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01 March 2021, Volume 54 Issue 5

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Scientia Agricultura Sinica. 2021, 54(5):  0. 

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Effect of Overexpression of OsRRK1 Gene on Rice Leaf Development

YinHua MA,KaiQin MO,Lu LIU,PingFang LI,ChenZhong JIN,Fang YANG

Scientia Agricultura Sinica. 2021, 54(5):  877-886.  doi:10.3864/j.issn.0578-1752.2021.05.001

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【Objective】OsRRK1 (RoP-Interacting receptor-like kinase 1) is a member of the cytoplasmic receptor kinases RLCK VI family. In this study, the leaf morphology of OE-OsRRK1 transgenic rice was observed to clarify the role of OsRRK1 in the leaf development of rice. 【Method】Primers were designed based on target sequences published in the rice genome database to construct OsRRK1 overexpression vector, then transformed to Japonica variety Hejiang 19 mediated by Agrobacterium tumefaciens. Positive plants were identified by PCR analysis. The copy number of inserted exogenous genes in transgenic plants was identified by Southern blot hybridization. The expression level of transgenic plants at RNA level was confirmed by Northern blot hybridization and qRT-PCR. T₂ generation homozygote plants at heading stage was selected to determine the leaf rolling index (LRI). Paraffin sections and aniline blue staining were used to observe the changes of rice bulliform cells after cross-cutting, and Image J software was used to calculate the area of the bubble cells. Chlorophyll content in plant leaves was measured by chlorophyll tester.【Result】44 positive transgenic plants were obtained and 17 lines were randomly selected for Southern blot analysis. Among the 17 lines, 8 lines were single-copy insertion and 9 lines were multi-copy insertion. 2 multi-copy lines (OE-1 and OE-4) and 5 single-copy lines (OE-5, OE-21, OE-22, OE-24 and OE-25) were randomly selected for subsequent analysis. qRT-PCR analysis revealed that OsRRK1 was overexpressed with different degree in different transgenic lines. Among them, OE-1, OE-4 and OE-25 lines with the highest OsRRK1 expression level, while OE-5 lines with the lowest and OE-21, 22 and 24 lines with the middle. Three single copy transgenic lines OE-5, OE-22 and OE-25 and WT control were selected for Northern blot analysis. The results were in consistent with the results of qRT-PCR results. Leaf rolling index of the seven selected lines and WT control revealed that the degree of leaf curl was positive correlated with the expression level of OsRRK1, with the higher the expression level, the higher the degree of rolling. Paraffin section and staining revealed the number and area of bulliform cells in the flag leaves of OE-OsRRK1 transgenic plants changed obviously compared to WT. OE-OsRRK1 transgenic plants showed less bulliform cell number than those in the control whose average is 4.6. OE-OsRRK1 transgenic plants with curlier leaf, with the more severe bulliform cell degeneration, less number, smaller area. Chlorophyll content determination revealed that the OE-22, OE-24 and OE-25 lines have higher chlorophyll content than those of the WT rice leaves.【Conclusion】The overexpression of OsRRK1affect the number and size of bulliform cells in rice leaves, which cause leaf rolling, and the degree of leaf rolling is positively correlated with the expression level of OsRRK1. The overexpression of OsRRK1 gene will lead to the increase of chlorophyll content in rice.

Construction and Application of Detection Model for the Chemical Composition Content of Soybean Stem Based on Near Infrared Spectroscopy

JiaJia LI,HuiLong HONG,MingYue WAN,Li CHU,JingHui ZHAO,MingHua WANG,ZhiPeng XU,Yin ZHANG,ZhiPing HUANG,WenMing ZHANG,XiaoBo WANG,LiJuan QIU

Scientia Agricultura Sinica. 2021, 54(5):  887-900.  doi:10.3864/j.issn.0578-1752.2021.05.002

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【Objective】The chemical components (cellulose, hemicellulose, lignin, crude fiber, etc.) in the stem are closely/intently linked with lodging resistance of soybean. However, due to the current detection and analysis of chemical components in the stem, the traditional chemical analysis technology is adopted, and the determination procedure is complex, time-consuming, labor-consuming, expensive, and lead to environmental pollution. Thus, the current study aimed to construct a low-cost, quick, scientific and pollution-free method for detection of chemical components in soybean stems, and provide a methodological basis for the study of the distribution of stem components in soybean germplasm resources and their relationship with soybean growth habits and lodging. 【Method】 In present study, a chemical component detection model of soybean stem based on near-infrared spectroscopy was established, and the model was used to detect neutral detergent fiber (NDF), acid detergent fiber (ADF) and crude fiber (CF) of soybean germplasm resource stem. CF and other chemical components were detected and analyzed. The intrinsic relationship between CF content of soybean stem and its growth habit and lodging resistance was elucidated by analysis of variance, multiple comparisons and violin plot analysis. 【Result】 The results showed that the correction correlation coefficient (R_C) of the NDF, ADF and CF components of the stem based on the rapid detection model constructed in this research was all above 0.90. The validity of the model was verified by using 16 soybean stem samples outside the model, and it was found that there was no significant difference between the results of routine chemical testing and the model testing (P > 0.05). This model was used to analyze the relationship between the CF content and growth habit of 393 soybean stems planted in 2017 and 2018. The findings showed that the CF content of soybean stems conforms to the normal distribution. Among the materials of the CF content is above 50.00%, the two-year data showed that the erect type (91.67% and 86.14%) was significantly higher than the sprawl type (8.33% and 13.86%), indicating that the CF content was significantly correlated with its growth habit of soybean stems (P < 0.01). 【Conclusion】 The Near-infrared Spectroscopy Model constructed in this study has the characteristics of low cost, fast, high efficiency and pollution-free. In addition, the plants of soybean cultivars with high CF content in the stem had stronger bending resistance, which could be used as an important index for screening lodging resistance breeding parents of soybean.

Color Difference Analysis in the Application of High Quality Foxtail Millet Breeding

Ting ZHANG,GenPing WANG,YanJie LUO,Lin LI,Xiang GAO,RuHong CHENG,ZhiGang SHI,Li DONG,XiRui ZHANG,WeiHong YANG,LiShan XU

Scientia Agricultura Sinica. 2021, 54(5):  901-908.  doi:10.3864/j.issn.0578-1752.2021.05.003

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【Objective】The color difference analysis for grain quality evaluation was conducted using the foxtail millet varieties participating national high-quality foxtail millet competition in recent years. The results, combined with the expert sensory evaluation, could improve high quality millet breeding.【Method】Millet varieties that participated in the past three high-quality millet competition in 2015, 2017, and 2019 were accounted, the L* values, a* values, b* values were determined by the color difference analysis, and the correlation analysis was performed with the commercial quality scores, cooking and eating quality scores and the total scores. 【Result】The number of millet varieties competed for high-quality increased by 82.3% in the last 3 sessions. summer-sowing varieties increased by 60.0% while spring-sowing was 100.0%. The number of first-class quality varieties increased by 133.3%, of which, 87.5% were bred by 7 scientific research institutes. 93.7% of the varieties were bred by cross breeding, 62.5% of which was herbicide resistant. Among all of the high-quality millets, the L* values ranged from 55.05 to 66.03 in summer-sowing region, which was from 53.12 to 68.84 in spring-sowing region. The a* values ranged from 1.63 to 8.74 in summer-sowing region, and that was from 2.13 to 8.59 in spring-sowing region. The b* values ranged from 54.50 to 75.35 in summer-sowing region, and that was from 40.07 to 71.40 in spring-sowing region. The L* values, a* values, b* values of all tested varieties and high-quality millets showed an increasing trend. Comparing with summer-sowing region, spring-sowing region had greater variation in b* value and the higher color richness. The commercial quality scores and the total scores were significantly correlated with L* values, a* values, b* values. The steamed millet scores were significantly correlated with L*, a* values. However, the millet congee scores were not significantly correlated with L* values, a* values, b* values. The correlation between commercial quality scores and total scores was the highest, with a correlation coefficient of 0.7528. The commercial quality scores were positively correlated with the steamed millet scores, but was not correlated with the millet congee scores. In the regression model of the first-grade high-quality millet, the L* values, a* values, b* values determined the 74.3% variation of the total score. The varieties with top 10 commercial quality scores and total scores in summer-sowing region were mainly divided into two types: one was the golden varieties with low a*, high L* and b * values, such as Jigu 39 and Yugu 33; the others were the light yellow varieties with high a*, low L* and b* values, such as Jigu 19 and Jigu 42. The varieties with top 10 commercial quality scores and total scores in spring-sowing region were mainly divided into three types: one was the dark yellow varieties with low L*, high a* and b * values, such as 2015KM826F7-75 and Fenghong 4 hao; one was the light yellow varieties with high L*, low a* and b* values, such as Yugukang 1 and Chihong 1 hao; one was the light yellow and red varieties with high a*, low L* and b* values, such as Zhangzagu 13 and Jinfen 106. 【Conclusion】In the last three national competitions for high-quality foxtail millet varieties, the number of millet varieties showed an increasing trend. Many scientific research institutes have focused on quality breeding. In the future, high grain quality would become an important goal for millet improvement. The commercial quality score and total score showed strong correlation, so it was important to improve the commercial quality of millet for quality breeding. It was an intuitive and convenient breeding method to select millet color by the color difference L* values, a* values, b* values. The commercial quality score correlated with L* values, a* values, b* values, so millet color selection should be based on the characteristics of the variety in quality breeding.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Dry Matter Accumulation, Allocation, Yield and Productivity of Maize- Soybean Intercropping Systems in the Semi-Arid Region of Western Liaoning Province

Qian CAI,ZhanXiang SUN,JiaMing ZHENG,WenBin WANG,Wei BAI,LiangShan FENG,Ning YANG,WuYan XIANG,Zhe ZHANG,Chen FENG

Scientia Agricultura Sinica. 2021, 54(5):  909-920.  doi:10.3864/j.issn.0578-1752.2021.05.004

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【Objective】The study investigated the mechanism of overyielding in maize-soybean intercropping systems and optimized maize-soybean intercropping configurations in semi-arid western Liaoning province by analyzing the dry matter accumulation and allocation of crops and the competition relationship between species in maize-soybean intercropping systems.【Method】The experiment was carried out in 2018-2019 at National Agricultural Environmental Station for Agricultural Environment in Fuxin. The cropping systems were: maize-soybean strip intercropping with 2 rows of maize and 2 rows of soybean (MS2:2), maize-soybean strip intercropping with 4 rows of maize and 4 rows of soybean (MS4:4), maize-soybean strip intercropping with 6 rows of maize and 6 rows of soybean (MS6:6), sole maize (M) and sole soybean (S). The dry matter accumulation and allocation of crops, interspecies competitiveness and its impact on crop yields and land productivity were analyzed. 【Result】The dry matter of intercropped maize in all the 3 intercropping systems in jointing and grain filling stages was increased by 16.58%-20.32% and 51.29%-52.56%, respectively, compared with that of the sole maize system. The effect of intercropping on the dry matter accumulation of soybean in the branching and grain filling stages was not significant, however the dry matter of soybean in the MS2:2 intercrop in the branching stage was significantly lower than that of sole stands. The dry matter allocation ratio of maize leaf in jointing stage was greater than that of stem, and the ratio of maize ear in filling stage was greater than that of maize stem and leaf. The allocation ratio of intercropped maize ear was 23.22%-31.70% higher than that of sole maize. The dry matter allocation ratio of soybean stem in branching stage was greater than leaf, and the ratio of soybean stem and leaf in grain filling stage was greater than that pod. The allocation ratios of intercropped soybean pod in MS2:2 and MS 4:4 were 19.30% and 17.22% lower than that of soled soybean, while the ratio under MS6:6 was only 6.1% (not significant) lower than that of soled soybean. Maize had a stronger interspecific competitiveness (A_MS>0) and yield nutrition competition ratio (CR_MS>1) than soybean in intercropping systems. MS6:6 and MS4:4 intercropping systems had significant land use advantages and the land equivalent ratio (LER) were 1.16 and 1.07, respectively, indicating that intercropping increased the land productivity by 7%-16%. MS2:2 intercropping system didn’t have a significant land use advantages, and the LER was 0.97.【Conclusion】The maize-soybean intercropping systems affected crop yield and land productivity by changing dry matter accumulation and allocation ratios and interspecific competition. Intercropping systems with wider strip width had a more significant yield advantage. MS6:6 intercropping systems performed the best, which significantly improved the land productivity and might be an option of cropping system in maintaining regional agricultural sustainability.

Research Progress and Development Prospect of Potato Growth Model

JianZhao TANG,Jing WANG,DengPan XIAO,XueBiao PAN

Scientia Agricultura Sinica. 2021, 54(5):  921-932.  doi:10.3864/j.issn.0578-1752.2021.05.005

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Potato (Solanum tuberosum L.) is the fourth food crop around the world, following maize, wheat and rice. Potato production is of significance for ensuring national food security. Potato growth models have been used widely in narrowing potato yield gap, enhancing water and nitrogen use efficiency, adapting to climate change, and optimizing agronomic management options. The study reviewed the development, application and prospect of potato growth models. Potato growth models were built since the 1980s by expressing the growth and development processes of potato in the mathematical models, which mainly included the modules of phenology and biomass accumulation. During this period, the potato growth models were mainly used to evaluate potato productivity. During the 1990s, the potato growth models could be used to optimize application of irrigation and nitrogen fertilizer with the soil water and nitrogen modules being added into the models. In the 2000s, the potato growth models were improved significantly by including the module of the response of potato growth to CO₂ concentration. Afterward, the potato models were used more widely in the estimation of potato potential yield, narrowing the yield gap, the optimization of cultivars and agronomic management practices, climate change impact assessment and adaptation, etc. Although the potato growth models have been used widely, there were still some limitations for potato growth models. Firstly, the simulation accuracy of potato growth models under water and nitrogen stresses should be further evaluated. Secondly, the potato growth models could not simulate the number and weight of tuber effectively. However, the number of marketable tuber had a significant impact on potato economic benefit. Thirdly, the current potato growth models could not reflect the impacts of diseases and pests on the growth and development of potato. In the future, the mechanism of potato growth models should be further enhanced based on the global big data, field and controlled experiments. Moreover, the application of potato growth models should be strengthened in the analysis of interaction of genotype, management and environment on potato production. For developing new generation models, potato growth models should be combined with remote sensing data and structure-functional models to realize the smart management of potato production.

Smart-Phone Application in Situ Grassland Biomass Estimation

HaiYu TAO,AiWu ZHANG,HaiYang PANG,XiaoYan KANG

Scientia Agricultura Sinica. 2021, 54(5):  933-944.  doi:10.3864/j.issn.0578-1752.2021.05.006

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【Objective】Biomass is the material and energy basis of grassland ecosystem and the most basic ecological parameter. In the past, the quantitative grassland biomass retrieval based on aerospace and aerial remote sensing was too specialized to be popularized among herders. Therefore, this paper proposed a method for estimating grassland biomass by using true color images taken on the phone near the ground, and constructing a grassland biomass estimation model, which provided a theoretical basis and technical support for herders to easily, quickly and non-destructively grasp the growth of grassland in their own pasture. 【Method】 Firstly, the feature sets of grassland biomass estimation were constructed based on vegetation index, texture features and combined vegetation index and texture features by using the ultra-high resolution true color images of mobile phones. Secondly, in order to prevent dimensional disaster caused by excessive feature extraction, this paper proposed a feature selection algorithm (XGB-SFS) that combined XGBoost and sequence forward selection to perform feature selection and optimal subset construction. Finally, random forest regression and leave-one-out cross-validation were used to compare the biomass estimation effects of different feature sets to build models, and analyze the role of different types of features and XGB-SFS algorithm in grassland AGB estimation.【Result】 (1) Compared with the model constructed by single-type features, the estimation model based on spatial texture features (R² = 0.76) was better than the estimation model based on spectral vegetation index (R² = 0.73), indicating that texture features had a certain role in the ultra-high-resolution grassland AGB estimation; (2) Compared with the model after feature selection, the combined spatial spectrum multi-type feature construction model was superior to any single-type feature model (R² = 0.83, RMSE = 127.57 g·m ^-2, MAE= 81.25 g·m ^-2), indicating that multi-type feature construction model could improve the accuracy of grassland AGB estimation to a certain extent. (3) Comparing the models building before and after feature selection, the model after feature selection by estimating the AGB effect was significantly better than the model without feature selection, and there was a high correlation between the selected features and grassland biomass, indicating that XGB-SFS could reduce the data dimension and improve the accuracy of grassland AGB estimation.【Conclusion】The ultra-high-resolution true color images of mobile phones could accurately estimate the grassland biomass. The XGB-SFS algorithm proposed in this paper could also select the features with high correlation with the grassland biomass from many features and improve the model estimation accuracy. Compared with the previous professional remote sensing quantitative inversion of grassland biomass, this method had the advantages of facing the public, low cost, and easy to use. The study combined the data collected on the phone with remote sensing and machine learning methods, which could open up new perspectives and support the development of agricultural informatization.

PLANT PROTECTION

Ligands Binding Characteristics of Chemosensory Protein CsasCSP16 of Carposina sasakii

XiaoHe LIU,GuiSheng QIU,ZhaoGuo TONG,HuaiJiang ZHANG,WenTao YAN,Qiang YUE,LiNa SUN

Scientia Agricultura Sinica. 2021, 54(5):  945-958.  doi:10.3864/j.issn.0578-1752.2021.05.007

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【Objective】Carposina sasakii is one of the most destructive pests on orchards in north China. The objectives of this study are to get the recombinant CsasCSP16 protein, characterize the binding profiles of CsasCSP16 with some small molecular volatiles from host plants, and predict the key residue between CsasCSP16 and putative ligands. In overall this research possibly lays a theoretical and practical foundation for the understanding of olfactory mechanism in C. sasakii.【Method】The recombinant protein CsasCSP16 was induced to express by constructing prokaryotic expression system, and purified by using the Ni-NTA agarose affinity column. Then, the fluorescence competitive assay was applied, and N-phenyl-1-naphthylamine (1-NPN) was selected as the fluorescence probe to measure the binding profiles of CsasCSP16 recombinant with 33 host plant volatiles and 2 sex pheromone compounds. The Modeller software was used to build three-dimensional model. CSPMbraA6 (PDB ID: 1N8U) was used as the template for homology modeling of CsasCSP16 protein structure. The modeled structure of CsasCSP16 was docked with 35 selected ligands by Autodock Vina. Then, the complexes from above step were introduced to GROMACS (2019.3) to conform the stability. Moreover, the binding energies of the complexes were calculated by g_mmpbsa and the key residues interaction between CsasCSP16 and host volatiles were predicted.【Result】The recombinant expression vector was successfully constructed, and the recombinant protein of CsasCSP16 with high purity was obtained by bacterial system. Further, competitive fluorescence binding assays with 35 candidate ligands showed that CsasCSP16 had high binding affinities against methyl salicylate, 6-methyl-5-hepten-2-one, pentadecane, butyl heptanoate and α-pinene, and the recorded Ki values were 6.59, 6.25, 3.50, 6.73 and 4.47 μmol·L^-1, respectively. Ligand docking results revealed that the Vina Scores of CsasCSP16-methyl salicylate, CsasCSP16-6-methyl-5-hepten-2-one, CsasCSP16-pentadecane, CsasCSP16-butyl heptanoate and CsasCSP16-α-pinene were -6.1, -5.3, -5.8, -5.2 and -6.6, respectively. Finally, the g_mmpbsa analysis demonstrated that the binding energies of CsasCSP16-methyl salicylate, CsasCSP16-6-methyl-5- hepten-2-one, CsasCSP16-pentadecane and CsasCSP16-butyl heptanoate were -50.264, -65.551, -136.035 and -93.805 kJ·mol^-1, respectively. Ile49, Val71, Ile72 and Tyr90 contribute energy more than 3 kJ·mol^-1, suggesting their key involvement in the function of CsasCSP16. 【Conclusion】The CsasCSP16 has a certain binding capacity with several odors from host plant volatiles, suggesting that it may play an important role in the localization of host plants. Ile49, Val71, Ile72, and Tyr90 may be the key residues involved in the function of CsasCSP16.

Comparison of Life Tables for Experimental Populations of Individual- Rearing and Group-Rearing Frankliniella occidentalis

XinHua LI,DengJie WANG,ZhongRen LEI,HaiHong WANG

Scientia Agricultura Sinica. 2021, 54(5):  959-968.  doi:10.3864/j.issn.0578-1752.2021.05.008

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【Objective】The method of individual-rearing (IR) was often used to study the life table of western flower thrip (Frankliniella occidentalis), and the results can be used to predict the population occurrence under natural conditions in the field. However, F. occidentalis often occur in groups rather than single head under natural conditions. The objective of this study to compare the life table parameters of individual-rearing and group-rearing (GR) F. occidentalis, and to explore the accuracy of population occurrence dynamics under natural conditions based on the data from individual-rearing and group-rearing.【Method】The age-stage, two-sex life table of the experimental population of F. occidentalis reared on bean pod was constructed by individual-rearing and group-rearing, respectively, and the life history and population parameters of F. occidentalis were compared under the two conditions. The means and standard errors of population growth parameters were calculated using the bootstrap method. The Mann-Whitney test (U test) was used to evaluate the differences in the population parameter, development period, and fecundity of individual-rearing and group-rearing F. occidentalis.【Result】Individual-rearing and group-rearing had significant effects on nymph stage, pupal stage, male longevity, total preoviposition period, per female oviposition, pupal weigh and adult body length, but not on egg stage, adult stage, adult preoviposition period, female longevity, pupal length, pupal width, adult width. The nymph stage (4.49 d), pupal stage (4.03 d), male longevity (22.82 d), total preoviposition period (11.37 d) of individual-rearing F. occidentalis were significantly longer than group-rearing ones (3.05, 3.32, 18.64 and 10.00 d, respectively). The pupal weigh (0.03 mg) of individual-rearing F. occidentalis was significantly lower than that of group-rearing F. occidentalis (0.07 mg). The adult body lengths of individual-rearing F. occidentalis (female: 203.72 μm, male: 149.74 μm) were significantly lower than those of group-rearing F. occidentalis (female: 288.81 μm, male: 203.39 μm). The per female oviposition of individual-rearing F. occidentalis (48) was significantly lower than that of group-rearing F. occidentalis (133.39). The intrinsic rate of increase (r), finite rate of increase (λ), net reproductive rate (R₀), gross reproductive rate (GRR) and mean generation time (T) of individual-rearing F. occidentalis were 0.161 d^-1, 1.175 d^-1, 20.730, 35.699, 18.70 d, respectively, while those of group-rearing F. occidentalis were 0.242 d^-1, 1.274 d^-1, 60.499, 102.342, 16.88 d, respectively. The population growth of individual-rearing F. occidentalis was slower than that of group-rearing F. occidentalis.【Conclusion】Compared with the individual-rearing F. occidentalis, the population of group-rearing F. occidentalis grew faster and produced more offspring per unit time. Using the individual-rearing feeding method to establish a life table to predict the population dynamics of F. occidentalis may delay the best control time. The life table established by the group-rearing method should predict the population dynamics more accurately.

Spread and Resistance Level of Aegilops tauschii to Mesosulfuron- Methyl in Winter Wheat Field of Shandong Province

XingXiang GAO,Jian LI,Shuai ZHANG,YueLi ZHANG,Feng FANG,Mei LI,LianYang BAI,ShuangYing ZHANG

Scientia Agricultura Sinica. 2021, 54(5):  969-979.  doi:10.3864/j.issn.0578-1752.2021.05.009

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【Background】Aegilops tauschii, a malignant weed, is a wild relative plant of wheat, which is one of the 10 most malignant weeds recognized in the world. At present, only the ALS inhibitor herbicide mesosulfuron-methyl can be used as treatment agent for post-seedling. The effect of mesosulfuron-methyl was decreased in some areas after years of application in wheat fields, which may be related to resistance. 【Objective】The objective of this study is to clarify the spread law and resistance level of A. tauschii to mesosulfuron-methyl in Shandong Province, and to provide a theoretical basis for the establishment of the precise regional control of A. tauschii in winter wheat field of Shandong Province. 【Method】The distribution and spread of A. tauschii in winter wheat field of Shandong Province was investigated three times in the decade of 2009-2019, and whole-plant dose response experiments were conducted to determine the resistance level of 62 A. tauschii populations collected in Shandong Province to mesosulfuron-methy in glasshouses.【Result】The results on the spread of A. tauschii showed that the distribution area was gradually expanding and the harm degree was more and more serious. In 2009 to 2010, A. tauschii mainly distributed in Shandong northwest plain region, middle mountain region and northern coastal region, with a total relative abundance 3.18; in 2013 to 2014, A. tauschii had been widely distributed in the northwest plain and other 4 regions, with a total relative abundance 7.33; in 2018 to 2019, this weed occurred in a large area throughout Shandong Province, with a total relative abundance 11.38. The results on resistance level showed that 20 populations had a certain degree of resistance to mesosulfuron-methyl, which distributed in Shandong northwest plain region, southwest plain region and middle mountain region, and no resistance population was found in other areas. The low resistance and middle resistance populations were 16 (RI ranged from 5.00 to 10.00) and 4 (RI ranged from 10.00 to 15.00), which accounted for 25.81% and 6.45% of the total population, respectively. The maximum RI was 12.63, and no high resistance population was found. Target site mutations were detected in 4 middle resistance populations, and no site mutation was detected.【Conclusion】The A. tauschii has been distributed all over Shandong Province, and its dominance and harmfulness are increasing year by year. Although there is no high resistance population to mesosulfuron-methyl, but there are 32.26% low or middle resistance populations. No site mutation was found in the target mechanism test. In view of the winter wheat field dominated by A. tauschii, it is not only necessary to rely on mesosulfuron-methyl for control, but also to promote the comprehensive control of A. tauschii combined with deep ploughing and other agricultural measures. The soil-treated herbicide is also a good way to reduce weed resistance.

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

Effects of Straw Addition on Soil Biological N₂-Fixation Rate and Diazotroph Community Properties

Xu LI,WeiLing DONG,ALin SONG,YanLing LI,YuQiu LU,EnZhao WANG,XiongDuo LIU,Meng WANG,FenLiang FAN

Scientia Agricultura Sinica. 2021, 54(5):  980-991.  doi:10.3864/j.issn.0578-1752.2021.05.010

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【Objective】The purpose of this study was to analyze the effects of different straw additions on soil N₂-fixation rate and diazotroph community structure, which was crucial for the management of crop residue and mineral fertilizer application in China.【Method】Using indoor incubation experiment, in addition to the control (C0: 0), a total of 5 straw addition gradients (C1: 0.2 mg·g^-1; C2: 1.0 mg·g^-1; C3: 2.0 mg·g^-1; C4: 4.0 mg·g^-1; C5: 10.0 mg·g^-1), using the ¹⁵N₂ labeling method, and soil samples were collected after 28 days of incubation in dark conditions. As a direct measure of N₂-fixation,¹⁵N₂ gas labeling method could quantify the rate of biological N₂-fixation, and destined for characterization of nifH gene marker and diazotroph community by using the Illumina PE250 sequencing and PCR techniques. 【Result】With the increase of straw addition, the content of NO₃^--N in soil decreased significantly, while the content of NH₄⁺-N did not change significantly. The pH of soil decreased and the rate of biological N₂-fixation increased significantly. C3, C4 and C5 treatments could reach 2.71-3.05 μg N·g^-1DW during the incubation period (28 d), which was about 87-96 kg N·hm^-2·a^-1, compared with C0, the rate of biological N₂-fixation increased by 38.1%-52.4%. The copy number of nifH gene ranged from 5.48×10⁷ to 9.20×10⁷ copies/(g soil) under all treatments. Compared with C0, the copy number of nifH gene was significantly increased under C4 and C5 treatments, and the number and activity of diazotroph were significantly increased (P<0.05). However, when the amount of straw added reached 4.0 mg·g ^-1, increasing the amount of straw had no significant effect on the copy number of nifH gene of soil diazotroph. With the increase of straw addition, Shannon-Weill index of C4 and C5 levels was significantly lower than that of the other four treatments (P<0.05), and the diversity of the other four treatments had no significant difference. The result of PCoA showed that diazotroph community structure was clustered into different groups depending upon the difference of straw addition. Diazotroph were divided into Proteobacteria, Cyanobacteria, Firmicutes, Actinobacteria and Spirochaetes at the phylum level. As the amount of straw added increases, the relative abundance of Cyanobacteria tends to increase first and then decrease. At the genus level, there were significant differences in the number of carbon sources among different species. Bradyrhizobium was the most abundant genus. With the increase of straw addition, the relative abundance of C5 was significantly different from that of C0, C1, C2 and C3 (P<0.05), increasing by 12.07%-14.13%. The relative abundance of Scytonema hofmanni, which was positively correlated with the rate of biological N₂-fixation, gradually increases with the increase of straw addition, but decreased when straw addition reaches C5 level (P<0.05). Meanwhile, PLS-PM analysis, RDA and correlation analysis revealed that the rate of biological N₂-fixation and soil diazotroph community were greatly affected by straw addition and soil NO₃^--N concentration. 【Conclusion】The rate of soil biological N₂-fixation increases with the amount of straw added. The effect of straw on N₂-fixation was mainly due to the reduction of NO₃^--N content in the soil by straw addition, which in turn promoted the diazotroph such as Bradyrhizobium, Scytonema hofmanni and Azospirillum grow. According to the calculation results of this experiment, compared with no straw, after the straw was added (4.0 mg·g ^-1), the rate of biological N₂-fixation could be increased by 26 kg N·hm^-2·a^-1, which significantly reduced the fertilizer demand.

Effects of Irrigation Regimes on N₂O and NO Emissions from Greenhouse Soil

LiYuan ZHANG,JinDong LÜ,XinYue SHI,Na YU,HongTao ZOU,YuLing ZHANG,YuLong ZHANG

Scientia Agricultura Sinica. 2021, 54(5):  992-1002.  doi:10.3864/j.issn.0578-1752.2021.05.011

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【Objective】 Reasonable irrigation in greenhouse is an effective measure to control N₂O and NO emissions and to improve utilization rate of nitrogen fertilizer. In order to provide a scientific basis for N₂O and NO emission reduction and soil irrigation management in greenhouse, the dynamics of soil N₂O and NO emissions under different irrigation regimes and its relationship with soil moisture and inorganic nitrogen and soluble organic nitrogen were studied, and the N₂O and NO emissions characteristics and its influencing factors were also analyzed.【Method】A seven-year long term field experiment was conducted in greenhouse, tomatoes were used as the experimental crop, and four irrigation regimes were conducted to control lower irrigation limits of 25 kPa (W₁), 35 kPa (W₂), 45 kPa (W₃) and 55 kPa (W₄), respectively. The N₂O and NO emission were monitored in-situ simultaneously by using closed static chamber-gas chromatography and NO_x analyzer, respectively. 【Result】 The soil N₂O and NO emission fluxes of different irrigation regimes varied between -34.46-1 671.78 μg N·m^-2·h^-1and 6.83-269.89 μg N·m^-2·h^-1 in tomato growing season, respectively. The peak periods of N₂O and NO emissions were synchronous and mainly occurred after fertilization and irrigation, and NO/N₂O was less than 1 for each treatment. The cumulative soil N₂O and NO emissions were the lowest under W₂ and W₁ treatments (P <0.01) respectively, and the total N₂O+NO emissions for each treatment were W₄ >W₃ >W₁ >W₂. Compared with W₁, W₃ and W₄ treatments, tomato yields under W₂ treatment was increased by 84%, 32.4% and 12%, respectively. The yield-scaled N₂O+NO emissions was the highest under W₄ treatment and the lowest under W₂ treatment (P <0.01). Repeated measurements anova of soil inorganic nitrogen and soluble organic nitrogen after fertilizations and harvest showed that except for the interaction of irrigation regime and measure time had no significant effect on the nitrite content, irrigation regimes, measure time and their interaction had a significant effect on soil inorganic nitrogen and soluble organic nitrogen (P <0.01). Redundancy and correlation analysis indicated that NO₂^--N, NH₄⁺-N and WFPS could explain 55%, 32.5% and 20.7% variations of N₂O and NO for greenhouse soil, which were the main influencing factors that affected N₂O and NO emissions very significantly under different irrigation regimes.【Conclusion】Comprehensive consideration of yield, N₂O and NO emission reduction effect, W₂ treatment with irrigation lower limit of 35 kPa was the most appropriate irrigation management measurement for this experiment condition.

Regenerative Agriculture-Sustainable Agriculture Based on the Conservational Land Use

MingHui HAN,BaoGuo LI,Dan ZHANG,Ying LI

Scientia Agricultura Sinica. 2021, 54(5):  1003-1016.  doi:10.3864/j.issn.0578-1752.2021.05.012

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With the intensified global ecological crisis and the increasing demand for food and food safety, it has become a widespread concern of the whole world as to how to realize the sustainable development of agriculture. In this context, the regenerative agriculture was proposed and developed to cope with new challenges. This paper discussed the content, ecological and economic benefits of regenerative agriculture and analyzed the application scenarios in China, aiming to provide theoretical guidance for the introduction and development of this new agricultural model in China. To sum up, regenerative agriculture is a nature-based solution which has at its core the intention to improve the health of soil through elevated soil organic content and nitrogen elements as well as reduced chemicals input and mechanical operations, so as to realize the sustainable development of agriculture, namely enhanced soil carbon sequestration, maximized biodiversity and improved quality of water, vegetation and land-productivity. Compared to several other agriculture models with respect to multiple dimensions, regenerative agriculture stands out by simultaneously achieving improvements in food security, soil health, ecological environment and economic benefits. The advantages of applying regenerative agriculture were further proved with specific cases. At last, suggestions on the development of regenerative agriculture in China were put forward.

HORTICULTURE

Response of Chloroplast Ultrastructure and Photosynthetic Physiology of Two Tomato Varieties to Low Light Stress

XianMin MENG,YanHai JI,WangWang SUN,ZhanHui WU,ZhaoSheng CHU,MingChi LIU

Scientia Agricultura Sinica. 2021, 54(5):  1017-1028.  doi:10.3864/j.issn.0578-1752.2021.05.013

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【Objective】 The chloroplast ultrastructure and photosynthetic physiological characteristics of two tomato cultivars under low light were studied to reveal the difference of the response of different tomato cultivars to low light and explore the potential of Solanum lycopersicum tolerance to low light. 【Method】 The Dutch low-light tolerance cultivar Glorioso and the Chinese low-light sensitive cultivar Jingdan No. 1 were selected as experimental materials, they exposed to normal light (CK, light intensity 300-350 μmol·m ^-2·s^-1) and 50% normal light (low light, light intensity 70-80 μmol·m ^-2·s^-1) for fifteen days. The plant chlorophyll fluorescence imaging, chlorophyll fluorescence and photosynthetic parameters of leaves were detected. We also observed stomatal morphology and chloroplast ultrastructure of leaves after treatment by the scanning electron microscopy (SEM) and transmission electron microscopy (TEM). 【Result】 Compared with the control, the P_n, q_P__Lss, the content of chl. (a+b), SOD activity, stomatal density and spatial scale of regular stomatal distribution of the two cultivars leaves were decreased, which also led to the decrease of dry weight and healthy index, the leaves NPQ__Lss, MDA content increased with plant height and maximum internode spacing. The variation range of Jingdan No. 1 was significantly larger than Glorioso, the QY__Lss, QY (ΦPSⅡ) and chloroplast structure of Glorioso remained unchanged, its P_n in leaves was significantly higher than Jingdan No. 1. In addition, the Glorioso improved the regular distribution of stomata by reducing the value of L(d) between stomata, chl.b content increased and chl.a/b decreased under low light. While the chl.b content of Jingdan No. 1 decreased, chl.a/b increased, the spatial scale and regularity of stomatal regular distribution reduced and the leaves were seriously affected by low light. To sum up, Glorioso has stronger light-harvesting ability than Jingdan No. 1 under low light, and uses more light energy absorbed by photosynthetic pigments for photochemistry transfer, reduced heat dissipation and improves the actual photochemistry rate, light energy conversion rate of PSII, so as to maintain operation of photosynthetic system under low light, which photosynthetic capacity and output are slightly higher than Jingdan No. 1, and has strong low light tolerance.【Conclusion】 The differences of response to low light between the two tomato cultivars were mainly reflected in chl.b content, stomatal spatial distribution pattern, chloroplast structure, SOD activity and photosynthetic fluorescence characteristics, which made the Dutch cultivar Glorioso maintain high photosynthesis efficiency under low light.

Physiological Response of Mitochondrial Function of Strawberry Roots to Exogenous Phenolic Acid

XiaoFeng LU,GuoDong DU,Jing SHAO,JingRu ZHANG,HaiLong SUN

Scientia Agricultura Sinica. 2021, 54(5):  1029-1042.  doi:10.3864/j.issn.0578-1752.2021.05.014

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【Objective】Continuous cropping obstacle is a serious problem in the facility production of strawberry (Fragaria × ananassa Duch.). Phenolic acid, a secretion of strawberry roots, is one of the main factors causing continuous cropping obstacle. The aim of this study was to explore the physiological response by which exogenous phenolic acids affected root mitochondrial function in strawberry roots under the effects of phenolic acids accumulated in the rhizosphere of long-term continuous cropping soil. The results were used to discuss the potential responding mechanism of mitochondria in strawberry roots to allelopathy effect mediating by phenolic acid, which would lay the foundation for the further study of continuous cropping obstacle of strawberry.【Method】In this study, strawberries were used as the test material, and syringic acid, phthalic acid and their mixed solution of 27.0 μg·g^-1 were exogenously applied. Scanning Electron Microscope (SEM), Histochemical Staining, Liquid-Phase Oxygen Measurement and other techniques were applied to observe the changes of ROS content in root system, to measure the changes of mitochondrial oxidative phosphorylation level, respiratory pathway and the activities of key enzymes in respiration, to study the effect of autotoxic phenolic acid-induced reactive oxygen species on mitochondrial function in root systems, and to explore the effects of root systems’ mitochondria on physiological functions, such as respiratory complex metabolism and energy metabolism. 【Result】Applying two exogenous phenolic acids or mixed solution treatment could significantly reduce the strawberry root vigor, affect the total root length, total surface area, total root volume and average diameter, and inhibit the occurrence and growth of roots, with the degree of changes: syringic acid treatment (D)>two solution mixing treatment (D+L)>phthalic acid treatment (L)>CK. The activity of H⁺-ATPase decreased significantly in the early stage with the phenolic acid stress time, and the decline amplitude was the largest at the third day. Compared with CK, D, L and D + L were decreased by 38.8%, 28.8% and 33.7%, respectively, which hindered the ion transport capacity of root cells and became stable in the later stage. The activities of K⁺Na⁺-ATPase and Ca²⁺Mg²⁺-ATPase in roots were also decreased, and the changes were similar. Exogenous phenolic acid could reduce SOD, POD, CAT and APX activity, increase the contents of H₂O₂ and $ \mathrm{O}_{2}^{\mathop{}_{.}^{-}}$, and also increase MPT in root mitochondria, and reduce Δψm and cytochrome Cyt c/a content in root mitochondrial inner membrane. Although the root respiratory pathway was still mainly the EMP-TCA pathway, the contribution rate was significantly reduced, while the PPP pathway showed an obvious increasing trend. In the late stage of applying exogenous phenolic acids, with the extension of stress time, all respiratory key enzyme activities were inhibited, which led to the obstruction of respiratory pathways, with the degree of changes: D>D+L>L>CK. 【Conclusion】 Exogenous phenolic acid treatment could reduce root vigor and hinder the operational capacity of ions inside and outside cells. The treatment could also reduce root antioxidant enzyme activity and root mitochondrial function, leading to the reduction of key enzyme activities in every respiratory pathways, and ultimately obstruct every respiratory pathways.

Metabolic Analysis of Aroma Components in Two Interspecific Hybrids from the Cross of F.ananassa Duch. and Fragaria nilgerrensis Schlecht.

AiHua WANG,HongYe MA,RongFei LI,ShiPin YANG,Rong QIAO,PeiLin ZHONG

Scientia Agricultura Sinica. 2021, 54(5):  1043-1054.  doi:10.3864/j.issn.0578-1752.2021.05.015

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【Objective】 Two interspecific hybrids PF (with honey peach aroma) and NF (without peach aroma) were obtained from the cross of Fragaria ananassa Duch. and F. nilgerrensis Schlecht. Fruit aroma compounds in PF and NF were compared to clarify the composition and content of honey peach aroma, aiming to provide a theoretical basis for research on strawberry aroma and utilization of wild strawberry resources. 【Method】 Matured fruits of PF and NF were harvested from greenhouse in February 18, 2018, respectively. The fruit traits comparison between two interspecific hybrids PF and NF were measured as Descriptors and Data Standard for Strawberry (Fragaria spp.). Fruit aroma compounds were extracted by using head solid-phase microextraction (HS-SPME), and then, detected by gas chromatograph tandem mass spectrometer technology (GC-MS). The mass spectra of the detected compounds were matched with NIST Library and also subjected to artificial qualitative analysis based on literatures. Multivariate statistics including principal component analysis (PCA) and supervised partial least squares-discriminant analysis (PLS-DA) were conducted to screen significantly differential metabolites. Variable Importance in the Projection (VIP) >1.0, log2FC >1.0 or log2FC<-1.0, and P value<0.05, indicates an increase and decrease by over 2-fold, respectively, in a comparison between PF and NF. The relative content of each significantly different metabolite (expressed as percentage) was calculated as the ratio between each peak area and the sum of all significantly different metabolite peak areas, multiplied by 100 [Relative Content. = (Areapeak/ΣAreaspeak) ×100]. Chemical Abstracts Service Registry Number (CAS#) was retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov). 【Result】 Fruits traits of PF was consistent with those of NF, except honey peach aroma. The fruits of both PF and NF were almost the same size, with red fruit color, small fruit cavity, soft texture and sweet and sour taste. Totally, 383 kinds of aroma compound were identified from the test samples by GC-MS, including 141esters, 41 alcohols, 40 ketones, 36 alkanes, 22 aldehydes, 17 olefins, 17 acids, 13 lactones, 10 naphthenes, 7 ethers, 6 furans and 16 other compounds. The main components were esters, kinds of which accounting for 36.81% of the total metabolites, followed by alcohol, ketone and alkane, accounted for 10.70%, 10.44% and 9.40%, respectively. A total of 67 significantly different metabolites were screened from the 383 detected metabolites, 58 of which were up-regulated and 9 were down-regulated. PF significantly up-regulated lactones. The top three up-regulated lactones were 2H-Pyran-2-one, tetrahydro-6-(2-pentenyl)-, (Z)-, 2H-Pyran-2-one, 6-hexyltetrahydro-, and 2H-Pyran-2-one, tetrahydro-6-pentyl-. NF down-regulated esters significantly. The top three down-regulated esters were 2-propenoic acid, 3-phenyl-, ethyl ester, sulfurous acid, 2-ethylhexyl isohexyl ester and butanoic acid, 3-hydroxy-, ethyl ester. The relative content of esters in PF (37.69%) was significantly lower than that of NF (57.20%), conversely, lactones in PF (20.91%) significantly higher than that of NF (6.12%). The relative content of ketones in PF (15.30%) was slightly higher than that of NF (9.12%). The relative content of alcohols, aldehydes, acids, olefins and other metabolites were almost equally present in PF and NF. The ester with the highest relative content in NF was butanoic acid, ethyl ester (17.92%), and the lactone with the highest relative content in PF was 2H-Pyran-2-one, tetrahydro-6-pentyl-(12.53%). The ketone with the highest relative content in PF was the same as NF, both were 2-heptanone. 【Conclusion】 Esters, such as 2-propenoic acid, 3-phenyl-, ethyl ester, butanoic acid, ethyl ester and butanoic acid, 3-hydroxy-, ethyl ester, might be the key aroma components of NF. Lactones, such as 2H-Pyran-2-one, tetrahydro-6-(2-pentenyl)-, (Z)-, 2H-Pyran-2-one, 6-hexyltetrahydro-, 2H-Pyran-2-one, and tetrahydro-6-pentyl-, might be the key aroma components to form the honey peach aroma in PF.

FOOD SCIENCE AND ENGINEERING

Determination of 5 Nitropolycyclic Aromatic Hydrocarbons in Roasted Meat Products by High Performance Liquid Chromatography- Fluorescence Detection

Xue BAI,Teng HUI,ZhenYu WANG,YunGang CAO,DeQuan ZHANG

Scientia Agricultura Sinica. 2021, 54(5):  1055-1062.  doi:10.3864/j.issn.0578-1752.2021.05.016

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【Objective】 This study was aimed to establish an approach to determine the content of five NPAHs (including 1-nitronaphthalene, 2-nitrofluorene, 3-nitrofluoranthene, 1-nitropyrene and 6-nitrobenzo[a]pyrene) in roasted meat based on solid-phase extraction (SPE) column and high performance liquid chromatography with fluorescence detection (HPLC-FLD).【Method】 1 g vacuum freeze-dried roasted meat sample was extracted by dichloromethane for ultrasonic extraction, and the filtrate was collected. Both the extraction and filtration processes were repeated three times. Then, the filtrate was dried by nitrogen and dissolved in 3 mL n-hexane. After that, the extract was loaded onto PAHs SPE column which was previously activated by dichloromethane and n-hexane. After loading, the sample tube was rinsed by n-hexane and loaded as well. The SPE column was washed by n-hexane and eluted by dichloromethane. The eluent was dried by nitrogen, and the residue was dissolved in acetonitrile to obtain NPAHs solution. Acidified methanol and iron powder were added to the NPAHs solution and heated in water bath with magnetic stirring for 40 min. After centrifugation and filtration, 20 μL sample was tested by HPLC-FLD at 40℃ by using Agilent ZORBAX Eclipse PAH analysis column. In the gradient elution system, water and acetonitrile were used as the mobile phases.【Result】 The result showed that five NPAHs were linear with correlation coefficients greater than 0.9940. Specifically, the detection limits (LOD) were within 0.12-2.17 μg·kg^-1, and the quantification limits (LOQ) were within 0.38-7.23 μg·kg ^-1. The average recoveries and the precision were within 53.16%-129.64% and 1.91%-30.73%, respectively. Furthermore, five kinds of typical Chinese roasted meat products were tested by this method, and the content of five NPAHs in all roasted meat samples were between 50.19-82.36 μg·kg ^-1. Among them, the content of 6-nitrobenzo[a]pyrene was the highest, about 31.01-35.89 μg·kg^-1, followed by 3-nitrofluoranthene, and the content was within the range of 9.99-23.06 μg·kg ^-1. 【Conclusion】 SPE column combined with HPLC-FLD was a suitable method to analyze NPAHs in roasted meat products; and NPAHs were widely contained in Chinese roasted meat products.

ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT

Molecular Epidemiological Investigation of Porcine Group A Rotavirus in Sichuan from 2017 to 2019

Qun ZHOU,XiaoFei CHEN,RuiCi KAN,Yu LI,Hui CAO,YanLing PENG,Bin ZHANG

Scientia Agricultura Sinica. 2021, 54(5):  1063-1072.  doi:10.3864/j.issn.0578-1752.2021.05.017

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【Objective】The aim of this study was to investigate the prevalence and molecular characterization of porcine group A rotavirus (RVA) in large-scale pig farms in Sichuan, to provid a theoretical basis for the development for porcine RVA vaccine. 【Method】 From 2017 to 2019, the 303 samples were collected from 40 pig farms in 14 regions of Sichuan province. Prevalence of RVA was detected by real-time RT-PCR method, and RVA positive samples were typed by RT-PCR. At the same time, the whole genomes of RVA VP4 and VP7 gene were amplified from some positive samples. The genotypes of the corresponding strains were determined by RotaC^2.0 classification tool. Sequence homology was analyzed by MegAlign. Phylogenetic tree was constructed by Neighbor-Joining method through MEGA 7.0. SimPlot and RDP4 softwares were applied for recombination analysis. 【Result】 Of the 303 samples examined, 32.34% (98/303, 95%CI=27.1%-37.9%) were positive for RVA. Among the 39 G types, G9 (41%) was the dominant genotype, while G4, G5, G26, and G3 were detected in 23%, 28.2%, 5.1% and 2.7%, respectively. The P[13] genotype (40.7%) was dominant among the 59 P types, followed by P[6], P[23] and P[1] in 30.5%, 23.7% and 5.1%, respectively. Furthermore, the 30 strains successfully identified the G/P combination genotype, and the dominant combination genotype was G9P[23] (23.3%), the other combinations were G4P[6] (16.7%), G9P[13] (13.3%), G5P[23] (10%), G5P[13] (10%), G9P[6] (6.7%), G26P[13] (6.7%), G4P[13] (6.7%), G4P[23] (3.3%) and G3P[13] (3.3%). Notably, the G5P[23], G4P[13], G9P[6], G26P[13] and G4P[23] were first identified in China. In addition, the recombination analysis showed that four strains had recombination on VP7 or VP4 genes. 【Conclusion】 The results demonstrated that the prevalence of RVA in diarrhea piglet feces was high and the genotypes were complex in Sichuan. The dominant genotype of RVAs was G9P[23]. The results of this study enriched the epidemiological data of RVA and provided an important reference for the prevention and control of porcine RVA in Sichuan province.

Development of a TaqMan Real-Time PCR Targeting the MGF360-13L Gene of African Swine Fever Virus

Tao WANG,Yu HAN,Li PAN,Bing WANG,MaoWen SUN,Yi WANG,YuZi LUO,HuaJi QIU,Yuan SUN

Scientia Agricultura Sinica. 2021, 54(5):  1073-1080.  doi:10.3864/j.issn.0578-1752.2021.05.018

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【Objective】The objective of this study is to develop a TaqMan real-time PCR for detection of African swine fever virus (ASFV) MGF360-13L gene, providing technical support for diagnosis, virus purification, differential diagnosis of MGF360-13L deletion ASFV strains, and gene function research of ASFV.【Method】In this study, the TaqMan real-time PCR primers and probes were designed based on the ASFV MGF360-13L (GenBank: MK333180.1), and the TaqMan real-time quantitative PCR assay of ASFV was established. A pair of specific primers, 13L-F/13L-R was designed to construct ASFV standard plasmid. The standard curve was generated for quantitative analysis using the ten-fold dilution of the ASFV standard plasmid, and the sensitivity and repeatability of the system were also evaluated. The ten-fold dilution of the ASFV standard was also detected by PCR to determine the sensitivity of the method. Thirty clinical samples collected from an outbreak ASF pig farm in Heilongjiang province were simultaneously tested by this TaqMan real-time PCR, and another one previously established in our laboratory to compare the coincidence rate of these two detection methods. Differential diagnosis of parent ASFV and MGF gene deletion strain after infection with PAM cell【Result】A specific band of about 800 bp was amplified using primer 13L-F/13L-R, and no band was found in the negative control, and the standard plasmid was successfully constructed. The standard curve had a good linear relationship between real-time PCR cycle threshold (Ct) and template copies. The linear regression equation was: y = -3.295 lg(x) + 45.995 with correlation coefficient of 0.997, and the minimum detection limit was 15.6 copies/μL of ASFV nucleic acid. There was no cross-reaction with classical swine fever virus, pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine transmissible gastroenteritis virus, porcine circovirus type 1, and porcine circovirus type 2. In the clinical sample test, the results of the two methods in McNemar test, P = 0.5>0.05, indicating that there was no statistical difference between the two methods and in Kappa test, Kappa = 0.867>0.75, P<0.001, suggesting it was a good agreement and could effectively identify parent ASFV or MGF gene deletion ASFV infection. 【Conclusion】The TaqMan real-time PCR for detection of ASFV MGF360-13L established in this study had highly specific, sensitive, reproducible, and high coincidence rate. It not only enriched the detection method of ASFV, but also provided technical support for researching the gene function of MGF360-13L, identification of the MGF360-13L gene-deleted ASFV, and differential diagnosis of related gene deletion vaccine strains.