Symbiotic mechanisms between peanut and bradyrhizobia: rhizobial crack infection and symbiotic signal exchange with peanut, peanut nodulation, nitrogen fixation, and nodule number regulation mechanisms; (3) Effects of environmental factors (soil nitrogen, pH, temperature and water content) on peanut nodulation, nitrogen fixation and yield. This review pointed out current problems in peanut bradyrhizobia, symbiosis between peanut and bradyrhizobia, and peanut field application, including few studies on genome functions of peanut bradyrhizobia, unknown interaction mechanisms between bradyrhizobia and peanut in details, as well as, poor utilization rate of peanut bradyrhizobia in the field, etc. Based on this analysis, the future researches should focus on genome omics analysis and gene functional analysis of peanut bradyrhizobia; signal communication pathways, nodule number regulation mechanisms, nutrient exchange systems between bradyrhizobia and peanut; rational application systems of nitrogen fertilizer that match with nodule nitrogen fixation rules, and obtain new peanut bradyrhizobia agents for peanut planting through synthetic biology. This article provided the theoretical basis for further understanding the symbiotic mechanisms of legumes and rhizobia, improving nodulation and nitrogen fixation efficiency of legume crops, reducing chemical nitrogen application, and improving agricultural ecological environment.

Effects of Nitrogen-Reducing Side Deep Application of Liquid Fertilizer at Tillering Stage on Yield and Nitrogen Utilization of Fragrant Rice

GUI RunFei, WANG ZaiMan, PAN ShengGang, ZHANG MingHua, TANG XiangRu, MO ZhaoWen

Scientia Agricultura Sinica. 2022, 55(8):  1529-1545.  doi:10.3864/j.issn.0578-1752.2022.08.005

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【Objective】 The effects of nitrogen-reduced side depth application of liquid fertilizer at tillering stage and conventional total application of solid fertilizer on nitrogen uptake and utilization, dry matter accumulation and physiological nitrogen metabolism in key growth stages of fragrant rice and its relationship with yield formation of fragrant rice were investigated, so as to provide the theoretical basis and guidance for mechanized side deep application of liquid fertilizer at tillering stage in south China fragrant rice growing region in the future.【Method】 Field experiments were conducted for two consecutive years from 2019 to 2020, two popular fragrant rice cultivars with large planting area in South China, including Yuxiangyouzhan and Xiangyaxiangzhan, were used as the test materials, and a randomized complete block design was adopted. Four treatments were set up, including no fertilization during rice growth season (T1), total fertilization of solid fertilizer (T2, total nitrogen application rate of 150 kg N·hm^-2), side depth application of liquid fertilizer with 10% nitrogen reduction at tillering stage (T3), and side depth application of liquid fertilizer with 20% nitrogen reduction at tillering stage (T4). The effects of different fertilization treatments on yield and its components, nitrogen absorption and utilization, dry matter accumulation and physiological nitrogen metabolism of fragrant rice were analyzed.【Result】 (1) In the two-year field experiments, the yield of both fragrant rice cultivars under T3 treatment was the highest, which was significantly higher than that under T1 treatment. Compared with T2 treatment, the grain yield of Yuxiangyouzhan in 2019 and 2020 under T3 treatment was increased by 1.84% and 15.20%, respectively; the grain yield of Xiangyaxiangzhan in 2019 and 2020 under T3 treatment was increased by 0.65% and 3.71%, respectively. The grain yield increment observed under T3 treatment was mainly related to the highest effective panicle. (2) In the two-year field experiments, the total dry matter accumulation, leaf area index, and total nitrogen accumulation of the two fragrant cultivars at panicle initiation stage and heading stage were generally the highest under T2 treatment, while no significant difference was observed between T3 treatment and T2 treatment. However, the total dry matter accumulation, leaf area index, and total nitrogen accumulation under T3 treatment were the highest for the two fragrant rice cultivars. Compared with T2 treatment, T3 treatment increased total dry matter accumulation, leaf area index, and total nitrogen accumulation of Yuxiangyouzhan by 25.14%, 26.57%, and 21.31% in 2019, respectively, and in 2020 the corresponding increases were 5.83%, 21.80%, and 46.47%, respectively. Compared with T2 treatment, the total dry matter accumulation, leaf area index, and total nitrogen accumulation under T3 treatment for Xiangyaxiangzhan were increased by 19.95%, 3.73%, and 13.41% in 2019, respectively, and in 2020 the corresponding increases were 21.17%, 1.78%, and 14.37%, respectively. In addition, the nitrogen recovery efficiency and nitrogen agronomic efficiency of the two fragrant rice cultivars were also the highest under T3 treatment. (3) At the panicle initiation stage and heading stage, T2 treatment showed higher in the activities of nitrogen metabolism-related enzymes in leaf of the two fragrant rice cultivars as compared with other fertilization treatments, and T3 treatment was observed with the highest activities of nitrogen metabolism-related enzymes in leaf of the two fragrant rice cultivars at maturity stage. 【Conclusion】 The two-year of field experiments showed that, compared with the total fertilization of solid fertilizer treatment, the side depth application of liquid fertilizer with 10% nitrogen reduction at tillering stage treatment could increase the yield of fragrant rice by ensuring the early growth, increasing total dry matter accumulation, leaf area index, nitrogen utilization and nitrogen metabolism-related enzymes activities during the late growth stage while reducing the application rate of nitrogen fertilizer.

Effects of Hybrid Rice on Grain Yield and Nitrogen Use Efficiency: A Meta-Analysis

LIAO Ping, MENG Yi, WENG WenAn, HUANG Shan, ZENG YongJun, ZHANG HongCheng

Scientia Agricultura Sinica. 2022, 55(8):  1546-1556.  doi:10.3864/j.issn.0578-1752.2022.08.006

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【Objective】Hybrid rice has a higher yield potential than inbred rice, but the difference in nitrogen (N) use efficiency between hybrid rice and inbred rice remains unclear. The objective of this study was to examine the effects of hybrid rice on yield and N use efficiency through meta-analysis techniques. 【Method】The peer-reviewed articles were collected, which included inbred rice as the control in comparison with a hybrid rice treatment. In total, the dataset included 56 studies involving 367 paired observations. Then, the meta-analysis was conducted to identify the response of grain yield and N use efficiency to hybrid rice as affected by hybrid type, N rate, the number of N application, soil total N content, the ratio of soil organic carbon to N, and soil texture. 【Result】Overall, the hybrid rice significantly increased rice yield (+11%) and biomass (+14%), but did not affect harvest index compared with inbred rice. Hybrid rice could improve rice yield relative to inbred rice under various N rates. However, the increase in rice yield under hybrid rice reduced with increasing N application rates. Moreover, the hybrid rice significantly increased N uptake, N physiological efficiency, and N recovery efficiency by 8.1%, 2.9%, and 3.6 units, respectively. 【Conclusion】Hybrid rice could improve yield and N use efficiency relative to inbred rice, which provided an insight to evaluate the effect of hybrid rice on grain yield and N use efficiency in China.

PLANT PROTECTION

Intelligent Forecasting Method of Rice Sheath Blight Based on Images

HAN XiaoTong,YANG BaoJun,LI SuXuan,LIAO FuBing,LIU ShuHua,TANG Jian,YAO Qing

Scientia Agricultura Sinica. 2022, 55(8):  1557-1567.  doi:10.3864/j.issn.0578-1752.2022.08.007

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【Objective】At present, the forecast of rice sheath blight relies on the number of diseased clusters, the number of rice plants and the severity of each plant to calculate the disease index based on manual surveys. The method is highly professional, time-consuming and laborious. The data is difficult to trace. The objective of this study is to propose a detection model of rice sheath blight lesions and a damage grading model of rice sheath blight based on images, and to provide a theoretical basis for the intelligent forecasting of rice sheath blight.【Method】Images of rice sheath blight in paddy field were collected by a portable image acquisition instrument. Different detection models (Cascade R-CNN and RetinaNet) and feature extraction networks (VGG-16 and ResNet-101) were developed to test the detection effect of disease lesions. The best model was chosen. However, the Cascade R-CNN model appeared some missing detection of sheath blight lesions. Because the sheath blight lesions are irregular in shape, and variable in size and location, the Cascade R-CNN model was improved through adding OHEM structure to balance the hard and easy samples in the network and choosing the bounding box regression loss function. The precision rate, missing rate, average precision and P-R curve were used to evaluate the detection effects of different models. Based on the detection results of the improved Cascade R-CNN-OHEM-GIOU model, two damage grading models based on the area and number of disease lesions were developed, respectively. The determination coefficient (R²) and Kappa value were used to choose the damage level model of rice sheath blight. 【Result】Under the same backbone network conditions, the Cascade R-CNN model had a better detection effect on rice sheath blight than the RetinaNet model. The Cascade R-CNN-ResNet-101 model had the best detection effect on sheath blight lesions. The precision rate was 92.4%, the average precision was 88.2% and the missing rate was 14.9%. The improved Cascade R-CNN-OHEM- GIOU model effectively solved the problem of sample imbalance, and effectively reduced the missing rate by adding a border regression loss function, which was 8.7% lower than the missing rate of the Cascade R-CNN-ResNet-101 model, and the average precision was increased to 92.3%. With the results of manual disease grading as the standard, the grading model of rice sheath blight at 0 to 5 grades based on the area of diseased lesions had the accurate rates of 96.0%, 90.0%, 82.0%, 76.0%, 74.0% and 96.0%, respectively. The average grading accuracy rate was 85.7%, and the Kappa coefficient was 0.83. The damage grade results of rice sheath blight based on images were consistent with the manual grading results.【Conclusion】The intelligent forecasting method of rice sheath blight based on images can automatically detect the disease lesions and calculate the damage grade. This method increases the intelligence level and the results are objective and traceable. It may also provide an idea for intelligent forecasting of other crop diseases.

Effect of Three Novel Compounds on Trehalose and Chitin Metabolism and Development of Spodoptera frugiperda

WANG SiTong,CHEN Yan,LUO YuJia,YANG YuanYuan,JIANG ZhiYang,JIANG XinYi,ZHONG Fan,CHEN Hao,XU HongXing,WU Yan,DUAN HongXia,TANG Bin

Scientia Agricultura Sinica. 2022, 55(8):  1568-1578.  doi:10.3864/j.issn.0578-1752.2022.08.008

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【Objective】Chitin is the main component of insect exoskeleton and peritrophic membrane. Its synthesis begins with trehalase and ends with chitin synthase. The process of molting and epidermal remodeling needs to be completed by chitinase. In this study, three novel compounds were injected to detect the activities of trehalase and chitinase, the expression levels of related genes in Spodoptera frugiperda, and their growth and development were also observed. This study aims to verify the inhibitory effects of novel compounds on trehalase and chitinase, screen the compounds with obvious effects, and to explore the mechanism of their regulation on the growth and development of S. frugiperda.【Method】Microinjection method was used to inject butenolactone analogues ZK-I-21, ZK-I-23 and piperine analogue ZK-PI-4 into the 3rd instar larvae of S. frugiperda. The changes of trehalase activity, chitinase activity and related sugar content were detected 48 h after injection, and the relative expression levels of SfTRE1, SfTRE2, SfCHS2 and SfCHT were measured at the molecular level by quantitative real-time PCR (qRT-PCR). The phenotypic changes of S. frugiperda were observed during the process from larvae to adults after injection. Besides, the mortality and deformities during developmental period were recorded.【Result】Compared with the control group, the membrane-bound trehalase activity of S. frugiperda decreased significantly after ZK-I-21 (P<0.01) and ZK-PI-4 (P<0.05) injection. The expression level of SfTRE1 was significantly increased and SfCHT was significantly decreased after ZK-I-21 injection. After ZK-I-23 injection, the expression level of SfTRE1 was significantly decreased, and the expression level of SfCHT was significantly increased. The expression level of SfCHT was significantly decreased after ZK-PI-4 injection. The observation results of developmental duration showed that ZK-I-21 significantly prolonged the developmental period of 6th instar larvae of S. frugiperda, and at the same time, the pupa weight became lighter and the pupa length became shorter. In addition, ZK-I-23 significantly shortened the length of 5th and 6th instar larvae, and ZK-PI-4 caused a significant reduction in adult emergence rate. All the three inhibitors could disrupt trehalose metabolism of S. frugiperda and then disrupt chitin metabolism, resulting in difficulties in molting and even death.【Conclusion】ZK-I-21 and ZK-PI-4 are membrane-bound trehalase inhibitor compounds, and ZK-I-23 can inhibit the expression of soluble trehalase gene. The three compounds all lead to the disturbances of chitin metabolism by affecting the process of trehalose metabolism, which results in difficulties in molting, deformities, and impaired growth and development of insects. The above results can provide a theoretical basis for the future use of novel inhibitors to regulate the growth of pests and thus control them, and support to promote the development of green and efficient pesticides.

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

Characteristics of Organic Nitrogen Mineralization in Paddy Soil with Different Reclamation Years in Black Soil of Northeast China

GAO JiaRui, FANG ShengZhi, ZHANG YuLing, AN Jing, YU Na, ZOU HongTao

Scientia Agricultura Sinica. 2022, 55(8):  1579-1588.  doi:10.3864/j.issn.0578-1752.2022.08.009

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【Objective】 The aim of this study was to analyze the mineralized nitrogen (N) content, net N mineralized rate and net N mineralized ratio (the ratio of mineralized N to total N) in paddy soil with different reclamation years in black soil, and to explore the soil N supply capacity and its characteristics, and to reveal the soil N evolution law, so as to provide the theoretical basis for rational utilization and fertilization of black soil in Northeast China.【Method】 The natural wasteland (0 years, as the control soil, original natural meadow vegetation) and paddy soils with different reclamation years (12, 35, 62 and 85 a) (topography and cropping system, fertilization, and water management, roughly the same) in black soil region were selected as the research object, and the characteristics of soil organic N mineralization after cultivated rice form natural wasteland in black soil were studied by the water-logged incubation method.【Result】 During the early stages of incubation (about 1 month), the cumulative mineralized N increased rapidly in each year, then showed a slow increase trend. At the end of incubation (297 d), the cumulative mineralized N ranged from 212.43 to 388.11 mg·kg^-1, and the order of cumulative mineralized N was 0, 12, 35, 85 and 62 a. The mineralization process of soil organic N could be well described by a hybrid model (Special model), and the soil organic N pools could be divided into the increment N pool (the N pool made available after a drying and rewetting event) and the resistant N pool. Compared with the control soil (0 a), the potentially mineralisable N (N_F) of the increment N pool in all paddy soils showed a decreasing trend in each year. There was no significant difference between paddy soils of 12 and 35 years, as well as 62 and 85 years, but the N_F in paddy soils of 12 and 35 years were significantly higher than that of 62 and 85 years (P<0.05). The rate constant (k_F) of mineralization of the increment N pool in paddy soils all showed an upward trend, but there was no significant difference between k_F of all paddy soils in each year (P>0.05). Compared with the control soil (0 a), the rate constant (k₀) of mineralization of the resistant N pool in paddy soils of 12 and 35 years did not change significantly (P>0.05), but k₀ in 62 and 85 years decreased significantly (P<0.05). The net N mineralization rate of the soils in each year were the largest at 4 days of incubation, and then decreased gradually. At the end of water-logged incubation (297 d), the order of the soil net N mineralization rate was consistent with that of the cumulative mineralization N. The net N mineralized ratio was relatively high at the beginning of incubation, and then increased slowly. At the end of incubation (297 d), the net N mineralized ratio ranged from 78.60 to 101.82 mg·g^-1, and the order was 0, 35, 12, 85 and 62 a. Soil total N and C/N were important factors affecting the amount of mineralization N and the net N mineralization rate in paddy soils with different reclamation years (P<0.05). The sum of initial mineral N and N_F could be used to characterize the N supply capacity of paddy soil in rice growing season; compared with the control soil (0 a), the N supply capacity of paddy soil in each year decreased significantly (P<0.05), and the soils of 12 and 35 years were significantly higher than that of 62 and 85 years (P<0.05).【Conclusion】 During 85 years of rice cultivation from natural wasteland in black soil, the N supply capacity in paddy soil have declined, and the decline was significant after 35 years rice cultivation. Therefore, the improvement of soil organic matter content should be paid attention in the soil fertility cultivation in paddy fields.

Annual Nutrients Balance and Economic Return Analysis of Wheat with Fertilizers Reduction and Different Rotations

MA XiaoYan, YANG Yu, HUANG DongLin, WANG ZhaoHui, GAO YaJun, LI YongGang, LÜ Hui

Scientia Agricultura Sinica. 2022, 55(8):  1589-1603.  doi:10.3864/j.issn.0578-1752.2022.08.010

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【Objective】The present study aimed to investigate the soil nutrients balance and economic benefits of different rotations with optimal chemical fertilizer application for wheat production, with the purpose of selecting a proper rotation for wheat production with lower fertilizer application and improvement of economic return for local farmers.【Method】The field experiments with randomized block design were carried out in Wugong county from 2018 to 2020, including four treatments: maize-wheat rotation with farmer accustomed fertilization (CWF), maize-wheat rotation with recommended fertilization (CWR), green manure-wheat rotation (GWR), and soybean-wheat rotation (BWR). Except for treatment of CWF, the fertilizers of rest three treatments were calculated according to the method of soil testing and determining fertilizer recommendations (STDFR). The plants tissues and soil with different depth were sampled for nutrients analysis.【Result】Compared with CWF, with 11.8% reduction of nitrogen and 41.7% reduction of phosphorus, the CWR treatment significantly increased wheat yield, with an average increase of 7.4%, and significantly increased the nitrogen and phosphorus uptake of wheat grain and aboveground shoot, with the nitrogen increase of 10.1% and 8.9% respectively, and the phosphorus increase of 9.8% and 6.4%, respectively. CWR treatment decreased the surplus rate of soil N, P and K by 25.1%, 72.3% and 54.4%, respectively, and increased the economic benefit by 36%. Among three different rotation, CWR rotation had the highest wheat yield, followed by soybean wheat rotation and green manure wheat rotation. GWR rotation had the lowest surplus ratio of NPK among three treatment with optimal fertilizer application, which was significantly lower than that of CWR and GWR rotation, with the decrease range of 87.3%-92.0%, 41.9%-67.7%, and 78.1%-85.3%, respectively, and obtained the highest economic return.【Conclusion】Fertilization recommendation by monitoring soil nutrients techniques could not only decrease fertilizer input and production cost, but also increase wheat yield, increase nutrient uptake of wheat grain, reduce surplus rate of nitrogen and phosphorus nutrient balance, and thus improve economic benefits. Compared with maize-wheat rotation, the soybean-wheat rotation not only reduced the input of chemical fertilizer, significantly reduced the surplus of nitrogen, phosphorus and potassium, but also achieved higher economic benefits. In conclusion, it was recommended that the fertilizers application rates should be optimized for maize-wheat rotation in Guanzhong Plain. However, taking into account of economic return and ecological benefit, the soybean-wheat rotation was prior choice for the purpose of decreasing soil nutrients surplus and enhancing the economic return of farmers.

Effect of Drip Fertigation Mode on Maize Yield, Nutrient Uptake and Economic Benefit

LI Qian, QIN YuBo, YIN CaiXia, KONG LiLi, WANG Meng, HOU YunPeng, SUN Bo, ZHAO YinKai, XU Chen, LIU ZhiQuan

Scientia Agricultura Sinica. 2022, 55(8):  1604-1616.  doi:10.3864/j.issn.0578-1752.2022.08.011

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【Objective】 To study the effects of different drip fertigation modes on maize yield, nutrient absorption, soil nitrogen balance, water use efficiency and economic benefits, in order to provide a theoretical basis for drip irrigation maize production in semi-arid area of Jilin province.【Method】Field experiment was carried out in Minle Village of Jilin Province for 2018-2019. Five treatments were set up: drip fertigation with plastic film (DFM), shallow buried drip fertigation (DF), shallow buried drip fertigation urea (DIU), shallow buried drip irrigation (DI), and farmer practices (FP). We collected plant samples at jointing stage, bell stage, silking stage, filling stage and mature stage, divided into stem, leaf and grain parts, measured the shoot dry matter weight and the contents of nitrogen, phosphorus and potassium, respectively. Based on these data, the nutrient absorption were calculated or evaluated. The soil samples from 0-100 cm soil depth were respectively collected before sowing and after harvest of maize to study soil nitrogen balance．The soil samples from 0-200 cm soil depth were respectively collected before sowing and after harvest of maize to study water use efficiency.【Result】The maize yield of drip fertigation treatments (DFM, DF, DIU and DI) were higher than farmers' conventional treatment 10.3%-20.6%, the rate of yield increase in the dry year (2018) (13.7%-27.9%) was higher than that in the rainy year (2019) (7.2%-13.7%), meanwhile, the accumulation of nitrogen, phosphorus and potassium in mature stage was increased by 15.7%-31.7%（P<0.05）, 11.0%-35.6% (P<0.05) and 5.2%-20.9%, especially increased the nitrogen, phosphorus and potassium uptake after silking by 63.1%-95.2% (P<0.05), 11.6%-63.0% and 40.0%-110.0% (P<0.05); it also significantly increased the water use efficiency (WUE) by 21.8%-33.9% and decreased the apparent nitrogen loss by 13.8%-92.0%. Compared with shallow buried drip fertigation (DF), DFM treatment increased the yield and water use efficiency in dry years, but differences were not significant in rainy years, DFM treatment significantly reduced soil nitrogen apparent loss by 74.2%, but there was no significant difference in net income between in two treatments, and the benefit/cost ratio of DFM treatment was lower than that of DF treatment significantly. Under shallow buried drip irrigation, there were no significant differences in maize yield, nitrogen apparent loss and water use efficiency between DF and DIU treatments. However, the accumulation of dry matter and P and K in DF treatment at mature stage were significantly higher than those in DIU treatment. There was no significant difference between DF treatment and DIU treatment in net income and benefit/cost ratio. The maize yield of DI treatment was 13.7% higher than that of FP treatment in dry years, but not significantly in rainy years. It also significantly increased N and P accumulation at mature stage and N and K accumulation after silking. The net income of DI treatment was not significantly different from that of FP treatment, but the ratio of production and input was significantly lower than that of FP treatment.【Conclusion】Drip fertigation mode could increase maize yield, N, P and K accumulation and water use efficiency, and reduce N apparent loss in semi-arid areas, and the effect was significant in dry years. The advantage of DFM treatment were greater than DF, but the benefit/cost ratio was lower than DF. There was no significant difference in the yield, nutrient uptake and water use efficiency, net income and the benefit/cost ratio between shallow buried drip fertigation urea technology and shallow buried drip fertigation technology, the low cost DIU treatment simplified the production process, and also had the obvious effect on increasing grain yield. In conclusion, shallow buried drip fertigation urea mode was suitable for the current situation of maize production in semi-arid area of Jilin Province.

HORTICULTURE

Metabolites Changes of Cucumber Xylem and Phloem Sap Under Low Phosphorus Stress

LI QingLin,ZHANG WenTao,XU Hui,SUN JingJing

Scientia Agricultura Sinica. 2022, 55(8):  1617-1629.  doi:10.3864/j.issn.0578-1752.2022.08.012

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【Background】 Xylem sap is responsible for the transport of water and nutrients for the aerial part, while phloem sap is mainly responsible for the transport of photosynthetic products. When plants are subjected to abiotic stress, the sap of xylem and phloem undergoes different changes. 【Objective】 The aim of this study was to investigate the content changes of various metabolites in cucumber xylem and phloem sap under low phosphorus stress, and to explore the metabolic changes of cucumber under low phosphorus stress, so as to provide new ideas and basis for the diagnosis of cucumber phosphorus deficiency. 【Method】 The xylem and phloem sap were collected from cucumber plants under normal phosphorus level (1.25 mmol∙L^-1) and low phosphorus stress (0.3125 mmol∙L^-1). These sap samples were analyzed qualitatively and quantitatively by GC-MS after trimethylsilyl derivatization. 【Result】In xylem sap under low phosphorus stress, there was no significant change in carbohydrates, and the content of most amino acids increased significantly. Among organic acids, the malic acid showed a significant downward trend, while others, such as palmitic acid and oleic acid, were significantly higher than the normal levels. In phloem sap under low phosphorus stress, most carbohydrates and amino acids were lower than the normal level, and the contents of organic acids, such as oxalic acid and succinic acid, were also significantly reduced. Further, in order to screen potential biomarkers in cucumber xylem and phloem sap, the detection data were further analyzed by OPLS-DA and Student's t test. Eight potential biomarkers (VIP>1, P<0.05) under low phosphorus stress were detected in cucumber xylem sap, which were cadaverine, malic acid, 4-aminobutyric acid, palmitic acid, myo-inositol, cellobiose acid, N, N-dimethyldodecamide, and glycerol monostearate. Seven potential biomarkers (VIP>1, P<0.05) under low phosphorus stress were detected in cucumber phloem sap, which were serine, linoleic acid, 9-(3-methyl-5-pentylfuran-2-yl) nonanoic acid, dehydroabietic acid, N, N-dimethyldodecamide, 2, 2'-methylenebis (6-tert-butyl-4-methylphenol), and L-2-piperic acid. 【Conclusion】 Under low phosphorus stress, the carbohydrates, amino acids and organic acids in the xylem and phloem sap of cucumber changed, but the change trends were not the same. Eight and seven biomarkers were screened from xylem and phloem sap, which could provide new ideas and basis for the diagnosis of cucumber phosphorus deficiency. Among the biomarkers, the detection methods of malic acid, 4-aminobutyric acid, palmitic acid, serine and linoleic acid have been relatively mature, and the cucumber phosphorus deficiency diagnosis method based on them was more practical.

Screening and Verification of CsHIPP26.1 Interaction Protein in Tea Plant

FAN YanGen,WANG Yu,LIU FuHao,ZHAO XiuXiu,XIANG QinZeng,ZHANG LiXia

Scientia Agricultura Sinica. 2022, 55(8):  1630-1641.  doi:10.3864/j.issn.0578-1752.2022.08.013

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【Background】 Huangjinya is a light sensitive chlorotic tea (Camellia sinensis) variety, the leaf color of which presents yellow under strong light and presents green under weak light, but the chlorotic mechanism of leaf color in response to light is not clear. Previous proteomic studies on etiolated leaves, shaded green leaves and evergreen leaves found that the expression of heavy metal-associated isoprenylated plant protein CsHIPP26.1 (TEA000549) responded to light intensity, indicating that CsHIPP26.1 may be involved in regulating the light response process of leaf color etiolation of Huangjinya. 【Objective】The proteins related to the light signal response interacting with CsHIPP26.1 were screened to provide a scientific basis for leaf color response to light signal changes. 【Method】The gene were cloned from one bud and two leaves of Huangjinya. And the screened target protein was further verified by yeast two hybrid point-to-point verification, in vivo bimolecular fluorescence complementarity (BiFC), and in vitro pull-down techniques. 【Result】 The tea cDNA library was screened by yeast two hybrid, and a total of 26 candidate interaction proteins were screened, which mainly played a role in cell components, binding and catalytic activity. Among them, the enrichment degree of biotin anabolism process was high, and the proteins related to light signal pathway and chlorophyll synthesis were only the bHLH30 transcription factor, and its gene ID was TEA026466.1. After cloning the gene of bHLH30 transcription factor, it was found that the transcription factor was in the same evolutionary tree branch as tea light signal transduction pathway protein PIF4, and contained the same HLH and ACT domains as tea PIF4 protein. Therefore, the bHLH30 transcription factor was named CsPIF4.2, GenBank registration number: MW16834. And through the pull-down protein interaction in vitro and bimolecular fluorescence complementarity (BiFC) in vivo, it was found that CsHIPP26.1 and CsPIF4.2 proteins could indeed interact, and the site of interaction was in the nucleus. 【Conclusion】 26 proteins interacting with CsHIPP26.1 were preliminarily screened, and it was found that CsHIPP26.1 could interact with one of the phytochrome interacting factors CsPIF4.2 in the nucleus.

FOOD SCIENCE AND ENGINEERING

Effects of Exogenous Sucrose on the Postharvest Quality and Chloroplast of Gynura bicolor D.C

XIE YiTong,ZHANG Fei,SHI Jie,FENG Li,JIANG Li

Scientia Agricultura Sinica. 2022, 55(8):  1642-1656.  doi:10.3864/j.issn.0578-1752.2022.08.014

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【Background】The physiological metabolism of G. bicolor is active after harvest, and it is sensitive to low temperature. After harvest, it is often stored in a dark environment with slightly lower than room temperature. However, the long-term dark storage of G. bicolor will lead to sugar starvation, which affects the quality of G. bicolor. The dark storage also inhibits the photosynthetic process, resulting in reduced photosynthetic assimilation products and aggravated postharvest sugar starvation, and sucrose is the main form of photosynthetic product transport in plants. 【Objective】The effects of exogenous sucrose treatment on postharvest quality, sucrose metabolism and chloroplast of G. bicolor were studied to explore the related mechanism of sucrose treatment on delaying postharvest senescence in this study.【Method】On the basis of screening out the optimal concentration, the contents of starch, soluble sugar, reducing sugar, soluble protein and chlorophyll in G. bicolor during storage were detected to study the effect of sucrose treatment on postharvest quality of G. bicolor. The contents of sucrose, fructose, glucose, and sucrose metabolism related enzyme activities, such as Amylase, SPS, AI, SS-s, and SS-c, were detected during storage, and then the effects of sucrose treatments on sucrose metabolism of G. bicolor was studied. The changes of chloroplast ultrastructure during storage were observed by TEM. The activity of LOX, the content of MDA, and the Fv/Fm and QY of chloroplast during storage were detected, then the effects of sucrose treatment on the physiology and function of chloroplast were studied. The effects of postharvest sucrose treatment on G. bicolor were studied at biochemical and subcellular levels.【Result】The screening of sucrose concentration in the earlier study showed that 12% sucrose had the best preservation effect. Especially in the late storage period, compared with the control (distilled water treatment), the respiratory intensity, weightlessness rate, and decay rate of 12% sucrose treatment decreased by 39%, 7.8%, and 15.87%, respectively. Further study found that in the late storage, compared with the control, the treated sucrose content ratio was 1.82, starch content ratio was 1.10, soluble sugar content ratio was 1.11, soluble protein content ratio was 2.20, and chlorophyll content ratio was 1.23, indicating sucrose treatment significantly delayed the degradation of carbohydrates and nitrogenous substances. Sucrose treatment significantly inhibited the activities of SPS, AI and Amylase, indicating that sucrose treatment inhibited sucrose metabolism, thereby reducing the decomposition of sucrose and starch. In the later study on the physiological functions of chloroplasts of G. bicolor, it was found that at the end of storage, compared with the control, the treated G. bicolor effectively maintained the structural integrity of chloroplasts, reduced the activity of chloroplast LOX by 53.13%, and reduced the content of MDA by 33.33%. The Fv/Fm and QY were 1.35 and 1.97 times that of the control, respectively, indicating that sucrose treatment significantly delayed the senescence of chloroplasts. Further analysis showed that chloroplast function was positively correlated with starch and soluble sugar content, indicating that carbon source deficiency caused by sugar starvation could affect chloroplast function.【Conclusion】Sucrose treatment inhibited postharvest quality deterioration and chloroplast senescence of G. bicolor by reducing respiratory intensity, weightlessness rate and decay rate, regulating sucrose metabolism, reducing the degree of chloroplast membrane lipid oxidation, and maintaining the integrity of chloroplast structure, thereby delaying the senescence of G. bicolor.

ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT

circ-13267 Regulates Egg Duck Granulosa Cells Apoptosis Through Let-7-19/ERBB4 Pathway

WU Yan,ZHANG Hao,LIANG ZhenHua,PAN AiLuan,SHEN Jie,PU YueJin,HUANG Tao,PI JinSong,DU JinPing

Scientia Agricultura Sinica. 2022, 55(8):  1657-1666.  doi:10.3864/j.issn.0578-1752.2022.08.015

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【Background】 Follicle development is a key factor for laying performance of egg ducks. Previous studies have shown that follicular development is a very complex biological process in poultry. At present, the pattern of follicular development in poultry has been understood. However, as an important factor determining egg production, the specific regulation mechanism of follicular development still needs further study. Granulosa cells are the main functional cells in follicles. They can regulate the growth, differentiation and maturation of theca cells and oocytes. They also regulate the growth and development of follicles, maintain normal ovarian function, such as induce ovulation, maintain maturation division, and provide substrates for oocytes. Circular RNAs (circRNAs) are a new type of endogenous specific non-coding RNA, which plays an important role in follicular development. 【Objective】The objective of this study was to explore the effects and regulatory mechanism of circ-13267 on apoptosis in egg duck granulosa cells, through regulating the expression of circ-13267 by constructing the overexpression vector, so as to provide the evidence for analysis the regulatory mechanism of egg duck follicular development. 【Method】Firstly, the expression levels of circ-13267 in cytoplasm and nucleus of granulosa cells was detected by Q-PCR. The overexpression vector circ-13267-pLCDH was constructed. After transfection of circ-13267 in egg duck granulosa cells, the expression levels of circ-13267, let-7-19, ERBB4, FAS and BCL2 were detected by Q-PCR. The proliferation of egg duck granulosa cells was detected by EdU method after transfection circ-13267-pLCDH and pLCDH-ciR. The linear sequence of circ-13267 or the 3'UTR of ErbB4 was cloned into pmirGLo vector. At the same time, let-7-19 binding site in the wild-type sequence was mutated to obtain the vector expressing the mutant sequence. The targeting relationships between circ-13267 and let-7-19, let-7-19 and ERBB4 were verified by dual luciferase reporter assay, respectively. Then, after transfection of circ-13267-pLCDH and pLCDH-ciR into egg duck follicular granulosa cells, the flow cytometry and Annexin V-FITC were utilized to explore the effects of circ-13267 on duck granulosa cells. 【Result】 In duck granulosa cells, circ-13267 was expressed in both cytoplasm and nucleus. The dual luciferase reporter gene assay confirmed that let-7-19 could bind to ERBB4 and down regulate the activity of luciferase; when the binding site of let-7-19 in ErbB4 sequence was mutated, let-7-19 could not inhibit the expression of luciferase, indicating that ERBB4 was a target gene of let-7-19. The results of Q-PCR showed that, after overexpression of circ-13267, the expression of BCL2 gene decreased significantly (P<0.05), while the expression of FAS and ERBB4 gene increased significantly (P<0.05); after overexpression of let-7-19, the expression of ERBB4 gene increased significantly (P<0.05), while after inhibition of let-7-19, the expression of ERBB4 gene decreased significantly (P<0.05). EdU test results showed that the number of follicular granulosa cells in egg ducks decreased significantly after overexpression of circ-13267, it was shown that circ-13267 promoted the apoptosis of follicular granulosa cells in egg ducks. However, after co-transfection of circ-13267 and let-7-19 into egg duck follicular granulosa cells, compared with the control group, there was no significant change in the expression of BCL2 and FAS (P>0.05); however, compared with overexpression of circ-13267, the expression of BCL2 gene decreased significantly (P<0.01) and FAS increased significantly (P<0.01). It was shown that circ-13267 could inhibit the apoptosis of egg duck follicular granulosa cells. In addition, flow cytometry was used to detect the transfected egg duck follicular granulosa cells. Compared with the co-transfection groups of circ-13267 and let-7-19, the number of late apoptotic cells and total apoptotic cells increased significantly (P<0.05), while the number of living cells decreased significantly (P<0.05). 【Conclusion】 circ-13267 was expressed in the cytoplasm and nucleus of egg duck follicular granulosa cells. circ-13267 could sponge let-7-19 and target ERBB4 gene, which promoted the apoptosis of egg duck follicular granulosa cells. This results provided a theoretical basis for analysis of the regulatory mechanism of egg duck follicular development.

Effects of Soy Isoflavones on the Proliferation and Apoptosis of Yak Ovarian Granulosa Cells

WANG JiaMin,SHI JiaChen,MA FangFang,CAI Yong,QIAO ZiLin

Scientia Agricultura Sinica. 2022, 55(8):  1667-1675.  doi:10.3864/j.issn.0578-1752.2022.08.016

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【Background】Soybean isoflavones, mainly including genistein and daidzein, could exert estrogen-like effects, scavenge free radicals and promote cell proliferation. Granulosa cells are an important cell population in follicles and its physiological state is directly related to ovarian function, and the apoptosis of granulosa cells causes oocyte atresia. Yak (Bos grunniens) is one of the endemic species in Qinghai-Tibet plateau, but its reproductive rate is so low, however, the mechanism is still unclear. Ovary granulosa cell is an ideal model to study the regulating mechanism of female animal reproduction. 【Objective】The aim this study was to investigate the effects of soybean isoflavones on the proliferation and apoptosis of granulosa cells of yak ovary, and to provide evidence for the mechanism of soybean isoflavones.【Method】The yak ovarian granulosa cells were isolated and cultured. Immunohistochemistry staining was used to check FSHR for ovarian granulosa cell authenticating. MTT assay was used to detect cell proliferation, then the growth curve was drawn. Ovarian granulosa cells treated with different concentration of genistein and daidzein (0, 1 000, 2 000, 3 000, 4 000 and 5 000 pg·mL^-1), the optimal concentration of genistein or daidzein for was selected to treat the second-generation granulosa cells for 48 h, then, the cell culture medium was collected and used to detect the concentration of estradiol and progesterone secreted by granulosa cells by ELISA. At the same time, the cells were collected to extract total RNA and to research the expression of proliferation-related gene AKT1 and apoptosis-related genes Bcl-2, Bax, Bad, Fas, FasL, Caspase-3 and p53 by qRT-PCR.【Result】The yak ovarian granulosa cells were typical epithelial-like cells. After inoculated for 2 h, the granulosa cells began to adhere to the wall. After 12 h, the cells appeared aggregation and growth. After 24 h, the cells were larger and showed long fusiform, star-shaped or polygonal. After cultured for 48 h, the cells looked like paving appearance. Immunohistochemistry staining showed FSHR positive indicated that the cultured cells were ovarian granulosa cells. MTT assay showed that the growth curve of the yak ovarian granulosa cells was S-shape: the growth was slow in 24 h and the cells were in the latent growth stage, and then, which was rapidly proliferated at 24-48 h and entered the exponential growth phase. The granulosa cells, in the plateau phase, steadily proliferated during 48-120 h. After 120 h, the density of living cells began to decline, and the cells entered the phase of degeneration. The second-generation of granulosa cells grew faster and the exponential proliferation phase at 24 h, so the second-generation of granulosa cells was selected for this experiment. MTT assay showed that treated with 3 000 pg·mL^-1 genistein or daidzein for 48h, the cell viability were significantly promoted (P<0.01), the secretion of estradiol were induced treated with daidzein, but the progesterone secretion were markedly inhibited treated with genistein. The results of qRT-PCR showed that 3 000 pg·mL^-1 genistein or daidzein significantly up-regulated the expression of AKT1, Bcl-2, Bad, p53, Fas and Bcl-2/Bax (P<0.01), down-regulated the expression of Bad and FasL. In addition, genistein significantly down-regulated the expression of Caspase-3 and daidzein significantly up-regulated it (P<0.01). 【Conclusion】The yak ovarian granulosa cells were isolated and cultured to provide an effective cell model for further study on the yak female reproduction regulating mechanism. The results indicated that genistein and daidzein inhibited the progesterone secretion of yak ovarian granulosa cells, promoted cell proliferation, and protected cells from apoptosis by up-regulating Bcl-2, p53, Fas and Bcl-2/Bax and down-regulating Bad and FasL.

Method Improvement and Its Application of Micro Complement Fixation Test for Brucellosis

JIANG Hui,FENG Yu,QIN YuMing,ZHU LiangQuan,FAN XueZheng,DING JiaBo

Scientia Agricultura Sinica. 2022, 55(8):  1676-1684.  doi:10.3864/j.issn.0578-1752.2022.08.017

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【Objective】 The complement fixation test is used as a confirmatory test for the serodiagnosis of brucellosis. The complement fixation tests are divided into constant complement fixation test (CFT) and micro complement fixation test (mCFT) in diagnostic techniques for animal brucellosis (GB/T 18646-2018). However, there are differences in the related technical parameters and judgment of the result between them, which lead to the inconsistency of CFT and mCFT results. In this study, By the improvement and optimization of the parameters of mCFT, this study made the detection results of mCFT equivalent to classical CFT, and achieved the high-throughput and accurate diagnosis of CFT for brucellosis. 【Method】 According to classical CFT reaction conditions and diagnostic criteria, the reaction time, diluent, titer determination, result judgment and other related parameters for the current national standard of mCFT were optimized and improved. Compared with the original method, the dilution of the tested serum was changed from 1:2-1:64 to 1:10. The dilution method was the same as classical CFT so that the detection critical value was consistent with the constant method. The OD₆₀₀ value was read by the microplate reader to determine the hemolysis intensity, which reduced the error caused by visual judgment and improved the efficiency and accuracy of result judgment. The reaction time was shortened from 30 min to 20 min in water bath at 37℃, which improved the detection efficiency without affecting the results. The diluent was changed from barbital buffer to normal saline, which used the same diluent of classical CFT and achieved satisfactory results. The 409 clinical bovine and sheep serum samples (163 bovine serum samples and 246 sheep serum samples) from Inner Mongolia, Shandong, Beijing and Jiangsu were detected by improved mCFT, the national standard mCFT, and classical CFT, respectively. The coincidence rate was analyzed and compared. 【Result】By detecting the 409 clinical samples, the result showed that 53 samples were positive, 11 samples were suspicious and 345 were negative by improved mCFT. The 53 positive samples, 9 suspicious samples and 347 negative samples were detected by classical CFT. Compared with the results of the two methods, only two bovine serum samples were suspicious by the improved mCFT, which were negative by classical CFT. The results of the other 407 samples were consistent. The coincidence rate of bovine serum samples was 98.77%, which of sheep serum samples was 100%, and the total coincidence rate was 99.51% between improved mCFT and classical CFT. However, the positive criteria of national standard mCFT were lower than that of classical CFT, and no suspicious interval was set. The results showed that 97 samples were positive and 312 samples were negative. Compared with the results of classical CFT, the coincidence rate of bovine serum samples was 88.34%, which of sheep serum samples was 89.84%; the total coincidence rate was 89.24%, which was far lower than 99.51% of improved mCFT and classical CFT. 【Conclusion】 By optimizing the parameters of mCFT, the mCFT method was established, which was highly consistent with the criterion and the results of the classical CFT.

Effects of Sublethal Doses of Imidacloprid on the Expression of Neurometabolic Genes in Apis cerana cerana

QIU YiLei,WU Fan,ZHANG Li,LI HongLiang

Scientia Agricultura Sinica. 2022, 55(8):  1685-1694.  doi:10.3864/j.issn.0578-1752.2022.08.018

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【Background】The target of neonicotinoid insecticides is the acetylcholine receptor in the nervous system of insects. Due to its good systemicity and low toxicity to humans and animals, it has been widely used in agricultural production. Thus there is still a low residue in plants, and this sublethal dose residue can still cause adverse effects on the behavior and nervous system of flower-visiting insects like bees.【Objective】The objective of this study is to clarify the effect of sublethal dose imidacloprid (a kind of neonicotinoid insecticides) on the nervous physiological and metabolism system of Apis cerana cerana.【Method】In this study, the worker bees were treated with two sublethal concentration gradient doses of 5 and 10 μg·L^-1 imidacloprid for 10 days (three biological replicates). After total RNA was extracted, the resulting library was analyzed by RNA-seq. Throughput sequencing, and the bioinformatics technology was used to assemble and annotate the sequence de novo, and the differentially expressed genes (DEGs) after sublethal doses imidacloprid treatment were analyzed by clustering and enrichment. Finally, real-time fluorescence quantitative PCR (RT-qPCR) technology was used to verify the DEGs related to the nervous and metabolic systems.【Result】A total of 9 sequencing libraries were obtained, the ratio of effective sequencing data exceeded 94.45%. From the obtained 37 364 unigenes, 571 DEGs were identified. The enrichment analysis of GO and KEGG found that the DEGs were mainly related to multiple pathways such as protein translation, redox, oxidative phosphorylation, and ribosome, indicating that sublethal doses of imidacloprid had an impact on multiple physiological processes and metabolic pathways of A. c. cerana. Nine DEGs (e.g. neuropeptide F (NPF), neuropeptide SIFamide receptor (SIFaR), phosphoinositide 3-dependent kinases (PDK1), A-kinase anchoring protein 1 (AKAP1), carbonic anhydrase 3 (CA3), superoxide dismutase 2 (SOD2), NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 10 (ND42), cuticle protein 12 (CP12), and odorant-binding protein (OBP17)) related to nerve signal transmission and metabolic function were selected for RT-qPCR verification. Their expression patterns were completely consistent with the transcriptome results.【Conclusion】The sublethal dose of imidacloprid can affect lots of aspects of A. c. cerana such as nerve signal transduction, cellular respiration, immune response, maintenance of homeostasis, and olfactory perception.