Table of Content

01 December 2024, Volume 57 Issue 23

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SPECIAL FOCUS: MINING AND UTILIZATION OF CROP DISEASE RESISTANCE AND INSECT-RELATED GENES

Mining and Utilization of Crop Disease Resistance and Insect-Related Genes

HE NiQing, LIN ShaoJun, CHENG ZhaoPing, YANG DeWei

Scientia Agricultura Sinica. 2024, 57(23):  4589-4592.  doi:10.3864/j.issn.0578-1752.2024.23.001

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The Mechanism of Cystathionine-β-Synthase OsCBSX4 in Rice Blast Resistance

DIAO ZhiJuan, CHEN LiZhe, WANG Xun, LU Ling, LIU Yan, ZHANG Jing, XIA Na, TANG DingZhong, LI ShengPing

Scientia Agricultura Sinica. 2024, 57(23):  4593-4606.  doi:10.3864/j.issn.0578-1752.2024.23.002

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【Objective】 Rice blast is one of the main diseases that threaten rice yield and quality. OsBSK1-2 has been found to be involved in rice blast resistance regulation. Previously, OsCBSX4, a cysteine sulfide-β-Synthase, was identified by screening the proteins that interact with OsBSK1-2. The purpose of this study was to verify the interaction between OsBSK1-2 and OsCBSX4, and clarify the function and molecular mechanism of OsCBSX4 in rice blast resistance, providing a theoretical basis for rice disease resistance breeding.【Method】 Co-immunoprecipitation, bimolecular fluorescence complementation and luciferase complementation assays were used to determine the interaction between OsBSK1-2 and OsCBSX4. Then, quantitative PCR and agrobacterium- mediated transient transformation in N. benthamiana were used to detect the gene expression pattern and protein localization of OsCBSX4. Subsequently, OsCBSX4-knockingout and OsCBSX4-overexpressing plants were generated via CRISPR/Cas9 technology and Agrobacterium-mediated genetic transformation, respectively, and their resistance to rice blast was determined by inoculation with M. oryzae. Moreover, the immune responses induced by chitin and M. oryzae in the oscbsx4 mutant were analyzed using ROS burst and DAB staining assays respectively. In the end, the interaction between OsCBSX4 and OsRbohB was verified via bimolecular fluorescence complementation and split-luciferase complementation imaging assays, and the impact of the metabolite of OsCBSX4 on rice blast resistance was determined using the detached leaf inoculation method. The studies above will reveal the immune function and molecular mechanism of OsCBSX4.【Result】 The interaction between OsCBSX4 and OsBSK1-2 was verified by CoIP, LCI and BiFC assays. Compared to the wild type, the oscbsx4 mutant showed more disease lesions after inoculation with M. oryzae by the spray method, as well as a larger lesion area and greater fungal growth after inoculation with M. oryzae by the punch method, suggesting that knocking out OsCBSX4 decreased rice blast resistance. Moreover, the expression of the pathogenesis-related genes, OsPR5 and OsPR10, and the H₂O₂ accumulation induced by M. oryzae infection and the ROS burst induced by chitin treatment were reduced in the oscbsx4 mutant. Compared with the wild type, OsCBSX4 overexpressing plants showed a smaller lesion area and less fungal growth after inoculation with M. oryzae by the punch method, suggesting that overexpression of OsCBSX4 increased rice blast resistance. In addition, we found that OsCBSX4 can interact with OsRbohB, a key regulator of rice ROS production and treating rice with L-cysteine, a metabolite of OsCBSX4, does not affect rice blast resistance. 【Conclusion】 OsCBSX4 is an important component of OsBSK1-2 signaling and positively regulates rice blast resistance. OsCBSX4 may mediate ROS production by interacting with OsRbohB, thereby regulating rice immunity.

Hydrolase Gene BGIOSGA023826 Involved in Regulation of Resistance Process to Rice Blast

ZHAO Jie, ZHAO LongYuan, PAN NingHui, GUAN LiRong, DU YunLong, LI ChengYun, WANG YunYue, XIE Yong

Scientia Agricultura Sinica. 2024, 57(23):  4607-4618.  doi:10.3864/j.issn.0578-1752.2024.23.003

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【Objective】 Acuce, currently the predominant cultivating rice landrace at Yuanyang Hani terrace, Yunnan Province, has been continuously planted for more than 100 years. Interestingly, there has never been a major outbreak of rice blast during the past decades. Clarifying the types, quantities, expression characteristics, and phenotypic effects of functional genes involved in the resistance response process so as to exploring the molecular mechanism of Acuce resistance to Magnaporthe oryzae will optimize facilitate the conservation and utilization of rice landrace. 【Method】 The present research focuses on the biological effect of disease resistance-related candidate gene BGIOSGA023826 which screened from Acuce -M. oryzae interaction transcriptome. Initially, biological function of this gene was analyzed by using bioinformatics software and conducted gene cloning. Subsequently, overexpression vector was constructed through Agrobacterium-mediated genetic transformation, then carry out positive identification and disease resistance phenotype analysis of overexpressed plants. Finally, Real-time fluorescence quantitative PCR was used to analyze the expression of PR genes in overexpressed plants, and enzyme-linked immunosorbent assay was used to determine changes in endogenous hormone content. 【Result】 The candidate gene BGIOSGA023826 encodes 354 amino acids figuring unstable hydrophobic characteristic without transmembrane domains, coding a non-membrane and non-secretory protein, classified as a hydrolase gene. The overexpression of BGIOSGA023826 in transgenic plants significantly enhances resistance to M. oryzae compared to Nipponbare. The results of real-time fluorescent quantitative PCR indicate that the relative expression levels of the PR genes PR1b, OsNPR1, and OsAOS2 as well as transcription factors WRKY45 significantly upregulated. Both of the PR1b gene and the key gene OsAOS2 functioning in the JA synthesis pathway continuously upregulated from 12 hpi. Meanwhile, the expression level of the transcription factor gene WRKY45 drastically increased at 24 hpi and reached its peak at 36 hpi, which were nearly two folds higher than that of Nipponbare. As to the results of endogenous hormone content measurement, the JA increased to 263.88 pmol·L^-1 at 12 hpi, reaching peak 304 pmol·L^-1 at 36 hpi; Similarly, the SA increased to 186.5 pmol·L^-1 at 24 hpi, reaching peak 198.88 pmol·L^-1 at 36 hpi; At the same time, the content of ROS also rises continually and reached its peak at 36 hpi, MDA also increased from 40.43 nmol·g^-1 to 53.94 nmol·g^-1; However, SOD did not exhibit significant differences at individual time points in transgenic plant, but showed an upward trend in Nipponbare. 【Conclusion】 Preliminary elucidating of the temporal expression relationship between the hydrolase gene BGIOSGA023826 and the PR genes as well as endogenous hormone levels, demonstrating it involved in the landrace Acuce resistance to M. oryzae infection process.

Fine Mapping and Analysis of Pyramiding Effects of Rice Brown Planthopper Resistance Genes QBPH1 and QBPH4

XIONG ShangYe, ZHANG Xiang, LIANG BaoHui, YE YangDong, LI YuYang, ZHU Xiao, ZHU ZhiHong, GUAN HuaZhong, ZHANG Shuai, WU JianGuo, HU Jie

Scientia Agricultura Sinica. 2024, 57(23):  4619-4631.  doi:10.3864/j.issn.0578-1752.2024.23.004

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【Objective】 This study aims to discover new quantitative trait loci (QTLs) for resistance to the brown planthopper (BPH) in rice by utilizing a genetic population and to assess the pyramiding effects of these QTLs, thereby providing crucial genetic materials and resources for breeding insect-resistant rice. 【Method】 A recombinant inbred line (RIL) population derived from a cross between susceptible NPB and resistant Jiafuzhan was used, combined with seedling stage resistance evaluation and extreme mixed pool sequencing (BSA-seq) to map BPH resistance QTLs. Further, fine mapping, candidate gene identification, pyramiding effect analysis, and mixed-pool transcriptome sequencing (BSR-seq) were employed to elucidate the physiological and molecular mechanisms mediated by these QTLs. 【Result】 BSA-seq identified two major resistance QTLs on chromosomes 1 (30-32 Mb) and 4 (5-7 Mb), named QBPH1 and QBPH4, respectively. Interval-linked molecular markers confirmed the authenticity of these QTLs. QBPH4 coincides with previously cloned BPH3 and BPH15, while QBPH1 represents a newly discovered QTL. High-density markers and recombinant analysis further narrowed the QBPH1 region to 30.61-30.65 Mb. This analysis identified Os01g53294 and Os01g53330 as reliable candidate genes, which code for a respiratory burst oxidase protein B and an anthocyanin 5,3-O-glucosyltransferase, respectively. In evaluations of seedling resistance, brown planthopper honeydew area and weight, insect weight gain, lethality, and preference, comparisons between QBPH1 and QBPH4 single genes and their polymerized lines revealed no significant enhancement in resistance. Both QBPH1 and QBPH4 mediated antibiosis and antixenosis mechanisms with varying effects. BSR-seq analysis highlighted significant enrichment of differentially expressed genes (DEGs) involved in transcriptional regulation, protein phosphorylation, and redox processes among different QBPH1 alleles. Additionally, genes associated with jasmonic acid (JA) synthesis and signaling pathways were significantly upregulated in resistant materials, confirmed by RT-qPCR experiments. 【Conclusion】 A novel BPH resistance QTL, QBPH1, was successfully identified on chromosome 1 of rice. QBPH1 mediated both antibiosis and avoidance against BPH, though its aggregation effect with another QTL was not significant. QBPH1 may mediate defense mechanisms against BPH through involvement in the JA pathway. Based on this, Os01g53294 and Os01g53330 have been validated as reliable candidate genes for QBPH1.

Cloning and Functional Analysis of GmRHF1 Gene Against Soybean Mosaic Virus

FENG WenMi, ZHOU FangXue, YU Zhe, MOU KeXin, JING Yan, LI HaiYan

Scientia Agricultura Sinica. 2024, 57(23):  4632-4643.  doi:10.3864/j.issn.0578-1752.2024.23.005

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【Objective】 Soybean mosaic virus (SMV) disease causes by SMV seriously restricts soybean yields and qualities. In this study, we cloned GmRHF1, which encodes a Ring-H2 type zinc finger protein (RHF), and identified its function in SMV resistance through the sequence variation analysis and the virus induced gene silencing (VIGS) assay. Yeast two-hybrid and luciferase complementation assay (LCA) were used to screen and verify the interacting proteins of GmRHF1. The research lay a foundation for further exploring the mechanism of GmRHF1 in soybean resistance to SMV.【Method】 Firstly, GmRHF1 was cloned from soybean varieties of Xudou 14 (highly resistant to SMV) and Shishanheidou (highly susceptible to SMV), and the consistency of sequence variation was then analyzed in representative resistant and susceptible soybean resources, so as to excavate the excellent alleles with SMV resistance. Secondly, the expression abundance of GmRHF1 in different soybean tissues and under SMV treatment were analyzed by real-time quantitative PCR (RT-qPCR), respectively. At the same time, the function of GmRHF1 in SMV resistance was identified by the VIGS assay. The interacting proteins of GmRHF1 were screened and verified by yeast cDNA library screening, point to point verification and LCA experiments. Finally, the function of GmClpP6 on SMV resistance was verified, and the biological significance based on the interaction of GmRHF1 and GmClpP6 for soybean resistance to SMV was clarified.【Result】 GmRHF1 contained a conserved RING-H2 domain of the E3 ubiquitin protein ligase, and belongs to the typical RING-H2 finger family. The results of sequence variation analysis showed that a natural non-synonymous mutation existed in the CDS region of GmRHF1 among various soybean varieties, providing a probable relevance to the SMV resistance or sensibility. After SMV inoculations, the expression level of GmRHF1 in resistant soybean was significantly higher than that in susceptible material by RT-qPCR assay. Through the VIGS experiments, GmRHF1 was effectively silenced and the soybean leaves showed a weakened SMV resistance than the normal plants, indicating that GmRHF1 could enhance soybean resistance to SMV. Based on the yeast cDNA library, 30 potential interacting proteins of GmRHF1 were screened. The interaction between GmRHF1 and GmClpP6 was confirmed by the two-hybrid and LCA experiments. Furthermore, the soybean resistance to SMV was also reduced in soybean leaves with the silenced GmClpP6 by the VIGS experiments.【Conclusion】 The non-synonymous mutation identified in the coding sequence of GmRHF1 may be a key SNP variation related to the SMV resistance and sensibility; and GmRHF1 can interact with GmClpP6. GmRHF1 plays an important role in SMV resistance.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Regulatory of Exogenous Melatonin on Floret Development and Carbon Nutrient Metabolism in Winter Wheat Under Drought Stress

ZHANG Rong, LIU LinRu, FU KaiXia, WU ZiJun, SONG YiFan, WANG LuYuan, HOU GeGe, HE Li, FENG Wei, DUAN JianZhao, WANG YongHua, GUO TianCai

Scientia Agricultura Sinica. 2024, 57(23):  4644-4657.  doi:10.3864/j.issn.0578-1752.2024.23.006

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【Objective】 This study aimed to clarify the regulatory effects of exogenous melatonin on floret development and carbon nutrient metabolism in winter wheat under drought stress. 【Method】 Two soil water conditions (drought stress treatment: D, and normal moisture treatment: W) were set up using multi-spike variety Yumai 49-198 and large-spike variety Zhoumai 22 as experimental wheat materials, with foliar spraying 100 μmol·L^-1 exogenous melatonin (MT) and clear water control (CK) before the peak of floret degradation (about 20 days after jointing) in 2021-2023, focusing on the effects of exogenous melatonin on the number and morphological characteristics of floret development, SPAD value, net photosynthetic rate, sucrose content and its metabolic enzyme activities of top spread leaves, and yield component factors of winter wheat under drought stress. 【Result】 The drought stress led to an increase in floret degradation and abortion in wheat, while spraying exogenous melatonin could effectively reduce floret degradation and abortion, and increase number of fertile florets of the two varieties, but could not completely counteract the negative effect of drought stress; exogenous melatonin also showed positive regulatory effect on normal water treatments of the two varieties. Spraying exogenous melatonin could effectively increase SPAD value, net photosynthetic rate, carbon metabolism- related enzyme activities of top spread leaves and spike sucrose content of the two varieties under drought stress and normal water treatment, and the increase range was higher in drought treatment than in normal water treatment; exogenous melatonin decreased sucrose content of stem and leaf organs of both varieties under drought stress, but the opposite was true under normal water treatment. Spraying exogenous melatonin significantly increased grain number per spike of two varieties under two moisture treatments, compared with no-spraying MT treatment, the grain number per spike of Yumai 49-198 with spraying MT treatment increased by 19.12% (D) and 6.65% (W), respectively; the grain number per spike of Zhoumai 22 with spraying MT treatment increased by 21.57% (D) and 8.73% (W), respectively; spraying MT showed some regulation effect on spike number and thousand grain weight of the two varieties under the same water treatment, but did not reach a significant level. Compared the differences between two varieties, the regulatory effect of spraying melatonin was overall higher in the large-spike variety Zhoumai 22 than in the multiple-spike variety Yumai 49-198. 【Conclusion】 Spraying exogenous melatonin before the peak of floret degradation could effectively increase the SPAD value, net photosynthetic rate, and carbon metabolism-related enzyme activities of top spread leaves in wheat, and promote synthesis of photosynthesis products and the distribution and transportation of sucrose from stem and leaf nutrient organs to spike organ, which could provide sufficient nutrient security for the development of florets to increase number of fertile florets, thereby increasing grain number per spike, and the regulating effect on the large-spike variety of Zhoumai 22 was more pronounced. The results of this study provided the theoretical basis and technical support for increasing grain number per spike, stabilizing yield and reducing disaster under drought stress through the application of exogenous melatonin.

Regulation Effects of Line-Spacing Expansion and Row-Spacing Shrinkage on Dry Matter and Nutrient Accumulation and Transport of Summer Maize Under High Plant Density

SHI DeYang, LI YanHong, WANG FeiFei, XIA DeJun, JIAO YanLin, SUN NiNa, ZHAO Jian

Scientia Agricultura Sinica. 2024, 57(23):  4658-4672.  doi:10.3864/j.issn.0578-1752.2024.23.007

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【Objective】 The aim of this study was to explore the effect of line-spacing expansion and row-spacing shrinkage on maize yield, dry matter and nutrient accumulation and transport under high-density planting conditions, and to clarify the optimal allocations of row-spacing, so as to provide the theoretical basis for the further increase of grain yield in Huang-Huai-Hai summer maize region.【Method】 For two consecutive maize growing seasons in 2019-2020, under the planting density of 82 500 plants/hm², a field comparison experiment was conducted with 5 equidistant row, including 60 cm (B1), 65 cm (B2), 70 cm (B3), 75 cm (B4) and 80 cm (B5), and 2 summer maize varieties, including Denghai 518 (DH518) and Denghai 605 (DH605). The effects of line-spacing expansion and row-spacing shrinkage on maize yield and its constituent factors, dry matter accumulation, distribution and transport, nutrient absorption and transport were studied, and the correlation between dry matter accumulation, nutrient absorption and yield was analyzed too. 【Result】 The increase of the yield of summer maize showed a trend of increasing first and then decreasing, reaching the extreme value under B4. In the 2-year experiment, the yields of DH518 and DH605 under B4 treatment increased by 9.59% and 13.18% on average compared with B1 treatment, respectively. The analysis of yield components showed that the yield of summer maize was affected mainly by the number of grains per ear, the grain number per spike of DH518 and DH605 increased by 8.30% and 11.1% under B4 treatment compared with B1 treatment, respectively. Line-spacing expansion and row-spacing shrinkage significantly affected the dry matter accumulation of maize plants after silking (R1), and the increase of trailing distance showed a trend of first increasing and then decreasing, which reached the maximum value under B4 treatment. Logistic regression equation was used to fit the growth curve, and it was found that the maximum dry matter accumulation rate of DH518 and DH605 under B4 treatment increased by 13.6% and 16.3% than that under B1 treatment, respectively, and the average growth rate increased by 15.9% and 17.5%, respectively. Appropriate increase of planting row spacing could improve dry matter accumulation after R1, and dry matter transfered from vegetative organs to grain before R1. The accumulation of N, P and K in the two varieties increased first and then decreased. The N, P and K accumulation of DH518 in R1 and physiological maturity (R6) were increased by 5.2%-25.2%, 9.8%-43.5%, 3.5%-26.1% and 6.3%-29.0%, 9.6%-49.9%, and 8.5%-31.0% compared with B1 treatment, respectively; DH605 increased by 6.0%-17.4%, 5.7%-28.9%, 5.2%-19.1% and 7.6%-28.4%, 8.7%-46.5%, and 6.6%-25.7%, respectively. The increase of row spacing significantly increased the volume of transshipment of N, P and K in the 2 varieties, and reached the extreme value under B4 treatment. The volume of transshipment of N, P and K in DH518 and DH605 under B4 treatment increased by 19.9%, 39.3%, 23.3% and 14.6%, 30.8%, 24.9% compared with B1 treatment, respectively. The correlation analysis of above-ground dry matter accumulation and N, P, K accumulation and yield in R1 and R6 showed that the dry matter accumulation and N, P, and K accumulation were significantly positively correlated with grain yield.【Conclusion】 Under high density planting conditions, line-spacing expansion and row-spacing shrinkage improved the maximum and average dry matter growth rate of DH518 and DH605, and promoted nutrient translocation amount and contribution rate of accumulation nutrients after the R1 stage, synergistically, thus increased maize yield and fertilizer utilization. Considering yield, accumulation and transport of dry matter and nutrients, 75 cm equal row spacing was beneficial to yield under the planting condition of 82 500 plants /hm² in Huang-Huai-Hai summer maize region.

Effects of Late Sowing on Yield, Quality, Photosynthetic Source Succession and Loadability Characteristics of Rape

LI Fei, XIONG Cai, GU JiaJia, CAO Xin, WANG ShanShan, HU Wei, ZHOU ZhiGuo, CHEN BingLin

Scientia Agricultura Sinica. 2024, 57(23):  4673-4685.  doi:10.3864/j.issn.0578-1752.2024.23.008

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【Objective】 This study aimed to explore the regulation mechanism of late sowing on succession of photosynthetic sources (leaf and silique shell), source loadability, yield and quality in rape. 【Method】 The experiment was conducted in the Dafeng District, Yancheng city of Jiangsu Province, and Nanjing of Jiangsu Province in 2022-2023. The main factors sowing dates were set up at 6 levels, October 17th (SD1), October 23rd (SD2), October 29th (SD3), November 4th (SD4), November 10th (SD5), and November 16th (SD6), and the secondary factors were two conventional varieties: Nannongyou 4 (strong cold resistance) and Zheyou 51 (medium cold resistance). The effects of different sowing dates on dynamic change characteristics of leaf area index (LAI), pod area index (PAI) and source succession, source loadability and rape yield and quality were investigated. 【Result】 (1) The later the sowing dates, the younger the pre-winter seedling age, and the lower the overwintering survival rate. The latest safe date of late-sowing rapeseed in the lower reaches of Yangtze River was the end of October, and the pre-winter seedling age was more than 5 leaves. The sowing date was postponed to November, the leaf age of rape decreased to 1.2-3.6 leaves, and the average overwintering survival rate was less than 30%. The average overwintering rate of Nannongyou 4 in November was 30.9%, which was 14.5% higher than that of Zheyou 51. (2) During the latest safe sowing period, the yield of rapeseed decreased significantly with the delay of sowing dates, and the decrease of plant silique number and 1000-seed weight were the most direct factors causing the decrease of oilseed yield. With the delay of sowing date, compared with SD1, the yield of SD2 and SD3 decreased by 9.6% and 29.0%, the number of plant silique number decreased by 6.1% and 23.9%, and the 1000-seed weight decreased by 4.4% and 6.5%, respectively. The average content of fat and protein in Nannongyou 4 was 42.9% and 25%, which in Zheyou 51 was 47.9% and 22.7%, respectively. (3) With the delay of sowing dates, the maximum LAI and PAI of rape decreased significantly, the slow growth rate before LAI reaching the peak and the fast decline rate after LAI reaching the peak both decreased, and the rapid growth rate of PAI slowed down. The later the sowing dates, the earlier the LAI and PAI succession points (the lower the effective accumulated temperature), and the lower the LAI and PAI succession points (the lower the LAI and PAI succession point). The photosynthetic source and source succession dynamic characteristic values of Nannongyou 4 were generally better than those of Zheyou 51. (4) With the delay of sowing date, the leaf area decreased faster than the seeds yield and pod area, caused the leaf loadability gradually increased, while the silique loadability gradually decreased. Insufficient leaf source and seeds sink were the main reasons for the yield reduction of late sowing. The leaf and shell loadability of Nannongyou 4 was both higher than that of Zheyou 51.【Conclusion】 Within the latest safe sowing period of rapeseed in the lower reaches of Yangtze River, it was advisable to select cold-resistant, high-yield and high-oil varieties with suitable photosynthetic source succession point and high source load, to reduce yield loss caused by late sowing.

PLANT PROTECTION

Pathogenicity and Epidemic Risk of Barley Yellow Striate Mosaic Virus

ZHANG AiHong, YANG Fei, ZHAO YuanYe, ZHAO YiHan, DI DianPing, MIAO HongQin

Scientia Agricultura Sinica. 2024, 57(23):  4686-4697.  doi:10.3864/j.issn.0578-1752.2024.23.009

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【Objective】 Barley yellow striate mosaic virus (BYSMV) is widely distributed in wheat producing areas of northern China. In order to evaluate the epidemic risk of the disease and provide a theoretical foundation for formulating strategies in prevention and control, the pathogenicity to gramineous crops was determined and its natural hosts were also identified.【Method】 The virus was inoculated on 151 varieties of Triticum aestivum, Zea mays, Setaria italica, Sorghum bicolor, Avena sativa, and Hordeum vulgare by planthopper (Laodelphax striatellus) carrying BYSMV, and the uninoculated materials as control. The latent period and symptom progression of the virus on different hosts were observed and analyzed. After 30 d of inoculation, one-step RT-PCR was used to detect the infection of the virus. The pathogenicity of BYSMV to main hosts was analyzed by biological and molecular biological detection. A total of 580 suspected virus-infected samples were collected from gramineous crops growing area in Hebei Province, and BYSMV was detected using one-step RT-PCR to assess the risk of BYSMV epidemic in the field.【Result】 After 9-10 d of inoculation with the virus, the leaves of T. aestivum, S. italica, H. vulgare and A. sativa showed chlorotic stripes, and the plants grew slowly and were seriously dwarfed. All tested varieties of these crops were susceptible to infection. The incidence of tested T. aestivum, S. italica, A. sativa and H. vulgare varieties was 60.00%-100%, 18.18%-100%, 55.56%-100% and 100%, respectively. Yellow stripes and mild dwarfing symptoms could be observed after 15-17 d of virus inoculation on some Z. mays var. saccharata and S. bicolor. Among the tested Z. mays var. saccharata varieties, three varieties showed typical symptoms of BYSMV inoculation and the incidence was 14.29%-21.43%. Eight S. bicolor materials were susceptible to BYSMV with the incidence ranging from 4.00% to 33.33%, but two of them remained asymptomatic. Field surveys revealed that the positive rate of BYSMV in T. aestivum, S. italica and Z. mays var. saccharata samples was 78.03%, 100% and 9.52%, respectively. The positive rate of BYSMV in Gramineae weeds, such as Setaria viridis, Eragrostis minor and Eleusine indica, was 75.68%, 65.38% and 8.33%, respectively. Artificial inoculation indicated that BYSMV could also infect Chloris virgata, Bromus japonica, Echinochloa crusgalli, Brachypodium distachyon and Avena fatua. The disease incidence was 100%, 82.61%, 20.00%, 60.71% and 10.00%, respectively. E. indica, E. crusgalli and A. fatua remained asymptomatic after being infected.【Conclusion】 BYSMV has strong pathogenicity to T. aestivum, S. italica, H. vulgare and A. sativa. Cultivated varieties are susceptible to infection, it is necessary to focus on monitoring the epidemic dynamics of BYSMV to prevent major disasters. The pathogenicity of BYSMV to Z. mays var. saccharata and S. bicolor is relatively weak, and varies in different varieties. Resistant varieties can be selected for prevention and control. The weeds such as C. virgata, S. viridis, E. minor, E. indica are the primary initial infection of BYSMV, it is crucial to remove weeds to prevent the spread of BYSMV.

Accurate and Rapid Identification of Event Purity for Transgenic Soybean Seeds Based on Duplex Real-Time Fluorescence PCR Method

LI YunJing, REN XueZhen, XIAO Fang, JIN Fang, GAO HongFei, JING Qi, WU YuHua, SUN Quan, LI Jun, WANG Pei, ZHAI ShanShan, JIN ShiQiao, WU Gang

Scientia Agricultura Sinica. 2024, 57(23):  4698-4711.  doi:10.3864/j.issn.0578-1752.2024.23.010

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【Objective】 Production and application safety certificates have been successively granted to herbicide-tolerant soybean, including SHZD3201, DBN9004, and ZH10-6. The objective of this study is to establish duplex real-time fluorescence PCR detection methods for these three events, which are applied to the accurate and rapid identification of the purity of soybean transgenic event seeds, and to provide technical support for the quality and safety supervision of soybean transgenic varieties seeds.【Method】 Three event-specific real-time PCR methods for herbicide-tolerant soybean: SHZD3201, DBN9004, ZH10-6, and four soybean-specific real-time PCR methods targeting the Lectin were collected. Through the comparison of ΔCt values, the most effective soybean reference gene detection approach for duplex real-time fluorescence PCR was chosen. The study further adjusted the concentration of primers and probes for both events and Lectin, optimizing the reaction system of duplex real-time fluorescence PCR. The duplex real-time fluorescence PCR methods underwent specificity testing using diverse types of samples. The limit of detection (LOD) was assessed using range of gradient samples containing 2 000, 200, 20, 10, 5, and 1 copies. Using one-step extraction solution for rapid grinding of individual seeds, the diluted crude extract was directly used for duplex real-time fluorescence PCR amplification, thereby conducting event purity testing of individual seeds for herbicide-tolerant soybeans SHZD3201, DBN9004, and ZH10-6.【Result】 Following the determination of the minimum ΔCt value through testing and calculation, the optimal soybean reference gene for detecting Lectin was selected in duplex real-time fluorescence PCR of SHZD3201, DBN9004, and ZH10-6 events. After optimization, the SHZD3201/Lectin duplex real-time fluorescence PCR reaction system exhibited optimal performance with the primer/probe concentration of 0.3 μmol·L^-1/0.15 μmol·L^-1 for SHZD3201 event and the primer/probe concentration of 0.3 μmol·L^-1/0.15 μmol·L^-1 for Lectin. In the DBN9004/Lectin duplex real-time fluorescence PCR reaction system, the primer/probe concentrations for both the DBN9004 event and Lectin were identical, setting at 0.4 μmol·L^-1 for the prime and 0.2 μmol·L^-1 for the probe. In the duplex real-time fluorescence PCR reaction system of ZH10-6/Lectin, the primer/probe concentration of ZH10-6 event was 0.4 μmol·L^-1/0.2 μmol·L^-1, the primer/probe concentration for the Lectin was 0.5 μmol·L^-1/0.25 μmol·L^-1. The three methods had good specificity with the LOD of 10 copies each. Using 100 simulated single-seed samples, after rapid grinding and a 5-fold dilution of the template, the direct duplex real-time fluorescence PCR was used for rapid amplification, and the event purity of transgenic seeds of SHZD3201, DBN9004, and ZH10-6 was successfully detected.【Conclusion】 This study successfully established three duplex real-time fluorescence PCR methods for herbicide-tolerant soybean SHZD3201, DBN9004, and ZH10-6. By combining a rapid DNA extraction method using one-step extraction solution, the research achieved precise and rapid detection of event purity in transgenic soybean seeds.

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

The Effects of Soil Residual Nitrogen from Wheat Season on Summer Soybean Root Nodules, Root System and Yield

WANG Jing, WANG TianShu, WANG Li, ZHOU XinYu, LI TingYu, MENG YiLi, HUANG XinYang, YAO ShuiHong

Scientia Agricultura Sinica. 2024, 57(23):  4712-4724.  doi:10.3864/j.issn.0578-1752.2024.23.011

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【Objective】 This study aims to investigate the impact of residual nitrogen from the wheat season on the soil nitrate content and distribution during the soybean growth season in a winter wheat-summer soybean rotation system. It aims to clarify the dynamic relationship between soil nitrate content and the development of root nodules and root system architecture in soybean and reveal the pathways through which residual nitrogen from wheat season and soil nitrogen dynamics during the soybean season affect soybean yield.【Method】 According to the residual nitrogen levels of wheat stubble soil in double cropping region of the Huang-Huai-Hai Plain, five residual nitrogen levels were set: 5.25 mg·kg^-1 (N₅), 10.00 mg·kg^-1 (N₁₀), 20.00 mg·kg^-1 (N₂₀), 40.00 mg·kg^-1 (N₄₀), and 60.00 mg·kg^-1 (N₆₀) in 2021 and 2022. The soil nitrate content in the root layer (0-40 cm) was measured before soybean sowing, at the six-leaf stage, the flowering stage, and the early pod-filling stage. The above-ground and below-ground biomass, root nodule dry weight, and root traits at the six-leaf stage and the early pod-filling stage, as well as the yield at the harvest stage were analyzed.【Result】 The residual nitrogen increased the soil nitrate content during the soybean growth season, and it rapidly leached with rainfall. Differences in soil nitrate content among treatments persisted until the early pod-filling stage and the six-leaf stage in 2021 and 2022, respectively. The soil nitrate content at the six-leaf stage was 23.44-24.42, 24.98-28.07, 16.99-28.21, 23.81-45.34, 33.37-53.78 mg·kg^-1 in 2021, and 7.63-7.84, 8.02-8.86, 8.32-8.71, 9.43-10.01, 15.40-17.92 mg·kg^-1 in 2022. The soil nitrate content in the root layer at the six-leaf stage was significantly correlated with soybean yield, when it fell within the range of 17.83-40.33 mg·kg^-1, the yield increased with its increase; when it was 7.63-17.83 or 40.33-53.78 mg·kg^-1, the yield decreased with its increase; the yield reached its maximum at 7.63 or 40.33 mg·kg^-1. At the six-leaf stage, an increase in soil nitrate content resulted in higher above-ground biomass, root area, and root width, while it reduced below-ground biomass, root nodule dry weight, lateral root number, and root tip number. At the early pod-filling stage, above-ground biomass, root area, taproot root length, and root tip number showed a decreasing trend after an initial increase and subsequent decrease with the soil nitrate content at the six-leaf stage. Among the root nodules and root traits significantly affected by soil nitrate content at the six-leaf stage, root nodule dry weight, root area, root width, and root tip number were the main factors influencing soybean yield.【Conclusion】 Residual nitrogen from the wheat season affects the soil nitrate content in the root layer during the soybean growth season both temporally and spatially. It regulates the nitrate content in soybean root layer at six-leaf stage, and thus impact above-ground biomass and yield by influencing the development of soybean root nodules and root systems. We recommend to apply nitrogen with appropriate amount and period based on residual nitrogen levels in order to control the nitrate content in the root layer of soybean during the seedling stage at around 7.63 or 40.33 mg·kg^-1. This will enhance nitrogen fertilizer utilization efficiency and achieve higher yields.

Study on the Influence of Rural Population Structure on Agricultural Green Total Factor Productivity

DENG YuanJian, LIU Peng

Scientia Agricultura Sinica. 2024, 57(23):  4725-4745.  doi:10.3864/j.issn.0578-1752.2024.23.012

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【Objective】 The development level of agricultural green total factor productivity in China was measured, and the mechanism and path of rural population structure on agricultural green total factor productivity were analyzed, so as to provide the theoretical basis and decision-making reference for promoting agricultural green development in China. 【Method】 Based on the input-oriented, constant return to scale and the super-efficient SBM model with unexpected output combined with Malmquist- Luenberger (ML) index, the agricultural green total factor productivity of 31 provinces and cities in China from 2000 to 2021 was measured by using the software of MaxDEA. 【Result】 (1) The level of green total factor productivity in Chinese agriculture from 2000 to 2021 exhibited significant temporal and spatial differences. The level of green total factor productivity in agriculture showed a trend of slow decline followed by steady increase and then rapid increase, with the lowest value being 0.5016 in 2005 and the highest value being 0.8872 in 2020, divided by 2005 and 2013; the most provinces were at a medium to high level, with 1 province at a low level, 15 provinces at a medium level, accounting for 48.39%, 12 provinces at a medium to high level, accounting for 38.71%, and 3 provinces at a high level. (2) The various indicators of rural population structure had a significant inhibitory effect on agricultural green total factor productivity. (3) The impact of various indicators, such as rural child dependency ratio, gender ratio, Engel coefficient of rural residents, and average years of education of rural population, on agricultural green total factor productivity mainly depended on the efficiency of agricultural green technology in the rural population structure. (4) The impact of rural population structure on agricultural green total factor productivity had obvious regional characteristics, and the influence coefficients of various indicators varied significantly in different regions. Among the negative impacts of various indicators on agricultural green total factor productivity, the influence of rural child dependency ratio was most significant in the central region, the influence of rural gender ratio was most significant in the eastern region, the influence of average education years of rural population was most significant in the western region, and the influence of Engel coefficient of rural residents was relatively significant nationwide. 【Conclusion】 There were significant differences in the green total factor productivity of agriculture among different provinces in China, and the agricultural population structure inhibited the green development of agriculture, with regional variations. Therefore, it was necessary to build a sustainable rural labor input system, explore and cultivate rural female human resources, build a long-term mechanism for increasing farmers' income, promote the orderly transfer of rural labor, fill the gaps in regional opening up, and improve the ecological oriented financial investment structure for supporting agriculture.

Supply and Demand Balance Analysis of Livestock and Poultry Manure Equivalent Substitution of Chemical Fertilizer in Henan Province

GAO HuiShan, LI GenMing, ZHANG JinCai, JI GuangXing, LI QingSong

Scientia Agricultura Sinica. 2024, 57(23):  4746-4760.  doi:10.3864/j.issn.0578-1752.2024.23.013

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【Objective】 This study aimed to determine the reasonable and appropriate amount and proportion of regional livestock and poultry manure nutrient resources to replace chemical fertilizer. The findings would provide valuable insights for mitigating pollution caused by livestock and poultry breeding, promoting efficient and rational use of organic fertilizers derived from livestock and poultry, enhancing fertilizer efficiency, and facilitating agricultural safety production. 【Method】 Following the concept of substituting chemical fertilizer nitrogen (N) with an equivalent amount of livestock and poultry manure organic nitrogen (N), this study conducted a comparative analysis of the total nutrient content of livestock and poultry manure as well as the purification amount of chemical fertilizers applied crops in Henan Province in 2020. It explored the variations in the fertilizer replacement ability of livestock and poultry manure in different cities. 【Result】 (1) In 2020, the yield of livestock and poultry manure in Henan Province was 1.21×10⁸ t, in which the organic nitrogen (N) and organic phosphorus (P₂O₅) nutrient content were 53.16×10⁴ t and 24.57×10⁴ t, respectively, accounting for 29.2% and 28.7% of the total consumption amount of N and P₂O₅ in Henan Province. (2) The actual N application amount on different crop types was as follows: grain crops (109.03×10⁴ t)>vegetables (34.14×10⁴ t)>oil crops (15.04×10⁴ t)>fruit trees (14.77×10⁴ t)>tea trees (2.72×10⁴ t)>cotton (0.19×10⁴ t), among which grain crops and vegetables took a dominated demand of the consumption, accounting for 81.4% of the total N consumption on crops in Henan Province. The actual N application amount in the 8 cities of Zhoukou, Nanyang, Xinyang, Xinxiang, Zhumadian, Shangqiu, Anyang, and Kaifeng was more than 10⁵ t separately, accounting for 74.2% of the total application amount in Henan Province. (3) The organic nitrogen (N) and organic phosphorus (P₂O₅) of livestock and poultry manure in Henan Province were generally in a state of supply and demand balance. For N nutrient supply, it was less than demand only in Xinyang; whereas the supply exceeded demands in the 10 cities of Kaifeng, Luoyang, Hebi, Puyang, Xuchang, Luohe, Sanmenxia, Shangqiu Zhumadian and Jiyuan; and it was in the balance state in all other cities in Henan. Regarding P₂O₅ nutrient supply from livestock and poultry manure, it was less than demand only in Zhengzhou, and the supply exceeded demands in the 6 cities of Hebi, Puyang, Xuchang, Luohe, Sanmenxia and Zhumadian; it achieved a balance between supply and demand in all other cities in Henan. 【Conclusion】 The livestock and poultry manure production was abundant in Henan Province. The organic nitrogen (N) and organic phosphorus (P₂O₅) of livestock and poultry manure in Henan Province were generally in a state of supply and demand balance, and all livestock and poultry manure could be returned to the field as fertilizers, however, there was an imbalance between regional supply and demand spatially. The fertilizer requirement of different crops varies greatly, and the breeding industry should be reasonably distributed according to the scale and structure of crop cultivation, so as to achieve a balance between supply and demand of livestock and poultry manure as a substitute for chemical fertilizer, and to realize the ecological agricultural cycle mode of combining planting and breeding.

HORTICULTURE

Genetic Diversity Analysis of Mandarin and Excellent Germplasm Screening Based on Whole-Genome Resequencing Data and Phenotypic Traits

LI Pei, HE ZhiLin, TAN YueXia, ZHAO WanTong, FENG JinYing, CHEN GuiHu, YAN Chi, WANG ZiHao, HUANG Ping, JIANG Dong

Scientia Agricultura Sinica. 2024, 57(23):  4761-4773.  doi:10.3864/j.issn.0578-1752.2024.23.014

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【Objective】 In this study, the whole-genome resequencing data and phenotypic identification information were used to reveal the genetic variation and population structure of mandarin germplasm resources and to explore elite citrus germplasms with these information, the aim was to promote citrus variety improvement in the direction of high fruit quality and diversification. 【Method】 In 2021 and 2022, 25 phenotypic traits of fruits and flowers of 473 mandarin accessions were identified in two consecutive years, and the young leaves were collected for DNA extraction, library construction and sequencing. Diversity analysis of phenotypic information was carried out using the variation coefficient and Shannon-Wiener index. To screen out excellent mandarin germplasm, a comprehensive evaluation on mandarin germplasm resources was conducted using correlation analysis, cluster analysis, and principal component analysis. Phylogenetic tree analysis was performed based on resequencing data to analyze the characteristics of the group to which the excellent germplasm belongs. 【Result】 There are extensive variations and rich genetic diversity in 473 mandarin accessions. The variation coefficient of 21 quantitative traits ranges from 9.36% to 76.98%, with an average of 34.95%. and the genetic diversity index from 1.71 to 2.08, with an average of 1.93. The diversity index of the four qualitative traits ranged from 0.35 to 0.98, which was significantly lower than the quantitative traits. Among them, the variation coefficient of segment membrane and juice sac wall weight is the largest, and peel color have the largest genetic diversity index. Principal component analysis was performed on 17 fruit phenotypes, and the top five principal components have the cumulative contribution rate more than 70%. Factor analysis was used to conduct a comprehensive evaluation, it was used to select elite mandarin accessions with the comprehensive evaluation score which ranged from -1.23 to 1.59, the cultivar with the highest score was LP31 (Nishinokaori×Jin Kui), followed by LP45 (Nishinokaori×Jin Kui), LP18 (Benimadona×American Tang Ju), LP3 (Tarocco Blood Orange×Jin Kui). Both the phenotypic cluster analysis results and the phylogenetic tree analysis divided the tested mandarin germplasm resources into 7 groups, different groups can reflect the differences in the origin, variety type and hybrid combination derived from different genetic backgrounds. The germplasms with high comprehensive evaluation scores mostly are concentrated in Group Ⅳ, which showed that these excellent germplasms have similar genetic component. 【Conclusion】 The 473 mandarin accessions used in this study has extensive phenotypic variation and rich genetic diversity. The phenotypic cluster analysis results and phylogenetic tree gave similar classification results, and the excellent germplasm selected based on comprehensive evaluation score have high genetic similarity.

Analysis of Aroma Characteristics and Origin Discrimination of White Peony Tea in Different Origins

LUO Qin, MAO FangHua, CHEN XieYong, CHEN Jing, HUANG Biao, YE NaiXing, ZHENG DeYong, WEI Hang, YAO QingHua

Scientia Agricultura Sinica. 2024, 57(23):  4774-4793.  doi:10.3864/j.issn.0578-1752.2024.23.015

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【Objective】 This study aimed to explore the origin discrimination technology for White Peony tea from small regions based on aroma characterization, so as to provide the technical support for tea origin traceability and brand building.【Method】 The aroma composition of White Peony tea from the main production areas (Zhenghe, Songxi, and Fu’an) was analyzed using the gas chromatography-mass spectrometry (GC-MS) with a headspace solid-phase microextraction method. A model for identifying the origin of White Peony tea was constructed by integrating mathematical and statistical methods to screen for characteristic compounds.【Result】 The aroma compounds in White Peony tea were comprised with aldehydes (24), ketones (23), alcohols (22), esters (14), acids (8), and other compounds (alkanes, alkenes, furans, ethers, anhydrides, phenols, amines, naphthalenes, etc.). The average aroma content in the tea samples was (3 487.93±1 014.04) μg∙kg^-1. The average aroma content in samples from Zhenghe County was significantly higher than that in samples from Songxi County and Fu’an City (P<0.01). For samples from Songxi and Fu’an, there was no significant difference. The total content of alcohols and acids in samples were not significantly different across the three regions (P>0.05), while the total content of ketones, aldehydes, and esters in samples from Zhenghe were significantly higher than those in samples from Fu’an and Songxi (P<0.01). Furthermore, 58 aromatic compounds, including hexanol, 1-octen-3-ol, and benzyl alcohol, showed no significant differences among the three regions (P>0.05). The PLS-DA and OPLS-DA models were well-validated with prediction indices of 0.732 and 0.728, respectively, and the discrimination accuracy of them was 98.89% and 96.67%, respectively. Based on two models, 38 and 46 characteristic aroma substances were identified with VIP values ≥1. The stepwise discriminant analysis also proved stable and reliable, screening 20 characteristic aroma compounds with 100% accuracy in both initial and cross-validation. Using combined OPLS-DA, PLS-DA, and stepwise discriminant analysis, 1H-Pyrrole-2- carboxaldehyde,1-ethyl-, Geranial, Undec-8-enal <cis->, Benzyl alcohol, Cyclohexanol, 2,6-dimethyl-, Hept-5-en-2-one <6-methyl->, Trans-3-Nonen-2-one, Cyclodecanone, Nonanoic acid, Ethyl ester, and Benzyl acetate were identified as the main characteristic aroma compounds in samples from Zhenghe County. Ionone <(E)-, beta-> was the main characteristic aroma compound for samples from Songxi County. The characteristic aroma compounds in samples from Fu’an City were Enanthaldehyde, Hexanol <n->, Nerol, 3-Nonen-5-one, Nonanoic acid, ethyl ester, and Geranyl acetate. 【Conclusion】 A method for discriminating the origin of White Peony tea from small regions based on aroma characteristics was developed.

FOOD SCIENCE AND ENGINEERING

Research Progress on Postharvest Disease and Its Control Techniques of Agaricus bisporus

WANG WenJun, JIANG HaiYan, TIAN Hao, MENG Kuo, GOU WenQing

Scientia Agricultura Sinica. 2024, 57(23):  4794-4805.  doi:10.3864/j.issn.0578-1752.2024.23.016

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Agaricus bisporus (A. bisporus) is one of the most widely cultivated, distributed, and highest-yielding edible fungi in the world. It has excellent qualities due to rich in nutrients and protein, while low in fat, as a food, and tastes tender and delicious. In some regions of China, the A. bisporus industry plays an important role in poverty alleviation and in promoting rural economic development. The mushroom is primarily consumed as fresh food, so appearance and freshness are the main factors determining its commercial value. However, due to its vigorous postharvest physiological activity and lack of a distinct protective structure on the surface, it is highy susceptible to enzymatic browning caused by external factors, such as impact, temperature, and humidity. Additionally, it is easy to be infected by pathogens, such as Pseudomonas, Trichoderma, and Verticillium, leading to browning, decay, and rot, which significantly diminish its appearance, edibility, and commercial value. Due to the large number and variety of pathogens, the current research on postharvest diseases of A. bisporus is mainly focused on disease symptoms, with limited studies on the pathogenic mechanisms of various pathogens. Disease prevention and control for edible fungi like A. bisporus primarily occur during the agricultural cultivation stage. However, postharvest diseases are also unavoidable. Currently, integrated management approaches using physical and chemical methods are commonly employed, such as low temperature and pressure, irradiation, modified atmosphere packaging, and the application of chemical agents like benzoic acid, ascorbic acid, and 1-Methylcyclopropene. Green and safe biological control technologies have become a research hotspot in recent years, including the use of antagonistic bacteria from the genera Pseudomonas and Bacillus. However, many studies on these antagonistic bacteria are still at the laboratory stage, and their practical applications are still a long way off. In recent years, with the expansion of A. bisporus cultivation and industry, the sector has faced numerous urgent issues, including the complexity and diversity of pathogens, which makes it difficult to take targeted measures to control the spread of pathogens promptly, and the lack of safe and effective control products and systematic control measures. To advance research on postharvest diseases, browning mechanisms, and disease control in A. bisporus, this review introduced the main infectious and physiological diseases affecting A. bisporus, systematically summarized the melanogenesis pathways involved in browning mechanisms, and reviewed the current research progress in disease control. This work aimed to provide a theoretical foundation for postharvest preservation techniques for A. bisporus and to contribute to the green, healthy, and sustainable development of the edible fungi industry.

ANIMAL SCIENCE·VETERINARY SCIENCE

The Model Establishment of Lipid Deposition in Primary Chicken Embryo Liver Cells Induced by Oleic Acid

WANG ChaoHui, ZHANG LiMin, SUN Xi, LI SiJing, YANG XiaoJun, LIU YanLi

Scientia Agricultura Sinica. 2024, 57(23):  4806-4814.  doi:10.3864/j.issn.0578-1752.2024.23.017

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【Background】 Fatty liver syndrome (FLS) is a common nutritional metabolic disease in laying hens during the laying period, which includes a multi-stage pathogenesis process, such as “simple steatosis, steatohepatitis, liver fibrosis, and cirrhosis”, causing substantial economic losses in poultry industry. The construction of fatty liver model is the key to exploring the pathogenesis of FLS, screening nutritional regulation measures and subsequent disease prevention and evaluation. However, there are few studies on poultry hepatocyte steatosis models. 【Objective】 Chicken embryo liver (CEL) cells were used to establish a liver lipid deposition model by oleic acid induction, aiming to explore the induction time required for different stages of FLS.【Method】 CEL at the age of 18 embryo was used and when CEL cells grew to about 80%, and the medium containing 400 μmol·L^-1 oleic acid was replaced for treatment. After 24 h, 48 h and 72 h of culture, the cells were stained with oil red O, and the lipid content was measured. At the same time, the mRNA and protein levels of genes related to lipid metabolism, inflammation and fibrosis were analyzed.【Result】 Compared with the Control group, there were a large number of red lipid droplets in 24 h group, 48 h group and 72 h group, and the red color gradually deepened with the increase of induction time (P<0.05), indicating that fat droplets in cells were increased. Meanwhile, the content of TG and TC in the liver were higher than those in the Control group (P<0.05). RT-PCR analysis revealed a significant upregulation of ACC, FAS, ELOVL6, and SREBP gene expression related to lipid synthesis following 24 h of oleic acid induction (P<0.05), indicating that oleic acid lipid deposited on cells after 24 h of processing. ELISA results showed that the content of IL-6, IL-1β and TNF-α were not significantly changed in 24 h (P>0.05). However, an upward trend was observed in IL-6 level with prolonged induction durations (P=0.052). Notably, following 72 h of oleic acid treatment, the levels of IL-6 and IL-1β were significantly increased in Control group (P<0.05), which indicated that acid treatment after 48 h cells began to inflammation reaction and increased with the extension of induction time. In addition, compared with the Control group, the expression level of IL-6 protein in the 48 h group was significantly increased, which was consistent with the ELISA results, while the protein expression levels of COL1A1 and FIBRONECTIN were increased (P<0.05). On the contrary, the protein expression levels of IL-6, COL1A1 and FIBRONECTIN were significantly higher than those in the Control group at 72 h (P<0.05). 【Conclusion】 The early lipid deposition model of hepatocytes could be established by 400 μmol·L^-1 oleic acid induction for 24 h. In addition, inflammation and fibrosis would occurr after 48 h and 72 h induction, respectively.

Detox Dynamics and Reproductive Performance of Langya Chickens Infected with ALV-J

ZHANG HuiYong, WU HuCong, ZHU GuoQiang, LI GuoHui, YU Yan, YIN JianMei, XUE Qian, ZHOU ChengHao, JIANG YiXiu, SU YiJun, HUANG HuaYun, HAN Wei

Scientia Agricultura Sinica. 2024, 57(23):  4815-4824.  doi:10.3864/j.issn.0578-1752.2024.23.018

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【Background】 China is rich in local chicken resources. The spread of J subtype avian leukemia (AL-J) in local chicken breeds has restricted the conservation, development and utilization of local chickens. 【Objective】 By analyzing the infection dynamics of ALV-J positive Langya chickens rooters, the detox dynamics of ALV-J positive Langya chickens hens from pre-opening to self-propagation, and the effects of ALV-J on reproductive performance (fertilization rate, hatching rate, egg number and egg production decline rate after peak laying), this study provided a reference for the AL-J purification of local chickens and was helpful to the protection, development and utilization of local chicken breeds in China. 【Method】 For the ALV-J negative and positive Langya chicken hens, the blood virus isolation and cloacal swab P27 antigen detection were performed every 30 days starting from the 145th day of age. Starting from the age of 330 days for the ALV-J positive rooster group, the semen virus isolation and cloacal swab P27 antigen detection were performed every 4 days. The semen quality and DNA damage degree of ALV-J negative and positive roosters were determined, as well as egg quality of ALV-J negative and positive hens. The insemination test of ALV-J negative and positive male and female chickens (G1 group: positive male chicken × positive hen; G2 group: positive male × negative hen; G3 group: negative male × positive hen; G4 group: negative male chicken × negative hen) was carried out, and the fertilization rate and hatching rate of each group of eggs were counted. The average number of eggs laid per week from the start of laying to 43 weeks of age in ALV-J negative and positive hen groups as well as the egg production decline rate after peak egg production were analyzed. 【Result】 Studies on the detox dynamics of ALV-J positive roosters showed that semen and cloacal swabs were not completely consistent each test, with some positive chickens testing positive only in semen or cloacal swabs, and five out of 20 positive cocks testing negative in consecutive tests. The detox dynamic results of ALV-J positive hens showed that the isolation and detection rate of blood virus was the lowest at 205 days (78.16%) and the highest at 325 days (90.79%), and gradually increased at 265, 295 and 325 days, and was similar at 295 and 325 days. Comparison results of the blood virus isolation and cloacal swab detection of ALV-J negative and ALV-positive chicken flocks showed that cloacal swab detection had a high false positive and missed detection rate. There was no significant difference in routine semen quality and DNA damage detection (DFI) between ALV-J negative and ALV-J positive roosters (P>0.05). The egg quality measurement results of ALV-J negative and ALV-J positive hens showed that there were extremely significant differences in egg weight, Hastelloy units, yolk weight, and albumen height (P<0.01). There was no significant difference in eggshell strength, egg shape index, egg yolk color, and eggshell thickness (P>0.05). The results of the breeding insemination experiment showed that there was no significant difference in the fertilization rate and hatchability of ALV-J-positive male eggs from those of negative chickens. The fertilization rate and hatching rate of chicken eggs were significantly lower than those of negative chickens, and there was no significant difference in the fertilization rate and hatching rate of ALV-J positive male and female chicken eggs. Analysis of the egg production performance of ALV-J negative and positive groups showed that the average number of eggs laid per week from the beginning of laying to 43 weeks of age in the ALV-J positive group was significantly lower than that in the ALV-J negative group (P<0.01). The egg production decline rate after the egg production peak (0.016) was lower than that of the ALV-J positive group (0.033). 【Conclusion】 The semen of ALV-J positive rooters has been tested negative for many times, which could be used as breeding roosters in local chicken breeds with high ALV-J positive rate. According to the results of ALV-J detox dynamic study of ALV-J infection in Langya chickens combined with self-propagation time, the best time period for ALV-J blood virus isolation and detection was around 300 days of hen age. Cloacal swab ALV testing had extremely high false positive and missed detection rates and was not recommended. ALV-J infected hens significantly reduced the number of eggs laid, the decline rate of egg production accelerated after the peak laying, and significantly reduced egg weight, yolk weight, protein height and other indicators, leading to a decrease in fertilization rate and hatching rate. The research results provided data support for the formulation of ALV-J purification plan for Langya breeder chickens.