Table of Content

01 November 2022, Volume 55 Issue 21

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Genome-Wide Association Study of Cold Tolerance at the Germination Stage of Rice

PANG HongBo, CHENG Lu, YU MingLan, CHEN Qiang, LI YueYing, WU LongKun, WANG Ze, PAN XiaoWu, ZHENG XiaoMing

Scientia Agricultura Sinica. 2022, 55(21):  4091-4103.  doi:10.3864/j.issn.0578-1752.2022.21.001

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【Objective】Rice is an important food crop, and its growth and development are most vulnerable at the germination stage. Under cold stress, direct-seeded rice exhibited significantly reduced germination rates (GRs) and yield compared with normally grown plants. Thus, a better understanding of genetic mechanisms regulating cold tolerance will enable to develop rice varieties with improved tolerance during germination. 【Method】238 representative rice germplasm resources from 14 countries worldwide were tested in phenotypic identification in Shenyang in 2021 and 2022; the low-temperature germination rate and relative low-temperature germination rate (LTGR and relative LTGR; 1-10 days under 15℃) were evaluated in an artificial climate incubator, and a 5-10 day LTGR histogram was constructed using R. The day suitable for GWAS was determined by phenotypic variation (Hill) and a mixed linear model combining LTGR and relative LTGR phenotype data with resequencing data. 【Result】LTGR histogram and phenotypic variation showed optimal GR on day 8 (Hill=0.84), i.e., it was higher than on other days (Hill=0.48-0.83), which could be used for GWAS. The principal component analysis results divided all germplasms into five groups—indica, aus, temperate japonica, tropical japonica, and aromatic. GWAS analysis of two indicators detected three identical significant single nucleotide polymorphisms (SNPs) related to cold tolerance in rice at the germination stage. These were located on chromosome 4, which could explain 11.9%-25.4% of the phenotype. In addition, 24 candidate genes were screened in the 50-kb region upstream and downstream of these three SNPs. Further linkage disequilibrium analysis and haplotype analysis were carried out and highly significant differences were found between different haplotypes of the LOC_Os04g24840 and LOC_Os04g25140 genes for cold tolerance. LOC_Os04g24840 was divided into five haplotypes by the coding region SNP, and Hap_3 was significantly more cold tolerant than Hap_1; LOC_Os04g25140 was divided into 18 haplotypes by the coding region SNP and the amino acid variation (S>L) at 77 bp was different in japonica and indica rice. These results showed that the genes encoding glycosyltransferases (LOC_Os04g24840) and F-box protein (LOC_Os04g25140) might be closely related to cold tolerance in rice.【Conclusion】 A total of three SNP loci were detected in 238 rice germplasm resources, and two candidate genes were screened for their association with cold tolerance during germination in rice.

Lipase Activity Difference of Oat Varieties and Prediction of Low Lipase Activity Variety with High Quality

XIANG YuTing, WANG XiaoLong, HU XinZhong, REN ChangZhong, GUO LaiChun, LI Lu

Scientia Agricultura Sinica. 2022, 55(21):  4104-4117.  doi:10.3864/j.issn.0578-1752.2022.21.002

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【Objective】This study explored the differences and causes of oat lipase activity of different varieties. Providing a theoretical basis for screening varieties with low lipase activity and achieving stable enzyme inactivation effect of oat products. 【Method】Six main varieties of three main oat planting regions were selected for the study, and their lipase activity, nutritional indexes, physical traits, and agronomic indexes were measured. To answer the differences in lipase activity of oat varieties, the indicators significantly related to oat lipase were screened by correlation analysis. Through cluster analysis, classified multiple oat samples by lipase activity. Transform data having correlations into composite variables for statistical analysis by principal component analysis. To derive a predictive model for lipase activity, an analytical method combining gray correlation and multiple stepwise regression was used. The indicators correlating with lipase activity were used as independent variables, and the lipase activity was used as dependent variables for quantitative model fitting. 【Result】Lipase activity was significantly positively correlated with crude fat content (r=0.32, P<0.05), and the various trends of fat content, unsaturated fatty acid content, lipase activity, and acid value were consistent. Lipase activity was significantly positively correlated with crude protein content (r=0.46, P<0.01), and the higher lipase activity was, the higher percentage of electrophoretic bands located in 31-43 kD were. It was significantly negatively correlated with grain test weight (r=-0.71, P<0.01) and positively associated with growth period (r=0.37, P<0.01). Baiyan 18 and Diyan 1 had low lipase activity and high nutrition varieties according to grey relational analysis, and the relevance value with ideal variety X₀ were 0.951 and 0.883, respectively. Stepwise regression analysis only retained the test weight and protein content as independent variables. The prediction model of lipase activity was established as Y=720.274-2.255×test weight (g·L^-1)+75.761× protein content (%), P<0.01, R²=0.658.【Conclusion】The varieties had significant effects on oat lipase activity. Protein content, fat content, test weight, and growth period were the main influencing factors of oat lipase activity. Grey relational analysis combined with stepwise regression analysis could be used to comprehensively evaluate oat varieties effectively and quickly select varieties with low lipase activity.

CROP GENETICS ＆ BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Evaluation of Resistance to Leaf Scald Disease in Different Sugarcane Genotypes

DU JinXia,LI YiSha,LI MeiLin,CHEN WenHan,ZHANG MuQing

Scientia Agricultura Sinica. 2022, 55(21):  4118-4130.  doi:10.3864/j.issn.0578-1752.2022.21.003

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【Objective】Sugarcane leaf scald disease is an important bacterial disease affecting sugarcane yield. Selection of disease-resistant genotypes can effectively reduce the incidence of this disease. This study aimed to explore the leaf-scald resistance of sugarcane genotypes, standardize resistance evaluation method, and provide a basis for the selection and utilization of germplasm resources of sugarcane. 【Method】Xanthomonas albilineans JG43 isolated from Guitang 46, was used as inoculum on 70 different sugarcane genotypes using the decapitation method by placing 500 mL of bacterial suspension on the surface previously cut above the apical meristem with scissors dipped in the inoculum suspension of 10⁸ CFU/mL. The disease incidence (IC) was calculated at 14, 28, 42, 56, and 70 days post-inoculation (Dpi). The disease index (DI) and the area under the disease progress curve (AUDPC) were calculated according to the disease severity of leaf scald in sugarcane. Variance, principal component, and discriminant analysis were performed using SPSS 25.0 software. Among them, a general linear model procedure (PROC) and the square sum model of type III were used to analyze the variance, with IC, DI and AUDPC as dependent variables, genotype, block and days post-inoculation as fixed factors. After the original data were processed by standardization (Z-score), principal component analysis was carried out by KOM and Bartlett sphere test. The Euclidean metric was calculated for cluster analysis using the WPGMA method of DPS 9.50 software. The discriminant analysis was performed to evaluate the clustering results according to Fisher’s criterion. 【Result】Some genotypes displayed white pencil lines at 14 dpi, then gradually expanded to the edge at 28 dpi. The leaves began yellowing or albinism from the edge to the veins at 42 dpi, then curled inward and died at 56 dpi. The severely infected plant withered and eventually died at 70 dpi. Variance analysis exhibited highly significant effects for IC, DI, and AUDPC among genotype (Gen), days post-inoculation (Dpi), and their interactions effect (Gen × Dpi) (P<0.01). Approximately 42% of the total sum of square was attributed to Dpi effect, indicating significant differences among genotypes resistance across the days post-inoculation. At 56 dpi, the disease reached a steady plateau, and the data in this period could be better divided among sugarcane genotypes. The results of discriminant and cluster analysis showed that 70 genotypes were divided into five different groups, including 15 highly resistant, 14 resistant, 15 moderate, 11 susceptible, and 15 highly susceptible genotypes. 【Conclusion】The resistance of sugarcane genotypes to leaf scald was assessed using the decapitation method, the IC, DI and AUDPC at 56 dpi were used as the evaluation indicators. The combined method of clustering and discriminant analysis could improve the accuracy of clustering results. Fifteen genotypes of high resistance to leaf scald were assessed and used for the sugarcane breeding program in China, including Zhongzhe 9, Zhongzhe 4, Zhongzhe 2, GUC19, GUC8, Yunrui 03-103, Yunrui 05-649, Yunrui 05-182, Yunrui 05-367, Yunrui 89-159, Funong 11601, Funong 09-4059, Guitang 02-467, Guitang 08-297, ROC22.

TILLAGE ＆ CULTIVATION·PHYSIOLOGY ＆ BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Coupling Effects of N-fertilizer Postponing Application and Intercropping on Maize Photosynthetic Physiological Characteristics

XU Ke,FAN ZhiLong,YIN Wen,ZHAO Cai,YU AiZhong,HU FaLong,CHAI Qiang

Scientia Agricultura Sinica. 2022, 55(21):  4131-4143.  doi:10.3864/j.issn.0578-1752.2022.21.004

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【Objective】The problem for film-mulched maize in the oasis irrigation region was an advanced nitrogen (N) requirement and led to the insufficient supply of N at the late growth stages. In this study, the effects of N-fertilizer postponing application on intercropped maize photosynthetic physiological characteristics and grain yield were studied, so as to reveal the photosynthetic mechanism of intercropped maize grain yield advantage in the experimental area. 【Method】From 2019 to 2021, the maize was used as experimental materials in Hexi oasis irrigation region. The split-plot experiment design was adopted, among which pea/maize intercropping and maize monoculture were the main factors, and three N fertilizer postponing application (postponing ration 20%, 10%, and traditional practice) were the secondary factors. Then, this field experiment was used to investigate the photosynthetic physiological characteristics and yield performance of maize under N-fertilizer postponing application and intercropping pattern. 【Result】The results demonstrated that the grain yield of intercropped maize under the postponing application of 20% N-fertilizer and 10% was increased by 28.5% and 13.8%, and biomass yield by 23.8% and 12.5%, respectively, compared with traditional N management practices. Similarly, compared with traditional N management practice, the grain yield of sole maize under the postponing application of 20% N-fertilizer and 10% was also increased by 29.7% and 13.3%, and biomass yield by 19.6% and 10.3%, respectively. Compared with the monoculture maize, intercropping could increase the grain yield by 33.2%-35.1% and biomass yield by 26.8%-31.5% under the same area. Furthermore, the postponing application of 20% N-fertilizer and 10% increased the population grain yield of intercropping pattern by 27.2% and 12.9%, respectively, compared with the traditional N management practice. The results showed that intercropping pattern could increase the grain yield of maize compared with the sole pattern, and the N fertilizer postponing application also boosted the improvement of grain yield in the intercropping system compared with the traditional N management practice. During the whole growth periods, the intercropping increased the net photosynthetic rate, stomatal conductance, transpiration rate, and decreased intercellular CO₂ concentration. Compared with traditional N management, the net photosynthetic rate under the postponing application of 20% N-fertilizer and 10% was increased by 12.8% and 6.0%, the stomatal conductance by 14.0% and 6.9%, and the transpiration rate by 20.5% and 9.5%, respectively, while the intercellular CO₂ concentration was decreased by 29.8% and 13.1%, respectively. The SPAD value under the postponing application of 20% N-fertilizer and 10% was increased by 7.5% and 3.7%, respectively. The principal component analysis results showed that the N-fertilizer postponing application and intercropping pattern could increase the grain yield via boosting the net photosynthetic rate, the stomatal conductance, the transpiration rate, and leaf SPAD value, and decreasing intercellular CO₂ concentration. 【Conclusion】N-fertilizer postponing application 20% treatment (36 kg·hm^-2 N fertilizer was topdressing at maize jointing stage and 108 kg·hm^-2 at 15 d post-silking stage) had the advantage of improving the photosynthetic characteristics of intercropped maize, thereby boosting the grain yield improvement.

Crop Classification with Time Series Remote Sensing Based on Bi-LSTM Model

HUANG Chong,HOU XiangJun

Scientia Agricultura Sinica. 2022, 55(21):  4144-4157.  doi:10.3864/j.issn.0578-1752.2022.21.005

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【Objective】Timely and accurate crop classification mapping is an important basis for agricultural situation monitoring. This study explores the potential of deep learning in time series remote sensing crop classification and early identification based on a bidirectional long short-term memory network model.【Method】In this paper, Yellow River Delta region was chosen as an example and a time-series NDVI dataset were constructed by using Sentinel-2 year-round available satellite images as the data source. A recurrent neural network architecture is used to build a bidirectional long short-term memory (Bi-LSTM) model for structured time-series remote sensing data to carry out crop classification, then the generalization ability of the model is evaluated. Through adjusting the length of time series, we explore the earliest identifiable time of different crops under the condition of satisfying certain mapping accuracy. 【Result】 Growth characteristics represented by time series remote sensing images have great potential to discriminate different crops. The overall accuracy of the Bi-LSTM model reached 90.9% with a Kappa coefficient of 0.892. By testing the effects of different time series lengths on crop classification, the earliest identifiable time of typical crops was obtained. The accuracy of crops such as winter-wheat and rice could improve significantly after the emergence of unique characteristics. Crops such as cotton and spring maize required complete growth sequences to ensure classification accuracy.【Conclusion】The structured feature information embedded in satellite image time series could effectively reduce crop spectral confusion at specific time periods. The Bi-LSTM model was able to consider both forward and backward temporal state information and could learn the spectral change characteristics of crops, which was excellent in the identification of confusing crops such as rice, cotton and spring maize. In addition, the deep learning model could effectively capture the variation trend on the sample in general, and showed better generalization ability and robustness in the crop multi-classification task. This study provided a feasible idea for regional crop mapping with high accuracy by integrating deep learning and remote sensing time series.

Development and Application of Rapid Investigation and Analysis Platform for Agricultural and Rural Information Based on Fission Model

ZHANG Jie,WANG Chuan,DONG XiaoXia,ZHU WenQi,YUE HuiLi,LIU ShengPing,ZHOU QingBo

Scientia Agricultura Sinica. 2022, 55(21):  4158-4174.  doi:10.3864/j.issn.0578-1752.2022.21.006

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【Objective】In order to effectively solve the problems existing in the process of collecting basic information of traditional agriculture and rural areas, such as long collection period, small sample size, inaccurate delivery, low data quality and difficulty in auditing, and to achieve rapid acquisition and visual analysis of massive data under the background of emergencies, a rapid survey platform for agricultural and rural information was designed and developed, which integrated accurate questionnaire delivery, rapid data collection and automatic analysis of results, so as to provide a convenient tool for agricultural management departments at all levels to make efficient decisions. 【Method】 (1) Overall design: The platform was designed based on the 5-layer architecture technology and the concept of “Mid-Plane”, and the new media, GIS and social network analysis (SNA) were used to build a questionnaire accurate delivery model, multi-level fission model, rapid review, and automatic analysis strategies. In order to deeply mine and present the value of platform data in multiple dimensions, this research used methods such as point centrality, betweenness centrality and spatial clustering to analyze the fission relationship, diffusion capacity, and community characteristics of the questionnaire dissemination network. (2) System realization: two subsystems were developed by adopting the front-end and back-end separation mode, namely, the rapid investigation collection system for survey users, and the rapid investigation management and analysis system for the information investigators. (3) Application: VMware vSphere integrated container and cluster server were used to deploy system and Nginx load balancing to improve system response speed. 【Result】 After six months of continuous operation, the system had strong universality. It has provided 12 large-scale agricultural and rural information survey services in 8 fields and collected a total of 76 953 questionnaires; In the agricultural and rural economic information survey cases 12 243 questionnaires have been collected within 5 hours, with a fission ratio of 88.23%, of which 20.24% formed diffusion and fission. The community effect was obvious, with high degree of fission, fast speed and wide range. The platform had strong comprehensive functions, covering the functions of questionnaire design, placement control, fission sharing, intelligent judgment of papers, reward distribution, statistical analysis, user classification, etc., which could provide one-stop service for rapid investigation of agricultural and rural information.【Conclusion】The rapid investigation and analysis platform of agricultural and rural information based on fission mode had the characteristics of fast data collection, true information acquisition, more sample collection, and strong functionality. It realized the function of obtaining basic information urgently and visualizing investigation results quickly under the background of emergencies, and formed a poll mode based on huge data and different from official investigation. It could provide strong technical support for efficient decision-making of agricultural management departments, and had reference significance for the information investigation of other industries.

PLANT PROTECTION

Pathotype Analysis of Xanthomonas oryzae pv. oryzae in Main Rice Producing Regions of China and Establishment of Differential Hosts of Near-Isogenic Lines

FENG AiQing,WANG CongYing,ZHANG MeiYing,CHEN Bing,FENG JinQi,CHEN KaiLing,WANG WenJuan,YANG JianYuan,SU Jing,ZENG LieXian,CHEN Shen,ZHU XiaoYuan

Scientia Agricultura Sinica. 2022, 55(21):  4175-4195.  doi:10.3864/j.issn.0578-1752.2022.21.007

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【Objective】The pathotypes of Xanthomonas oryzae pv. oryzae (Xoo) in diverse rice regions in China were analyzed, and the differential hosts of near-isogenic lines (NILs) were established to provide a scientific basis for accurate field monitoring of Xoo population, application of resistant varieties and breeding of resistant varieties against rice bacterial blight.【Method】To explore virulence diversity and distribution of pathotypes of Xoo, the pathotypes of 954 single colony strains collected from Guangdong, Guangxi, Hainan, Zhejiang, Hunan, Liaoning and Yunnan provinces (Autonomous Region) from 2018 to 2021 were identified by artificial leaf-cutting inoculation on 21 differential hosts including Chinese differential hosts, IR24 and 15 bacterial blight NILs. Based on the resistant and susceptible interactions among the tested Xoo strains, 15 NILs and IR24, the principal component analysis (PCA) was used to analyze the variable factors of which help abstraction of the candidate differentials. Based on the resistant and susceptible interactions between resistance genes and tested strains, the pyramiding effect of resistance genes was analyzed.【Result】The tested 954 Xoo strains were divided into 11 pathotypes including SRRRR (I), SSRRR (Ⅱ), SSSRR (Ⅲ), SSSSR (Ⅳ), SSRRS (Ⅴ), SRSRR (Ⅵ), SSSSS (Ⅸ), SSSRS (new pathotype 1), SRSRS (new pathotype 2), SRSSS (new pathotype 3) and SSRSS (new pathotype 4) based on Chinese differential hosts. The percentage of each pathotype mentioned above was 11.53%, 4.82%, 7.34%, 6.18%, 7.23%, 1.05%, 59.96%, 1.57%, 0.10%, 0.10% and 0.10%, respectively. Pathotype Ⅸ, with broad pathogenicity and more virulence, had become the predominant race in the South China rice region and the Yangtze River rice region (Hunan, Zhejiang), and the predominant pathotypes in Yunnan (Southwest China) and Liaoning (Northeast China) were pathotype Ⅳ and pathotype I, respectively. The resistance and susceptibility of 15 rice NILs to 954 Xoo strains were analyzed. The results showed that the 15 NILs could be divided into five types, highly susceptible lines with IRBB1, IRBB2, IRBB10, IRBB11, IRBB4, the moderately susceptible lines with IRBB3, IRBB203, IRBB14, moderately resistant lines with IRBB8, IRBB13, resistant lines with IRBB21, highly resistant lines with IRBB5, IRBB7, CBB23, GDBB23. Among the tested Xoo strains, 42 strains could infect xa5, 34 strains could infect Xa7, and 31 strains could infect Xa23. Factors were extracted from the interaction variable data matrix among 954 strains and 16 varieties (15 NILs and its recurrent parent IR24). With the total explained variable >85.0% as the boundary, eight principal components were extracted, and ten varieties (lines) mainly with NILs were constructed as differential hosts. Accord to their contribution to interaction variances, these differential hosts were IRBB10 (Xa10), IRBB4 (Xa4), GDBB23 (Xa23), IRBB5 (xa5), IRBB7 (Xa7), IRBB21 (Xa21), IR24 (Xa18), IRBB13 (xa13), IRBB3 (Xa3), Jingang30. With new differential hosts, 954 tested Xoo strains could be divided into 55 pathotypes, the newly developed differential hosts showed good discrimination ability for monitoring the virulence of Xoo in the tested rice regions. The results of gene pyramiding combined resistance analysis showed that the resistance frequency of different resistance genes pyramiding to Xoo was increased, and different resistance genes had diverse complementarity to the resistance of the test strains. 【Conclusion】The Xoo strains in the monitoring rice regions tended to be diversified, and the virulence differentiation was obvious. Pathotype Ⅸ of the virulent strain had become the prevailing races in some rice production regions, and the number of the strains compactible with broad-spectrum resistance genes xa5, Xa7 and Xa23 showed an increasing trend. Resistance gene polymerization can broaden the resistance spectrum of varieties to pathogen lines, which is an effective way to breed broad-spectrum resistant varieties. The establishment and application of NILs differential host could provide technical support for precise monitoring of the occurrence and early warning of epidemic of bacterial blight disease in the field.

Characteristics of Cysteine Proteinase Gene Family in Nicotiana benthamiana and Its Function During TMV Infection

PEI YueHong,LI FengWei,LIU WeiNa,WEN YuXia,ZHU Xin,TIAN ShaoRui,FAN GuangJin,MA XiaoZhou,SUN XianChao

Scientia Agricultura Sinica. 2022, 55(21):  4196-4210.  doi:10.3864/j.issn.0578-1752.2022.21.008

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【Objective】The objective of this study is to identify the effects of Nicotiana benthamiana cysteine proteinase (CP) induced by the antiviral compound chloroinconazide (CHI) on the infection of tobacco mosaic virus (TMV) and its gene family characteristics, and to provide a theoretical basis for understanding the antiviral molecular mechanism and chemical regulation of Solanaceae crops.【Method】Genome-wide analysis of NbCP genes was identified from Sol Genomics Network and the evolutionary relationships, motifs and promoter cis-acting elements of NbCP gene family were analyzed by bioinformatics. According to the results of bioinformatics analysis, the expression of NbCP genes after TMV infection was analyzed by real-time fluorescence quantitative PCR (qRT-PCR), so as to screen out potential disease-resistant protein. The effect of this key protein on TMV-GFP infection was verified by gene silencing mediated by tobacco rattle virus (TRV) and gene overexpression mediated by potato virus X (PVX). qRT-PCR and biochemical methods were combined to determine the antiviral mechanism of this key protein.【Result】There were 24 NbCP genes in N. benthamiana, which were named NbCP1-NbCP24 according to their chromosome location. The phylogenetic analysis showed that the NbCPs were divided into five subfamilies, of which Group V contained seven NbCPs, while Group I contained only two NbCPs. Motif analyses showed that NbCPs of different branches had similar motif distribution. Promoter cis-acting elements analysis showed that NbCP family genes were all regulated by light, and several NbCP family gene promoters contained hormone response elements such as methyl jasmonate (MeJA), methyl salicylate (MeSA), abscisic acid (ABA), gibberellin (GA) and auxin (IAA). Combined with bioinformatics analysis, five key potential disease-resistant related NbCP genes (NbCP8, NbCP12, NbCP13, NbCP18, and NbCP22) were screened from the above subfamilies, and the expression of these NbCP genes under TMV infection at the 5th day was analyzed by qRT-PCR, and it was found that the expression difference of NbCP8 was the greatest, which increased 2.6 times. The expression of NbCP8 was highest in leaves, followed by stems and roots, and lowest in flowers. TRV-mediated NbCP8 silencing significantly increased TMV-GFP infection, while PVX-mediated NbCP8 overexpression significantly inhibited TMV-GFP infection, suggesting that NbCP8 acts as a positive regulatory factor in plant to inhibit virus infection. Furthermore, silencing NbCP8 significantly inhibited the expression of salicylic acid (SA) signaling pathway related gene PR1 and jasmonic acid (JA) signaling pathway related gene MYC2, while the expressions of NPR1, COI1, and ABA related genes ABA1, NCED1 were not significantly affected. The overexpression of NbCP8 enhanced the expression of PR1 and ABA1, but had no significant effect on expression of NPR1, MYC2, COI1 and NCED1. 【Conclusion】N. benthamiana NbCPs are involved in plant stress defense, while NbCP8 plays a key role in TMV defense, which can induce SA signaling pathway to participate in the defense. The research results will provide evidence for the molecular mechanism of antiviral in Solanaceae crops.

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

Integration of Agricultural Machinery and Agronomic Techniques for Crop Nutrient Management in China

MI GuoHua,HUO YueWen,ZENG AiJun,LI GangHua,WANG Xiu,ZHANG FuSuo

Scientia Agricultura Sinica. 2022, 55(21):  4211-4224.  doi:10.3864/j.issn.0578-1752.2022.21.009

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The efficient nutrient management is essential for agricultural green development. With the increase of farm land scale and the development of agricultural mechanization, the mismatch between agricultural machinery and agronomy in nutrient management becomes increasingly obvious. There is a requirement to coordinate agronomic techniques and machinery operation. This paper analyzed the current situation and improvement direction in nutrient management techniques and agricultural machinery in the cropping system of spring maize in northeast China, winter wheat-summer maize in north China, and rice planting system in south China. It is indicated that efficient fertilizer application technology needs suitable fertilizer application machinery as guarantee, new fertilizer products need new fertilizer application machinery, changing cultivation and tillage methods generates new demand for agricultural machinery, and fertilizer application mechanization to be upgraded by using information and automatic intelligent techniques. At the same time, the fertilizer products and fertilizer application technology innovation need to take into consideration of the feasibility of mechanization. This paper described the research progress of starter fertilizer, synchronized fertilizer application and sowing technology, and straw mulching strip tillage technology in maize, within-season mechanized fertilizer application technology in wheat, mechanized side-depth fertilizer application technology in rice, and mechanized variable fertilizer application technology in China. The suggestions were provided to enhance the integration of agricultural machinery and agronomic technology, so as to upgrade the level of nutrient management of field crops.

Effects of Chelating Agent on Dissolution, Fixation and Fertisphere Transformation of Diammonium Phosphate

DONG ZeKuan,ZHANG ShuiQin,LI YanTing,GAO Qiang,ZHAO BingQiang,YUAN Liang

Scientia Agricultura Sinica. 2022, 55(21):  4225-4236.  doi:10.3864/j.issn.0578-1752.2022.21.010

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【Objective】Promoting the dissolution of phosphate fertilizer and reducing its fixation are important ways to ensure the phosphate supply and to improve the utilization rate of phosphate fertilizer. In this study, the test products of phosphate fertilizer by adding hydroxyethylidene diphosphonic acid (HEDP) and sodium gluconate (SG) into diammonium phosphate were prepared to investigate its dissolution, anti-fixation and fertisphere transformation characteristics, so as to provide a theoretical and technical basis for the efficient utilization of phosphate fertilizer. 【Method】 HEDP and SG were physically mixed with powdered diammonium phosphate at the addition ratio of 0, 0.5%, 1%, 3% and 5%, respectively, and the extrusion granulation was used to obtain the nine granular diammonium phosphate test products, specifically, common diammonium phosphate (P), HEDP_0.5%+P, HEDP_1%+P, HEDP_3%+P, HEDP_5%+P, SG_0.5%+P, SG_1%+P, SG_3%+P, and SG_5%+P. The dissolution rate and fixation rate of water-soluble phosphorus in different diammonium phosphate particles were analyzed and compared by using water dissolution rate method and CaCl₂ precipitation method, respectively. A soil cultivation was conducted to investigate the transformation characteristics of diammonium phosphate in the fertisphere. 【Result】 (1) Adding HEDP and SG increased the dissolution rate of diammonium phosphate on average by 27.7% and 20.0%, respectively. When HEDP and SG were added at 5% and 0.5%, the dissolution rates were 5.1%·min^-1 and 4.8%·min^-1, respectively, higher than other additive rate, and also higher than P by 39.2% and 32.6%, respectively. (2) When HEDP and SG were added, the fixation rates of water-soluble phosphorus in diammonium phosphate decreased by 10.3% and 6.6%, respectively. When the additive rate of chelating agent was at 5% and 3%, the fixation rate of water-soluble phosphorus was lower than P by 58.1% and 61.3% for HEDP, 17.0% and 11.8% for SG, respectively. (3) The content of available phosphorus in the fertisphere soil treated with HEDP+P and SG+P significantly increased by 39.5% and 21.2%, respectively, and HEDP+P had a better performance. When the additive rate of HEDP and SG was at 3%, the available phosphorus content in fertisphere soil was 2.9 g·kg^-1 and 2.5 g·kg^-1, respectively, higher than that of P by 53.3% and 31.9%, respectively. (4) The Ca₂-P contents in the fertisphere soil treated with HEDP+P and SG+P were higher than that of P by 38.2% and 43.0%, respectively, and both of HEDP and SG had a better performance when their additive amount at 3%. The Ca₈-P content in the 5 mm fertisphere soil treated with HEDP+P and SG+P was lower than that of P by 33.6% and 14.5%, respectively. When the additive amount of HEDP and SG was at 0.5% and 3%, the Ca₈-P content of fertisphere was 0.9 g·kg^-1 and 1.5 g·kg^-1, respectively, lower than that of P by 53.4% and 25.8%, respectively. (5) The contents of available phosphorus and Ca₂-P in the fertisphere soil were positively correlated with the dissolution rate of phosphorus fertilizer and negatively correlated with the fixation rate of water-soluble phosphorus.【Conclusion】The addition of HEDP and SG could effectively enhance the dissolution of diammonium phosphate and reduce phosphorus fixation. HEDP had a better performance, and especially when its addition amount was at 3%, HEDP could more improve the available phosphorus content and decrease the transformation from Ca₂-P to Ca₈-P in the fertisphere soil.

The Characteristics of Soil Nutrients and Soil Enzyme Activities During Wheat Growth Stage Under Different Tillage Patterns

ZHU ChangWei,MENG WeiWei,SHI Ke,NIU RunZhi,JIANG GuiYing,SHEN FengMin,LIU Fang,LIU ShiLiang

Scientia Agricultura Sinica. 2022, 55(21):  4237-4251.  doi:10.3864/j.issn.0578-1752.2022.21.011

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【Objective】This study was aimed to select the optimum tillage pattern by investigating the dynamic changes of different tillage patterns on soil nutrients, microbial biomass, and enzyme activities during different wheat growth stages in fluvo-aquic soil in Huang-Huai Plain.【Method】The field experiment was carried out from 2016 to 2019. Five rotation tillage modes were set as: (1) Continuous Rotary Tillage (RT-RT-RT); (2) Deep tillage-Rotary tillage-Rotary tillage (DT-RT-RT); (3) Deep tillage-Rotary tillage-Shallow rotary tillage (DT-RT-SRT); (4) Deep tillage-Shallow rotary tillage-Shallow rotary tillage (DT-SRT-SRT); (5) Deep tillage-Shallow rotary tillage-Rotating tillage (DT-SRT-RT). Three years is a cycle. In the third year of the 3-year cycle, i.e. 2019, 0-40 cm soil layer samples were taken at the wheat greening stage (GS), jointing stage (JS), filling stage (FS), and maturity stage (MS). The contents of alkali hydrolyzed nitrogen (AN), available phosphorus (AP), available potassium (AK), soil microbial biomass carbon (SMBC), soil microbial biomass nitrogen (SMBN), and the activities of urease, invertase, and neutral phosphatase in different soil layers were measured and analyzed.【Result】All soil indicators were decreased with the soil depths during the whole wheat growth stage. Compared with RT-RT-RT, the increment of available nutrients contents at 20-40 cm soil layer were significantly higher than those at 0-20 cm soil layer under the treatments with deep tillage combined with rotary tillage or shallow rotary tillage, but which presented few effects on soil microbial biomass and enzyme activities under soil layers compared with the effects on soil available nutrients. The dynamic changes of each soil indicators under different treatments during the wheat main growth stages were consistent with the growth and fertilizer requirements of crops. The AP, AK, SMBC, SMBN, urease, neutral phosphatase activities were showed an “N” type trend and reached to peak at the jointing stage. The sucrase activity was gradually increased with the growth stage. In 0-20 cm soil layer, the contents of AN, AP and AK under DT-SRT-RT treatment at the jointing stage were significantly higher than that under other treatments, and the highest value were 91.74 mg·kg^-1, 27.17 mg·kg^-1 and 139.81 mg·kg^-1, respectively. The AN and AP were significantly affected by rotation tillage patterns and soil depths. While the AK was affected by wheat growth stages, soil layers, and rotation tillage patterns, but the interaction among them was not obvious. During the whole growth period, compared with RT-RT-RT, in 0-40 cm soil layer, the SMBC and SMBN content under DT-RT-RT and DT-SRT-RT treatments were higher. The DT-SRT-RT treatment could significantly increase the activities of soil urease, invertase and neutral phosphatase, and their growth rates were between 3.79%-27.69%, 12.29%-36.10% and 8.61%-35.91%, respectively. In the whole wheat growth period, the soil microbial biomass and enzyme activities were significantly affected by different soil depth and rotation tillage mode, but the interaction between them on SMBN content, invertase, and neutral phosphatase activity was not significant. The wheat yield under the other treatments in 2019 was higher than that under RT-RT-RT, and the highest one was 6 557 kg·hm^-2 under DT-SRT-RT.【Conclusion】During this experimental period, in Fluvo-aquic soil in Huang-Huai Plain, the DT-SRT-RT treatment had the best effect on improving available nutrients contents, SMBC, SMBN, and soil enzyme activity, thereby further ensuring the high yield of wheat.

HORTICULTURE

Geographical Origin Tracing of Fuji Apple Based on Mineral Element Fingerprinting Technology

GAO XiaoQin,NIE JiYun,CHEN QiuSheng,HAN LingXi,LIU Lu,CHENG Yang,LIU MingYu

Scientia Agricultura Sinica. 2022, 55(21):  4252-4264.  doi:10.3864/j.issn.0578-1752.2022.21.012

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【Objective】 The aim of this study was to explore the geographical characteristics of the mineral element content in Fuji apple peel and the feasibility of geographical origin tracing, to screen out effective discriminant indicators by combining multivariate statistical analysis, and to establish an origin tracing model, so as to realize Fuji apple geographical origin identification. 【Method】 The 124 Fuji apple samples were collected from the two main production areas in China, namely, the Bohai Bay production area and the Loess Plateau production area, which were taken as the research object. Inductively coupled plasma-mass spectrometry (ICP-MS) was applied to determine the contents of 31 mineral elements in the peel, including macroelements (Sodium (Na), Magnesium (Mg), Potassium (K), and Calcium (Ca)), microelements (Vanadium (V), Chromium (Cr), Manganese (Mn), Iron (Fe), Cobalt (Co), Nickel (Ni), Copper (Cu), Zinc (Zn), Arsenic (As), Molybdenum (Mo), Cadmium (Cd), Antimony (Sb), Barium (Ba), Lead (Pb), and Uranium (U)), and rare earth elements (Yttrium (Y), Lanthanum (La), Caesium (Ce), Praseodymium (Pr), Neodymium (Nd), Samarium (Sm), Europium (Eu), Gadolinium (Gd), Terbium (Tb), Dysprosium (Dy), Holmium (Ho), and Erbium (Er)). The independent samples t-test, principal component analysis (PCA), linear discriminant analysis, and orthogonal partial least squares discriminant analysis were conducted for geographical origin tracing. 【Result】 The mineral elements Mg, Ca, Na, Fe, Mn, Cu, Ba, Ni, Nd, Pb, V, Ce, Pr, La, Dy, U, Ho and Co in the peel samples from the Bohai Bay and Loess Plateau production areas were significantly different (P<0.05). The results of the PCA showed that the cumulative variance contribution rate of the 12 extracted principal components was 81%, which allowed the preliminary clustering of the samples from the two major production areas. After linear discriminant analysis, 10 mineral elements (Mg, Ca, Cr, Mn, Fe, Ni, Gd, Tb, Dy, and U) were screened as the ideal indicators to discriminate the geographical origin of Fuji apples in the two major production areas. The discriminant rate of the established discriminant model for the original whole was 92%, and the cross-validation discriminant rate was 89.5%. The orthogonal partial least squares discriminant analysis showed that five mineral elements, Co, Ba, Ho, Dy and Pr, played the key roles in the sample classification, and the accuracy of origin identification by the model could reach 98%, which realized the origin traceability of Fuji apples in the two production areas. 【Conclusion】 The peel of Fuji apple could be used as an effective site for origin tracing. The contents of rare earth elements Dy, Ho, Pr, Gd and Tb were important indicators for the geographical origin tracing of Fuji apples. This study provided a theoretical basis and technical support for Fuji apple origin tracing.

Analysis Reveals the Differential Expression of Genes Related to Starch Accumulation in Chloroplast of Leaf with Different Ages in Pinot Noir Grape

YOU JiaLing,LI YouMei,SUN MengHao,XIE ZhaoSen

Scientia Agricultura Sinica. 2022, 55(21):  4265-4278.  doi:10.3864/j.issn.0578-1752.2022.21.013

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【Objective】The accumulation of starch in chloroplast of grape leaves increased with leaf maturation, but the related molecular mechanism has not been reported. In this study, the potential related genes were screened in order to clarify the molecular regulation mechanism of starch accumulation in chloroplast of grape leaves with different leaf ages.【Method】 Two years old ‘Pinot Noir’ grapes (Vitis vinifera L.) were used as plant materials to investigate the dynamic changes of starch accumulation in chloroplast of leaves with different ages. The RNA sequencing technology was used to characterize the differently expressed genes (DEGs) involved in starch and sucrose metabolism in leaves with different ages. The expression levels of several key candidate genes were detected by using real-time quantitative qPCR.【Result】The starch grains in NL (undeveloped leaves) were smallest in volume and lowest in number compared with these in GL (growing leaves) and ML (mature leaves). With the increase of leaf ages, the volume and amount of starch grains in GL and ML also increased. A total of 58.57 GB valid data was obtained by RNA-sequencing. Differentially expressed genes mainly involved in starch and sucrose metabolism pathway for the leaves with different ages. The genes involved in the process of sucrose conversion into starch synthesis precursor adenosine diphosphate glucose (ADP) showed up-regulated expression level with the increase of leaf ages, including cell wall invertase (CWINV), phosphoglucomutase (PGM), and ADP-glucose pyrophosphorylase (AGPase). The genes involved in starch synthesis and hydrolysis also presented up-regulated expression level with the increase of leaf ages, including soluble starch synthase (SSⅠ), starch branching enzyme (SBE), α-amylase (AMY), and β-amylase (BAM). AGPase, SSⅠ and SBE played an important role in synthesis of amylopectin with semi-crystalline structure.【Conclusion】With the increase of leaf ages, the starch accumulation in chloroplast also increased. AGPase, SSⅠ and SBE genes could be the key genes involved in the regulation of starch accumulation in chloroplast of grape leaves.

Optimizing Management Mode of Disease and Nutrient During the Entire Fruit Development for Improving Postharvest Storability of Longan Fruit

HAN DongMei,HUANG ShiLian,OUYANG SiYing,ZHANG Le,ZHUO Kan,WU ZhenXian,LI JianGuang,GUO DongLiang,WANG Jing

Scientia Agricultura Sinica. 2022, 55(21):  4279-4293.  doi:10.3864/j.issn.0578-1752.2022.21.014

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【Objective】In order to obtain an optimized preharvest management mode to improve the longan fruit storability, the effects of disease control and nutrient management during fruit development on the characteristic quality and storability of longan fruit were explored. 【Method】 Using the cultivar Shixia as material, a field-based orthogonal trial (L₁₂ (4×3³)) was conducted with 12 combinations (labeled as BR1-BR12), among which BR1 was the control (CK). Disease control (DCP), fertilization type (FT), and hormone regulation (HR) were used as factor A (FacA), factor B (FacB), and factor C (FacC), respectively. The fruit quality and mineral nutrient contents were determined at harvest time, and the storage effects for the fruit of 12 treatments stored at 5℃ were also observed regularly to screen the factors and levels with significant optimization effects.【Result】Descriptive statistical analysis showed that the variation coefficients of 11 fruit quality and 20 mineral nutrition (in pericarp and aril) indexes ranged from 2.19% to 49.50% and from 5.14% to 77.43%, respectively. Except for the organoleptic trait indicators including CDFS (cleanliness degree of fruit surface), RPTC (rust powder in the turtle cracks on the fruit surface) and ACD (aril crispy degree), the other indexes showed significant differences between treatments (P<0.01). With the prolonging of the storage period, the MFR (mouldy fruit rate), EBI (endocarp browning index), ABI (aril breakdown index) of each treatment fruit increased, while the EFR (edible fruit rate) and storability decreased, and the difference between treatments was extremely significant (P<0.01). Moreover, the clustering results showed that BR11 and BR12 had the best storage effect with the lowest EBS (endocarp browning score), ABS (aril breakdown score) and MFRS (score of mildew fruit rate), and the highest EFRS (score of edible fruit rate) and CSES (score of comprehensive storability throughout the entire storage), and their storage life was about 40 days and about 15 days longer than that under the control (BR1). On the contrary, BR1 and BR2 had the worst storability with about 25 days of storage life. Correlation analysis showed that disease control (facA) was closely associated with EBS, ABS, MFRS, EFRS and CSES during the fruit storage (P<0.01), while FSb^* (b^* value of fruit surface) and ACD, total Zn and Mn contents in pericarp were closely related to fruit storage effects (P<0.01). Besides these, the contents of K, Ca, and Mg mainly affected the SFM (single fruit mass), EPRF (edible portions rate in a fruit), TSS (content of total soluble solid substance) and RPTC of fruit, while the contents of Zn, Mn, and B mainly affected CDFS and RPTC. The estimated marginal means of each level of three trial factors showed that, compared with no disease control (1-DCK) and young fruit stage (2-YS), the disease control during the entire fruit period (4-EP) and expansion stage (3-ES) could significantly improve the FSb^*, FSC^* (C^* value of fruit surface, P<0.05) and CDFS (P<0.01), reduce the EBS, ABS and MFR, and ultimately improve the EFRS and CSES. Among the 4 kinds of disease control methods, the control effect of 4-EP was the best (P<0.01). The full nutrition fertilizer (3-FF) significantly improved the ACD, CDFS, and CSES and reduced the MFRS, otherwise, hormone regulation (HR) had no obvious effect.【Conclusion】The scientific and reasonable disease control and nutrient management before harvest could significantly improve the characteristic quality and storability of longan fruit. It was recommended to optimize the comprehensive management mode during fruit development as “disease control throughout the entire fruit period + full nutrient fertilization”.

ANIMAL SCIENCE·VETERINARY SCIENCE

Heritability Estimation and Correlation Analysis of Longevity and Milk Yield of Holstein Cattle in Xinjiang Region

DONG MingMing,ZHAO FanFan,GE JianJun,ZHAO JunLiang,WANG Dan,XU Lei,ZHANG MengHua,ZHONG LiWei,HUANG XiXia,WANG YaChun

Scientia Agricultura Sinica. 2022, 55(21):  4294-4303.  doi:10.3864/j.issn.0578-1752.2022.21.015

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【Objective】 Milk production and longevity are important traits in dairy cattle breeding. In recent years, the high milk yield has been pursued in dairy cattle breeding, while longevity has been declining. With the development of balanced breeding, the longevity traits were gradually included into the comprehensive selection index of dairy cows in developed countries. The purpose of this study was to explore the relationship between milk yield and longevity of dairy cows, and to provide the reference for Xinjiang region to develop the comprehensive selection index of Holstein dairy cows in China, so as to achieve balanced breeding and high-quality healthy development of dairy industry. 【Method】 The production records of three large-scale dairy farms in Xinjiang from 1997 to 2020 were collected, and the herd life and production life were calculated. The longevity records of 7 206 dairy cows and first parity 305 d milk yield of 15 218 first fetuses were obtained. A total of 18 183 pieces of pedigree information from three generations of individuals were collected, including 903 bulls and 20 883 cows. Firstly, the GLM process of SAS 9.2 software was used to analyze the effects of field, birth year, birth season and age at first calving on the herd days, productive life and first parity 305 d milk yield of Chinese Holstein cattle in Xinjiang region, and the least square mean values of the herd days, productive life and the first parity 305 d milk were calculated. SPSS 19.0 software was used to calculate the Pearson correlation coefficient between the number of herd days, production life and first parity 305 d milk yield of Chinese Holstein cattle in Xinjiang. Secondly, the genetic variance and covariance of herd days, productive life and first parity 305 d milk yield of Holstein dairy cows in Xinjiang were estimated by using AI-REML in DMU software combined with EM algorithm and multi-trait animal model, and the genetic correlation between herd days, productive life and first parity 305 d milk yield was calculated. Finally, the breeding values of the herd days, productive life and first parity 305 d milk yield of Chinese Holstein cattle in Xinjiang region were estimated by animal model BLUP method, and the genetic trend chart was drawn. 【Result】 The results of basic statistical analysis on the days in the herd days, productive life and first parity 305 d milk yield of Chinese Holstein cows in Xinjiang showed that the average herd days of Chinese Holstein cows in Xinjiang was 1 754.7 days, the average production life was 937.33 days, and the average first parity 305 d milk yield was 9 362.94 kg. GLM program analysis showed that the effects of different fields, birth year, birth season and age at first calving had significant effects on herd days, productive life and first parity 305 d milk yield (P<0.01). The heritability of herd days, productive life and first parity 305 d milk yield of Chinese Holstein cattle in Xinjiang was 0.11 (0.03), 0.11 (0.03) and 0.33 (0.03), respectively. The genetic and phenotypic correlations between herd days span and productive life span were 0.99 and 0.98, respectively. There was a positive correlation between first parity 305 d milk yield and the phenotypes of herd days and production life, with correlation coefficients of 0.079 and 0.077, respectively, while genetic correlation was negative, with correlation coefficients of -0.18 and -0.20, respectively. The genetic trend chart of the herd days, the productive life and the first parity 305 d milk yield of Holstein in Xinjiang showed that the breeding values of the herd days and the productive life changed greatly with the increase of the number of cattle tested, and the overall trend decreased. The first parity 305 d milk yield of Holstein cattle in the first foetus also changed greatly with the increase of the number of cattle tested, but showed an overall upward trend.【Conclusion】 Therefore, the longevity of dairy cows should be considered while improving milk yield, so as to avoid the decline of longevity of dairy cows with the increase of milk yield.

Effects of Feeding Levels on Muscle Tissue Structure and Muscle Fiber Composition Related Genes in Sheep

SONG ShuZhen, GAO LiangShuang, LI Hong, GONG XuYin, LIU LiShan, WEI YuBing

Scientia Agricultura Sinica. 2022, 55(21):  4304-4314.  doi:10.3864/j.issn.0578-1752.2022.21.016

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【Objective】Improving meat quality is one of the main goals of the modern mutton sheep production. The objective of this study was to compare the muscle tissue structure and the genes expression related muscle development of longissimus dorsi muscle in Altay sheep, so as to provide the reference for regulating meat quality and improving production efficiency of meat-fat type sheep by feeding level. 【Method】 Thirty Altay female lambs with a similar month ((3.0±0.5) months), similar body weight ((19. 16±0.54) kg), uniform tail size were randomly divided into 3 groups with 10 lambs per group. After 60 days of ad libitum feeding, three test sheep groups were fed with 50% (0.25 MJ/W^0.75 ×d^-1, NL group), 100% (0.5 MJ/W^0.75 ×d^-1, NM group), and 150% (0.75 MJ/W^0.75 ×d^-1, NH group) maintenance metabolizable for 30 days, respectively, and then slaughtered. The longissimus dorsi muscle was stained with Hematoxylin Eosin (HE staining) and 2% uranyl acetate-lead citrate double staining, respectively, and then the expression of myosin heavy chain I (MyHC I) and peroxisome receptor activator-activated gamma (PPARγ) was determined by immunohistochemistry. 【Result】(1) When Altay sheep were fed 50%, 100% and 150% metabolizable energy, respectively, the difference of average daily gain was significant (P<0.05), and the difference of three groups were -90.67 kg, 13.33 kg and 203.00 kg, respectively. The diameter of longissimus dorsi muscle fibers from NL group and NH group was significantly larger than that from NL group (P<0.05), while the thickness of muscular intima from NH group was significantly smaller than that in NL group (P<0.05), but there were no significant differences in the amount of muscle fiber, the area of muscle fiber, the thickness of muscle fascicle, the area of muscle fiber and the density of muscle fiber (P>0.05). (2) The feeding levels affected the area of terminal cisterna, lipid droplets and the area ratio of terminal cisterna, myofibril and sarcoplasm. The area and area ratio of terminal cisterna from NH were significantly larger than those from NM group and NL group (P<0.05), while the area of lipid droplets from NH group was significantly larger than that from NL group (P<0.05). The area ratio of sarcoplasm from NM group was significantly larger than that from NH and NL Group (P<0.05), and mitochondria and glycogen had no significant difference among the three groups (P<0.05). (3) The feeding levels affected the expression of gene related muscle fiber, PPARγ expression intensity, positive area and positive rate from NL, and NM groups were significantly lower than those from NH group (P<0.05). The expression intensity of MyHC Ⅰ protein increased with the increase of feeding level, but there was no significant difference among three groups (P>0.05).【Conclusion】The feeding level had a significant effect on the muscle tissue structure and the expression of gene protein related to muscle fiber type of the Longissimus dorsi muscle in Altay sheep, and it could be used to control the muscle tissue structure and muscle fiber type composition during fattening period, in order to regulate the quality of meat according to production needs.

Effect of Matrine on NLRP3 Inflammasome Signaling Pathway in H9N2 AIV Infected Mice

WEI JingJie,JIANG NingBo,LIANG Yan,ZHANG Qian,SUN YingJian,HU Ge

Scientia Agricultura Sinica. 2022, 55(21):  4315-4326.  doi:10.3864/j.issn.0578-1752.2022.21.017

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【Objective】 Influenza virus infection causes inflammatory response and imbalance of immune homeostasis. The resulting cytokine storm (CS) is the main cause of death of infected hosts. The aim of this study was to explore the protective effect of Matrine on H9N2 AIV infected mice as well as the characteristics and preliminary mechanism of regulating NLRP3 inflammatory body signal pathway, so as to further improve the theoretical basis of antiviral of traditional Chinese medicine and lay a foundation for the development of new antiviral drugs.【Method】72 8-week-old BALB/c mice were randomly divided into blank control group (0.1 mL sterile chicken embryo allantoic fluid) and virus group (0.1 mL 4×10⁵ PFU/mL H9N2 AIV), amantadine group (0.1 mL 4×10⁵ PFU/mL H9N2 AIV + 100×10^-6 99% amantadine), matrine high concentration treatment group (0.1 mL 4×10⁵ PFU/mL H9N2 AIV + 40 mL·kg^-1 matrine), medium concentration treatment group (0.1 mL 4×10⁵PFU/mLH9N2 AIV + 20 mL·kg^-1 matrine), and low concentration treatment group (0.1 mL 4×10⁵PFU/mL H9N2 AIV + 10 mL·kg^-1 matrine), and allantoic fluid nasal drops were used to construct the mouse model of viral pneumonia. The matrine was administered by gavage or drinking water for 5 consecutive days, and the test was conducted for 7 days. The weight changes of mice in different groups were observed. On the 1st, 3rd, 5th and 7th days, three mice were aseptically collected and killed, and the lung tissue was taken for histopathological observation, the expression of H9N2 and NLRP3 gene in mouse lung tissues was detected by RT-PCR, the expression of NLRP3 inflammatory body signal pathway related protein in lung tissue of H9N2 infected mice after matrine treatment was detected by Western blot, and the cytokine TNF in mouse serum was measured by ELISA- α and IL-1 β changes in the expression of IL-18 and IL-10.【Result】Compared with the virus group, the area of pulmonary edema and the number of inflammatory cells in the pathological section of H9N2 AIV infected mice in the high concentration matrine treatment group were significantly reduced, and the exudation of red blood cells decreased, while the effect was close to that in the amantadine group. At day 7, the alveolar wall of mice in the high concentration matrine treatment group was intact, the boundary between alveoli was clear, and the number of inflammatory cells and plasma cells in lung tissue decreased significantly, which was almost the same as that in the blank group; in the treatment group with medium concentration of matrine, there was a small amount of bleeding in the lung tissue, there was no swollen fluid in the alveoli, and the alveolar septum was intact; in the low concentration matrine treatment group, the red blood cells exuded from the alveoli, a large number of plasma cells were recruited, and there was fusion between the alveoli. The expression of H9N2 virus gene in lung tissue of mice treated with Matrine and amantadine decreased significantly on day 3, 5 and 7 (P<0.01). After 3 and 5 days of treatment, the expression of NLRP3 gene, protein and TNF- α, IL-1 β in matrine high concentration treatment group and amantadine group decreased significantly (P<0.01); on day 7, the expression of NLRP3 gene in lung tissue of mice in matrine high, medium and low treatment groups decreased significantly (P<0.01); the expression of NLRP3 protein, caspase-1 protein in low concentration group and caspase-1 protein in medium concentration group decreased significantly on day 5 (P<0.05); the expression of TNF- α and IL-1 β in matrine medium and low concentration groups on days 3 and 5 decreased significantly (P<0.01).The expression of IL-10 in Shegan medium concentration treatment group decreased significantly.【Conclusion】Matrine could inhibit the expression of H9N2 AIV in vivo, reduce the expression of TNF-α, IL-1β and IL-10 by down regulating NLRP3 inflammatory body signal pathway related proteins, and reduce the inflammatory response, which had good antiviral and anti-inflammatory effects.