2023 Vol. 22 No. 5 Previous Issue   

    Crop Science
    Plant Protection
    Animal Science · Veterinary Medicine
    Agro-ecosystem & Environment
    Food Science

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    Review on the fully mulched ridge–furrow system for sustainable maize production on the semi-arid Loess Plateau
    WANG Jin-bin, XIE Jun-hong, LI Ling-ling, ADINGO Samuel
    2023, 22(5): 1277-1290.  DOI: 10.1016/j.jia.2022.09.023
    Abstract ( )   PDF in ScienceDirect  

    The fully mulched ridge–furrow (FMRF) system has been widely used on the semi-arid Loess Plateau of China due to its high maize (Zea mays L.) productivity and rainfall use efficiency.  However, high outputs under this system led to a depletion of soil moisture and soil nutrients, which reduces its sustainability in the long run.  Therefore, it is necessary to optimize the system for the sustainable development of agriculture.  The development, yield-increasing mechanisms, negative impacts, optimization, and their relations in the FMRF system are reviewed in this paper.  We suggest using grain and forage maize varieties instead of regular maize; mulching plastic film in autumn or leaving the mulch after maize harvesting until the next spring, and then removing the old film and mulching new film; combining reduced/no-tillage with straw return; utilizing crop rotation or intercropping with winter canola (Brassica campestris L.), millet (Setaria italica), or oilseed flax (Linum usitatissimum L.); reducing nitrogen fertilizer and partially replacing chemical fertilizer with organic fertilizer; using biodegradable or weather-resistant film; and implementing mechanized production.  These integrations help to establish an environmentally friendly, high quality, and sustainable agricultural system, promote high-quality development of dryland farming, and create new opportunities for agricultural development in the semi-arid Loess Plateau.

    Crop Science
    Development and characterization of a novel common wheat–Mexico Rye T1DL·1RS translocation line with stripe rust and powdery mildew resistance
    LI Jiao-jiao, ZHAO Li, LÜ Bo-ya, FU Yu, ZHANG Shu-fa, LIU Shu-hui, YANG Qun-hui, WU Jun, LI Jia-chuang, CHEN Xin-hong
    2023, 22(5): 1291-1307.  DOI: 10.1016/j.jia.2022.08.039
    Abstract ( )   PDF in ScienceDirect  

    Rye (Secale cereale L., 2n=2x=14, RR) is a significant genetic resource for improving common wheat because of its resistance to multiple diseases and abiotic-stress tolerant traits.  The 1RS chromosome from the German cultivated rye variety Petkus is critical in wheat breeding.  However, its weakened disease resistance highlights the need to identify new resources.  In the present study, a novel derived line called D27 was developed from common wheat and Mexico Rye.  Cytological observations characterized the karyotype of D27 as 2n=42=21 II.  Genomic in situ hybridization indicated that a pair of whole-arm translocated Mexico Rye chromosomes were inherited typically in the mitotic and meiosis stages of D27.  Experiments using fluorescence in situ hybridization (FISH) and gliadin electrophoresis showed that D27 lacked wheat 1DS chromosomes.  They were replaced by 1RS chromosomes of Mexico Rye, supported by wheat simple-sequence repeat markers, rye sequence characterized amplified region markers, and wheat 40K SNP array analysis.  The wheat 1DS chromosomes could not be detected by molecular markers and wheat SNP array, but the presence of rye 1RS chromosomes was confirmed.  Agronomic trait assessments indicated that D27 had a higher tiller number and enhanced stripe rust and powdery mildew resistance.  In addition, dough properties analysis showed that replacing 1DS led to higher viscosity and lower dough elasticity in D27, which was beneficial for cake making.  In conclusion, the novel cytogenetically stable common wheat–Mexico Rye T1DL·1RS translocation line D27 offers excellent potential as outstanding germplasm in wheat breeding programs focusing on disease resistance and yield improvement.  Additionally, it can be valuable for researching the rye 1RS chromosome’s genetic diversity. 

    Revealing the process of storage protein rebalancing in high quality protein maize by proteomic and transcriptomic
    ZHAO Hai-liang, QIN Yao, XIAO Zi-yi, SUN Qin, GONG Dian-ming, QIU Fa-zhan
    2023, 22(5): 1308-1323.  DOI: 10.1016/j.jia.2022.08.031
    Abstract ( )   PDF in ScienceDirect  

    Quality protein maize (QPM) (Zea mays L.) varieties contain enhanced levels of tryptophan and lysine, exhibiting improved nutritive value for humans and livestock.  However, breeding QPM varieties remains challenging due to the complex process of rebalancing storage protein.  This study conducted transcriptome and proteome analyses to investigate the process of storage proteins rebalancing in opaque2 (o2) and QPM.  We found a weak correlation between the transcriptome and proteome, suggesting a significant modulating effect of post-transcriptional events on non-zein protein abundances in Mo17o2 and QPM.  These results highlight the advantages of proteomics.  Compared with Mo17, 672 differentially expressed proteins (DEPs) were identified both in Mo17o2 and QPM, and several of them were associated with storage protein, starch, and amino acid synthesis.  We identified 178 non-zeins as DEPs in Mo17o2 and QPM kernels.  The up-regulated non-zein DEPs were enriched in lysine, tryptophan, and methionine, which affected the protein quality.  Co-expression network analysis identified regulators of storage protein synthesis in QPM, including O2, PBF1, and several transcription factors.  Our results revealed how storage protein rebalancing occurs and identified non-zein DEPs that may facilitate superior-quality QPM breeding. 

    Association mapping of lignin response to Verticillium wilt through an eight-way MAGIC population in Upland cotton
    TIAN Xiao-min, HAN Peng, WANG Jing, SHAO Pan-xia, AN Qiu-shuang, Nurimanguli AINI, YANG Qing-yong, YOU Chun-yuan, LIN Hai-rong, ZHU Long-fu, PAN Zhen-yuan, NIE Xin-hui
    2023, 22(5): 1324-1337.  DOI: 10.1016/j.jia.2022.08.034
    Abstract ( )   PDF in ScienceDirect  

    Lignin metabolism plays a pivotal role in plant defense against pathogens and is always positively correlated as a response to pathogen infection.  Thus, understanding resistance genes against pathogens in plants depends on a genetic analysis of lignin response.  In the study, eight upland cotton lines were used to construct a multi-parent advanced generation intercross (MAGIC) population (n=280), which exhibited peculiar characteristics from the convergence of various alleles coding for advantageous traits.  To measure the lignin response to Verticillium wilt (LRVW), artificial disease nursery (ADN) and rotation nursery (RN) were prepared for MAGIC population planting in four environments.  The stem lignin contents were collected, and the LRVW was measured with the lignin value of ADN/RN in each environment, which showed great variation.  A total of 9323 high-quality single-nucleotide polymorphism (SNP) markers obtained from the Cotton-SNP63K array were employed for genotyping the MAGIC population.  The SNPs were distributed through the whole genome with 4.78 SNP/Mb density, ranging from 1.14 (ChrA06) to 10.08 (ChrD08).  A genome-wide association study was performed using a mixed linear model (MLM) for LRVW, and three stable quantitative trait loci (QTLs), qLRVW-A04, qLRVW-A10 and qLRVW-D05, were identified in more than two environments.  Two key candidate genes, Ghi_D05G01046 and Ghi_D05G01221, were selected within the QTLs through the combination of variations in the coding sequence, induced expression patterns, and function annotations, both of which presented nonsynonymous mutations in coding regions and were strongly induced by Verticillium dahliae. Ghi_D05G01046 encodes a leucine-rich extensin (LRx) protein, which is involved in Arabidopsis cell wall biosynthesis and organization.  Ghi_D05G01221 encodes a transcriptional co-repressor novel interactor of jaz (NINJA), which functions in the jasmonic acid (JA) signaling pathway.  In summary, the study creates valuable genetic resources for breeding and QTL mapping and opens up a new perspective to uncover the genetic basis of VW resistance in upland cotton.

    Dry matter production and panicle characteristics of high yield and good taste indica hybrid rice varieties
    LI Min, ZHU Da-wei, JIANG Ming-jin, LUO De-qiang, JIANG Xue-hai, JI Guang-mei, LI Li-jiang, ZHOU Wei-jia
    2023, 22(5): 1338-1350.  DOI: 10.1016/j.jia.2022.08.033
    Abstract ( )   PDF in ScienceDirect  

    Indica hybrid rice (Oryza sativa) production aims to achieve two crucial targets: high yield and good taste.  This study selected three types of indica hybrid rice according to grain yield and taste value, including high yield and good taste (HYGT), low yield and good taste (LYGT), and high yield and poor taste (HYPT), to analyze yield components, corresponding growth characteristics, and rice taste quality.  When values were averaged across varieties and years, there were almost no differences in taste value between HYGT and LYGT; HYGT showed a significant increase in yield, owing to a higher number of panicles and spikelets per panicle, with a respective increment of 16.2 and 20.6%.  The higher grain yield of HYGT compared with LYGT was attributed to three key factors: a higher leaf area index (LAI) during heading, a higher ratio of grain to leaf, and a higher biomass accumulation at maturity.  HYGT and HYPT achieved similar high yields; however, HYGT had more panicle numbers and lower grain weight.  In addition, HYGT showed a significantly higher taste value than HYPT, attributed to its significantly lower protein and amylose contents, with reductions of 8.8 and 15.7%, respectively.  Lower protein and amylose contents might be caused by a proper matter translocation from vegetative organs to panicle.  This study suggested that reasonable panicle characteristics and translocation efficiency from vegetative organs to panicle during heading to maturity are the key factors in balancing yield and rice taste quality.  These results will provide valuable insights for rice breeders to improve the grain yield and quality of indica hybrid rice.

    Late sowing enhances lodging resistance of wheat plants by improving the biosynthesis and accumulation of lignin and cellulose
    DONG Xiu-chun, QIAN Tai-feng, CHU Jin-peng, ZHANG Xiu, LIU Yun-jing, DAI Xing-long, HE Ming-rong
    2023, 22(5): 1351-1365.  DOI: 10.1016/j.jia.2022.08.024
    Abstract ( )   PDF in ScienceDirect  

    Delayed sowing mitigates lodging in wheat.  However, the mechanism underlying the enhanced lodging resistance in wheat has yet to be fully elucidated.  Field experiments were conducted to investigate the effects of sowing date on lignin and cellulose metabolism, stem morphological characteristics, lodging resistance, and grain yield.  Seeds of Tainong 18, a winter wheat variety, were sown on October 8 (normal sowing) and October 22 (late sowing) during both of the 2015–2016 and 2016–2017 growing seasons.  The results showed that late sowing enhanced the lodging resistance of wheat by improving the biosynthesis and accumulation of lignin and cellulose.  Under late sowing, the expression levels of key genes (TaPAL, TaCCR, TaCOMT, TaCAD, and TaCesA1, 3, 4, 7, and 8) and enzyme activities (TaPAL and  TaCAD) related to lignin and cellulose biosynthesis peaked 4–12 days earlier, and except for the TaPAL, TaCCR, and TaCesA1 genes and TaPAL, in most cases they were significantly higher than under normal sowing.  As a result, lignin and cellulose accumulated quickly during the stem elongation stage.  The mean and maximum accumulation rates of lignin and cellulose increased, the maximum accumulation contents of lignin and cellulose were higher, and the cellulose accumulation duration was prolonged.  Consequently, the lignin/cellulose ratio and lignin content were increased from 0 day and the cellulose content was increased from 11 days after jointing onward.  Our main finding is that the improved biosynthesis and accumulation of lignin and cellulose were responsible for increasing the stem-filling degree, breaking strength, and lodging resistance.  The major functional genes enhancing lodging resistance in wheat that are induced by delayed sowing need to be determined.

    Effects of sowing date and ecological points on yield and the temperature and radiation resources of semi-winter wheat
    ZHANG Zhen-zhen, CHENG Shuang, FAN Peng, ZHOU Nian-bing, XING Zhi-peng, HU Ya-jie, XU Fang-fu, GUO Bao-wei, WEI Hai-yan, ZHANG Hong-cheng
    2023, 22(5): 1366-1380.  DOI: 10.1016/j.jia.2022.08.029
    Abstract ( )   PDF in ScienceDirect  

    Exploring the effects of sowing date and ecological points on the yield of semi-winter wheat is of great significance.  This study aims to reveal the effects of sowing date and ecological points on the climate resources associated with wheat yield in the Rice–Wheat Rotation System.  With six sowing dates, the experiments were carried out in Donghai and Jianhu counties, Jiangsu Province, China using two semi-winter wheat varieties as the objects of this study.  The basic seedlings of the first sowing date (S1) were planted at 300×104 plants ha−1, which was increased by 10% for each of the delayed sowing dates (S2–S6).  The results showed that the delay of sowing date decreased the number of days, the effective accumulated temperature and the cumulative solar radiation in the whole growth period.  The yields of S1 were higher than those of S2 to S6 by 0.22–0.31, 0.5–0.78, 0.86–0.98, 1.14–1.38, and 1.36–1.59 t ha–1, respectively.  For a given sowing date, the growth days increased as the ecological point was moved north, while both mean daily temperature and effective accumulative temperature decreased, but the cumulative radiation increased.  As a result, the yields at Donghai County were 0.01–0.39 t ha–1 lower than those of Jianhu County for the six sowing dates.  The effective accumulative temperature and cumulative radiation both had significant positive correlations with yield.  The average temperature was significantly negatively correlated with the yield.  The decrease in grain yield was mainly due to the declines in grains per spike and 1 000-grain weight caused by the increase in the daily temperature and the decrease in the effective accumulative temperature.

    Novel models for simulating maize growth based on thermal time and photothermal units: Applications under various mulching practices

    LIAO Zhen-qi, ZHENG Jing, FAN Jun-liang, PEI Sheng-zhao, DAI Yu-long, ZHANG Fu-cang, LI Zhi-jun
    2023, 22(5): 1381-1395.  DOI: 10.1016/j.jia.2022.08.018
    Abstract ( )   PDF in ScienceDirect  

    Maize (Zea mays L.) is one of the three major food crops and an important source of carbohydrates for maintaining food security around the world.  Plant height (H), stem diameter (SD), leaf area index (LAI) and dry matter (DM) are important growth parameters that influence maize production.  However, the combined effect of temperature and light on maize growth is rarely considered in crop growth models.  Ten maize growth models based on the modified logistic growth equation (Mlog) and the Mitscherlich growth equation (Mit) were proposed to simulate the H, SD, LAI and DM of maize under different mulching practices based on experimental data from 2015–2018.  Either the accumulative growing degree-days (AGDD), helio thermal units (HTU), photothermal units (PTU) or photoperiod thermal units (PPTU, first proposed here) was used as a single driving factor in the models; or AGDD was combined with either accumulative actual solar hours (ASS), accumulative photoperiod response (APR, first proposed here) or accumulative maximum possible sunshine hours (ADL) as the dual driving factors in the models.  The model performances were evaluated using seven statistical indicators and a global performance index.  The results showed that the three mulching practices significantly increased the maize growth rates and the maximum values of the growth curves compared with non-mulching.  Among the four single factor-driven models, the overall performance of the MlogPTU Model was the best, followed by the MlogAGDD Model.  The MlogPPTU Model was better than the MlogAGDD Model in simulating SD and LAI.  Among the 10 models, the overall performance of the MlogAGDD–APR Model was the best, followed by the MlogAGDD–ASS Model.  Specifically, the MlogAGDD–APR Model performed the best in simulating H and LAI, while the MlogAGDD–ADL and MlogAGDD–ASS models performed the best in simulating SD and DM, respectively.  In conclusion, the modified logistic growth equations with AGDD and either APR, ASS or ADL as the dual driving factors outperformed the commonly used modified logistic growth model with AGDD as a single driving factor in simulating maize growth.


    Cassava MeRS40 is required for the regulation of plant salt tolerance

    MA Xiao-wen, MA Qiu-xiang, MA Mu-qing, CHEN Yan-hang, GU Jin-bao, LI Yang, HU Qing, LUO Qing-wen, WEN Ming-fu, ZHANG Peng, LI Cong, WANG Zhen-yu
    2023, 22(5): 1396-1411.  DOI: 10.1016/j.jia.2023.04.003
    Abstract ( )   PDF in ScienceDirect  

    Soil salinity affects the expression of serine/arginine-rich (SR) genes and isoforms by alternative splicing, which in turn regulates the adaptation of plants to stress.  We previously identified the cassava spliceosomal component 35 like (SCL) and SR subfamilies, belonging to the SR protein family, which are extensively involved in responses to abiotic stresses.  However, the post-transcriptional regulatory mechanism of cassava arginine/serine-rich (RS) subfamily in response to salt stress remains to be explored.  In the current study, we identified 37 genes of the RS subfamily from 11 plant species and systematically investigated the transcript levels of the RS40 and RS31 genes under diverse abiotic stress conditions.  Subsequently, an analysis of the conserved protein domains revealed that plant RS subfamily genes were likely to preserve their conserved molecular functions and played critical functional roles in responses to abiotic stresses.  Importantly, we found that overexpression of MeRS40 in Arabidopsis enhanced salt tolerance by maintaining reactive oxygen species homeostasis and up-regulating the salt-responsive genes.  However, overexpression of MeRS40 gene in cassava reduced salt tolerance due to the depression of its endogenous gene expression by negative autoregulation of its own pre-mRNA.  Moreover, the MeRS40 protein interacted with MeU1-70Ks (MeU1-70Ka and MeU1-70Kb) in vivo and in vitro, respectively.  Therefore, our findings highlight the critical role of cassava SR proteins in responses to salt stress in plants. 

    Identification of key genes involved in flavonoid and terpenoid biosynthesis and the pathway of triterpenoid biosynthesis in Passiflora edulis
    XU Yi, HUANG Dong-mei, MA Fu-ning, YANG Liu, WU Bin, XING Wen-ting, SUN Pei-guang, CHEN Di, XU Bing-qiang, SONG Shun
    2023, 22(5): 1412-1423.  DOI: 10.1016/j.jia.2023.03.005
    Abstract ( )   PDF in ScienceDirect  

    Passion fruit (Passiflora edulis Sims) is a vine of the Passiflora genus in the Passifloraceae family.  The extracted components include flavonoids and terpenoids, which have good anti-anxiety and anti-inflammatory effects in humans.  In this study, we analyzed the transcriptomes of four tissues of the ‘Zixiang’ cultivar using RNA-Seq, which provided a dataset for functional gene mining.  The de novo assembly of these reads generated 96 883 unigenes, among which 61 022 unigenes were annotated (62.99% yield).  In addition to its edible value, another important application of passion fruit is its medicinal value.  The flavonoids and terpenoids are mainly derivatives of luteolin, apigenin, cycloartane triterpenoid saponins and other active substances in leaf extracts.  A series of candidate unigenes in the transcriptome data that are potentially involved in the flavonoid and terpenoid synthesis pathways were screened using homology-based BLAST and phylogenetic analysis.  The results showed that the biosynthesis of triterpenoids in passion fruit comes from the branches of the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate (MEP/DOXP) pathways, which is different from the MVA pathway that is used in other fruit trees.  Most of the candidate genes were found to be highly expressed in the leaves and/or flowers.  Quantitative real-time PCR (qRT-PCR) verification was carried out and confirmed the reliability of the RNA-Seq data.  Further amplification and functional analysis of these putative unigenes will provide additional insight into the biosynthesis of flavonoids and terpenoids in passion fruit.

    Plant Protection
    Vacuolar processing enzyme positively modulates plant resistance and cell death in response to Phytophthora parasitica infection
    GAO Xian-xian, TANG Ya-ling, SHI Qing-yao, WEI Yu-shu, WANG Xiao-xue, SHAN Wei-xing, QIANG Xiao-yu
    2023, 22(5): 1424-1433.  DOI: 10.1016/j.jia.2022.08.124
    Abstract ( )   PDF in ScienceDirect  

    Oomycete, particularly Phytophthora species, causes the most devastating crop diseases, such as potato late blight, and threatens the sustainable crop production worldwide.  Our previous studies identified Resistance to Phytophthora parasitica 1 (RTP1) as a negative regulator of Arabidopsis resistance to multiple biotrophic pathogens and RTP1 loss-of-function plants displayed rapid cell death and reactive oxygen species (ROS) production during early colonization of Pparasitica.  In this study, we aim to decipher the mechanism of RTP1-mediated cell death, and identify a member of vaculoar processing enzymes (VPEs), γVPE, playing a role in rtp1-mediated resistance to Pparasitica and cell death occurrence.  Our results showed up-regulation of the expression of γVPE as well as increased VPE/caspase 1-like protease activity in Pparasitica-infected rtp1 mutant plants.  Besides, we found that the VPE/caspase 1-like protease activity was required for the cell death occurrence in Arabidopsis plants during the infection of Pparasitica as well as rtp1-mediated resistance to Pparasitica.  Further pathogenicity assays on either Arabidopsis γvpe mutant plants or leaves of Nicotiana benthamiana with transient overexpression of γVPE demonstrated γVPE could positively affect plant resistance to Pparasitica.  Together, our studies suggest that γVPE might function as an important regulator of plant defense and cell death occurrence in response to Pparasitica infection, and VPE/caspase 1-like protease activity is required for rtp1-mediated resistance to Pparasitica.

    Herbicidal activity and biochemical characteristics of the botanical drupacine against Amaranthus retroflexus L.

    YU Hua-long, TIAN Ci, SHEN Rong-yan, ZHAO Han, YANG Juan, DONG Jin-gao, ZHANG Li-hui, MA Shu-jie
    2023, 22(5): 1434-1444.  DOI: 10.1016/j.jia.2022.08.120
    Abstract ( )   PDF in ScienceDirect  

    Botanical herbicide has been a hot topic in the research and development of novel pesticides.  The herbicidal activity and biochemical characteristics of the botanical compound drupacine were studied by evaluating its effects on seed germination, seedling growth, morphological and physiological characteristics of Amaranthus retroflexus.  Drupacine inhibited seed germination and seedling growth, and had a median inhibition concentration (IC50) value of 38.99 mg L−1 against Aretroflexus root.  The α-amylase activity and soluble sugar content in treated plants were significantly lower than that of the control.  The expression of α-amylase gene was dosage-dependently inhibited compared to the untreated control.  This suggested that inhibition of α-amylase activity was a mode of action on seed germination.  The root hairs were significantly decreased and part of the root cap fell off after treatment with drupacine.  The ultrastructure observation showed that cell damage of root tips increased with the treatment time.  Drupacine also increased the relative conductivity and malondialdehyde (MDA) content.  Peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) activities were significantly enhanced in the treatment compared to the control.  These findings indicated that the physiological and biochemical reaction changes leading to morphological and membrane injuries were the main effects of drupacine on the inhibition of seedling growth.  Drupacine can be developed as a botanical herbicide. 

    mgr-mir-9 implicates Meloidogyne graminicola infection in rice by targeting the effector MgPDI

    TIAN Zhong-ling, ZHOU Jia-yan, ZHENG Jing-wu, HAN Shao-jie
    2023, 22(5): 1445-1454.  DOI: 10.1016/j.jia.2022.08.127
    Abstract ( )   PDF in ScienceDirect  

    MicroRNAs (miRNAs), a class of small non-coding RNAs, are crucial endogenous gene regulators in a range of animals, including plant-parasitic nematodes.  Meloidogyne graminicola is an obligate sedentary endoparasite of rice and causes significant yield losses.  A number of studies focused on the roles of Mgraminicola effectors during the parasitic process; however, how nematode miRNAs regulate its effectors needs elucidating.  In this research, we analyzed a cluster of Mgraminicola miRNAs obtained at the second-stage juveniles (J2s) stage that are closely linked to the regulation of Mgraminicola effectors.  There are 49 767 105 total clean reads obtained from three libraries.  A total of 233 known miRNAs and 21 novel miRNAs were identified.  Among the known miRNAs, mgr-lin-4, mgr-mir-1, mgr-mir-100, mgr-mir-86, mgr-mir-279, mgr-mir-87, mgr-mir-71, mgr-mir-9, mgr-mir-50, mgr-mir-72, and mgr-mir-34 are the most abundant 11 miRNAs families.  Moreover, the expression levels of selected miRNAs were validated by real-time quantitative PCR.  We hypothesized that these miRNAs might regulate the expression of secreted effectors during the J2s stage to facilitate its infection.  Consistent with this, we found that mgr-mir-9 targets MgPDI, an important Mgraminicola effector mRNA.  In addition to that, J2s treated with mgr-mir-9 mimics showed down-regulation of MgPDI expression and reduced reproductive ability, alluding mgr-mir-9 is involved in nematode infection.  These results provide novel insight into the regulatory functions of Mgraminicola miRNAs during the infection and identify miRNAs and their effector targets as potential key management targets to limit parasite survival during the early stages of infection.

    Brown planthopper E78 regulates moulting and ovarian development by interacting with E93

    ZHENG Shi-wen, JIANG Xiao-juan, MAO Yi-wen, LI Yan, GAO Han, LIN Xin-da
    2023, 22(5): 1455-1464.  DOI: 10.1016/j.jia.2022.08.106
    Abstract ( )   PDF in ScienceDirect  

    The brown planthopper (Nilaparvata lugens) is the main migratory pest in many rice growing areas in Asia.  E78 is a member of the nuclear hormone receptor superfamily which plays an important role in egg development and maternal regulation of early embryogenesis.  In this study, brown planthopper E78 (NlE78) was cloned, and the predicted amino acid sequence showed that it contains two conserved domains: NR-LBD and DBD.  qRT-PCR showed that the expression of NlE78 is high in the fifth instar nymphs and the ovaries of females.  After downregulation of NlE78, the rate of moulting failure (33.2%) increased significantly, and ovarian development was delayed.  However, when NlE78 was downregulated together with NlE93, the emergence rate increased significantly (78.79%), and ovarian development was similar to that when NlE78 was downregulated but not delayed.  A co-immunoprecipitation experiment showed that NlE78 interacts with NlE93, a crucial downstream transcription factor of the ecdysone signalling pathway.  Cellular localization by immunofluorescence revealed that NlE78 and NlE93 are expressed in the nucleus.  This study indicates that NlE78 regulates ovarian development and moulting, possibly through its interaction with NlE93.  This study is of great significance for the development of new pesticides and control methods based on newly discovered targets.

    Toxicity and horizontal transfer of bifenthrin and dimefluthrin against the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae), and the efficacy of their dust applications in the field

    LIANG Ming-rong, SHUANG You-ming, DENG Jie-fu, PENG Li-ya, ZHANG Sen-quan, ZHANG Chen, XU Yi-juan, LU Yong-yue, WANG Lei
    2023, 22(5): 1465-1476.  DOI: 10.1016/j.jia.2022.12.010
    Abstract ( )   PDF in ScienceDirect  

    The red imported fire ant, Solenopsis invicta Buren, poses a significant threat to biodiversity, agriculture, and public health in its introduced ranges.  While chemicals such as toxic baits and dust are the main methods for Sinvicta control, toxic baits are slow, requiring approximately one or two weeks, but dust can eliminate the colony of fire ants rapidly in just three to five days.  To explore more active ingredients for fire ant control using dusts, the toxicity of bifenthrin and dimefluthrin, the horizontal transfer of bifenthrin and dimefluthrin dust and their efficacy in the field were tested.  The results showed that the LD50 (lethal dose) values of bifenthrin and dimefluthrin were 3.40 and 1.57 ng/ant, respectively.  The KT50 (median knockdown time) and KT95 (95% knockdown time) values of a 20 μg mL–1 bifenthrin dose were 7.179 and 16.611 min, respectively.  The KT50 and KT95 of a 5 μg mL–1 dimefluthrin dose were 1.538 and 2.825 min, respectively.  The horizontal transfers of bifenthrin and dimefluthrin among workers were effective.  The mortality of recipients (secondary mortality) and secondary recipients (tertiary mortality) were both over 80% at 48 h after 0.25, 0.50 and 1.00% bifenthrin dust treatments.  The secondary mortality of recipients was over 99% at 48 h after 0.25, 0.50 and 1.00% dimefluthrin dust treatments, but the tertiary mortality was below 20%.  The field trial results showed that both bifenthrin and dimefluthrin exhibited excellent fire ant control effects, and the comprehensive control effects of 1.00% bifenthrin and dimefluthrin dusts at 14 d post-treatment were 95.87 and 85.70%, respectively.

    Photosensitivity and a precise combination of size-dependent lambda-cyhalothrin microcapsules synergistically generate better insecticidal efficacy 
    GAO Yue, LUO Jian, SUN Yue, ZHANG Hua-wei, ZHANG Da-xia, LIU Feng, MU Wei, LI Bei-xing
    2023, 22(5): 1477-1488.  DOI: 10.1016/j.jia.2022.08.081
    Abstract ( )   PDF in ScienceDirect  

    In this study, lambda-cyhalothrin (LC) loaded polyurea microcapsules (MCs) with different particle sizes were fabricated.  All of the MCs showed varying degrees of physical collapse, which was more obvious among those with smaller particle sizes.  MCs with particle sizes of 1.38 μm (MC-S), 5.13 μm (MC-M) and 10.05 μm (MC-L) had shell thicknesses of 39.6, 50.3 and 150.1 nm, respectively.  MCs with smaller particles tended to have significantly faster release profiles, and the MC-S group had much higher bioactivity against Agrotis ipsilon and better foliar affinity on the peanut leaves (indicated by rainfastness) than MC-M and MC-L.  All of the MCs exhibited light-enhanced release profiles and had much slower degradation compared with the emulsifiable concentrate (EC) group, among which MC-L had the slowest degradation.  To generate MCs with both favorable quick efficacy and long-lasting efficacy, binary mixtures of MC-S, MC-M and MC-L were produced by mixing them in pairs at ratios of 2:1, 1:1 and 1:2.  The mixture of MC-S:MC-L at 1:2 showed the best comprehensive efficacy in the peanut foliar spray scenario among the nine tested combinations, and its effective duration was three times longer than that of EC.  Overall, the precise combination of MCs with different particle sizes can regulate the efficacy of pesticide control and serve as a strategy for the better utilization of pesticides.

    Animal Science · Veterinary Medicine
    Effects of carcass weight, sex and breed composition on meat cuts and carcass trait in finishing pigs
    XIE Lei, QIN Jiang-tao, RAO Lin, CUI Deng-shuai, TANG Xi, XIAO Shi-jun, ZHANG Zhi-yan, HUANG Lu-sheng
    2023, 22(5): 1489-1501.  DOI: 10.1016/j.jia.2022.08.122
    Abstract ( )   PDF in ScienceDirect  

    Pork cutting is a very important processing in promoting economic appreciation across the swine business chain.  The goal of this research is to determine the proportion and weight of meat cuts, as well as to analyze the effects of carcass weight, sex and breed composition on meat cuts.  Simultaneously, we investigate the correlation between meat cuts, carcass traits and meat quality traits.  To assess 17 meat cut traits, 12 carcass traits and 6 meat quality traits, we sample 2 012 pigs from four breeds, including Landrace (LD), Yorkshire (YK), Landrace Yorkshire (LY), and Duroc Landrace Yorkshire (DLY).  The results showed that carcass weight, sex and breed composition have significant effects on the weight and proportion of most meat cuts.  The proportion of cuts for muscle and bone decrease as carcass weight grows, whereas the proportion of cuts for fat increases.  Moreover, the thickness of four-point backfat was significantly increasing (P<0.001) with increase of carcass weights, indicating that large amount of intaking energy in the late finishing stage was used for fat deposition.  Besides, the proportion of Shoulder cut (SC) and Back fat (BF) in barrows was significantly higher (P<0.001) than that in sows, whereas the Leg cut (LC) showed the opposite trend.  The Loin (LO) proportion and Loin muscle area (LMA) of barrows were significantly lower (P<0.001), but the proportion of fat areas in the image (PFAI) and visual marbling score (VMS) were significantly higher (P<0.001) than those of sows, respectively.  In terms of breeds, LD had the longest straight carcass length, significantly longer (P<0.001) than the other three breeds, which partially explains why LD had the largest proportion of the Middle cut (MC).  Moreover, the proportion of SC in DLY was the highest.  Last but not least, the correlations between the proportions of most meat cuts, and also between meat cuts and meat quality or carcass traits were low or not significant (P>0.05).  The effects of carcass weight, sex and breed composition on the meat cuts, meat quality and carcass traits are breed and growth stage dependent.  It also reflects the asynchrony of the growth curve between different sexes.  Our results laid an important foundation for breeding pig carcass cuts and composition.

    MicroRNA transcriptome of skeletal muscle during yak development reveals miR-652 regulates myoblasts differentiation and survival by targeting ISL1
    ZHOU Xue-lan, GUO Xian, LIANG Chun-nian, CHU Min, WU Xiao-yun, YAN Ping
    2023, 22(5): 1502-1513.  DOI: 10.1016/j.jia.2022.08.116
    Abstract ( )   PDF in ScienceDirect  

    The growth and development of skeletal muscle also determine the meat production of yak, ultimately affecting the economic benefits.  Hence, improving growth performance is a top priority in the yak industry.  Skeletal muscle development is a complex process involving the regulation of several genes, including microRNAs (miRNAs).  However, the transcription of miRNAs in yak skeletal muscle during prenatal to postnatal stages is unknown.  We used small RNA sequencing (small RNA-Seq) to determine the global miRNAs of longissimus dorsi muscle from yak (the samples were collected from three fetuses and three adults).  Totally 264 differently expressed miRNAs (|log2(fold change)|>1 and P-value≤0.05) were detected between the two groups.  Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that differently expressed miRNAs-targeted genes participated in pathways associated with muscle development, such as MAPK, PI3K-Akt, and Hippo signaling pathways, etc.  MiR-652, which was up-regulated in the fetal group, was transfected into C2C12 myoblasts to examine its role.  miR-652 promoted (P≤0.05) proliferation and differentiation, but inhibited (P≤0.001) apoptosis at early period.  Furthermore, miR-652 reduced (P≤0.001) the proportion of C2C12 myoblasts in the G1 phase while increasing (P≤0.01) the proportion of cells in the S and G2 phases.  Dual-luciferase reporter assays indicated that ISL1 served as a target of miR-652.  In general, these findings expand our understanding of yak skeletal muscle miRNAs, and suggested that miR-652 probably regulated myogenesis by regulating ISL1.

    Molecular epidemiology, characterization of virulence factors and antibiotic-resistance profile of Streptococcus agalactiae isolated from dairy farms in China and Pakistan
    Ambreen LEGHARI, Shakeel Ahmed LAKHO, Faiz Muhammad KHAND, Khaliq ur Rehman BHUTTO, Sameen Qayoom LONE, Muhammad Tahir ALEEM, Iqra BANO, Muhammad Ali CHANDIO, Jan Muhammad SHAH, LIN Hui-xing, FAN Hong-jie
    2023, 22(5): 1514-1528.  DOI: 10.1016/j.jia.2022.10.004
    Abstract ( )   PDF in ScienceDirect  

    Streptococcus agalactiae is one of the most common pathogens that cause bovine mastitis worldwide. Identifying pathogen prevalence and virulence factors is critical for developing prevention and control approaches. Herein, 1161 milk samples from various dairy farms in China (n=558) and Pakistan (n=603) were collected between 2019-2021 and were subjected to S. agalactiae isolation. Prevalence, serotyping, virulence genes, and antibiotic-resistant genes of S. agalactiae were evaluated by PCR assay. All isolates were characterized for haemolysis, biofilm production, cytotoxicity, adhesion, and invasion on bovine mammary epithelial cells. The prevalence of S. agalactiae-induced mastitis in cattle was found to be considerably higher in Pakistan than in China. Jiangsu and Sindh provinces had the highest area-wise prevalence in China and Pakistan, respectively. Serotypes Ia and II were prevalent in both countries, whereas serotype III was found only in Pakistan. Moreover, all isolates tested positive for PI-2b gene but negative for PI-1 and PI-2a genes. All isolates harboured cfb, cylE, hylB, and fbsB virulent genes, whereas many of them lacked bibA, rib and bca. However, the absence of bac and scp genes in Chinese isolates and cspA in Pakistani isolates was noted, while spb1 and lmb were not detected in isolates of both countries. Pakistani isolates, particularly serotype Ia-positive, had a considerably higher ability to produce biofilm, haemolysis, cytotoxicity, adhesion, and invasion than Chinese isolates. Most of the isolates were phenotypically resistant to tetracycline, erythromycin, and clindamycin and genotypic resistance was confirmed by the presence of ermA, ermB, tetM and tetO genes. Our study highlights the antimicrobial resistance profile and virulence-related factors contributing to the epidemiological spread of mastitis-causing S. agalactiae in China and Pakistan. The findings may facilitate future studies designed to develop improved treatment and control strategies against this pathogen. 

    Agro-ecosystem & Environment
    The effects of co-utilizing green manure and rice straw on soil aggregates and soil carbon stability in a paddy soil in southern China
    ZHANG Zi-han, NIE Jun, LIANG Hai, WEI Cui-lan, WANG Yun, LIAO Yu-lin, LU Yan-hong, ZHOU Guo-peng, GAO Song-juan, CAO Wei-dong
    2023, 22(5): 1529-1545.  DOI: 10.1016/j.jia.2022.09.025
    Abstract ( )   PDF in ScienceDirect  
    The co-utilization of green manure (GM) and rice straw (RS) in paddy fields has been widely applied as an effective practice in southern China. However, its effects on soil aggregate and soil organic carbon (SOC) stability remain unclear. In the present study, the effect of GM, RS, and co-utilization of GM and RS on particle size distribution of soil aggregates and SOC density fractions were measured in a field experiment. The experiment included six treatments, i.e., winter fallow (WF) without RS return (Ctrl), WF with 50% RS return (1/2RS), WF with 100% RS return (RS), GM without RS return (GM), GM with 50% RS return (GM1/2RS) and GM with 100% RS return (GMRS). The results showed that the proportion of small macro-aggregates (0.25–2 mm) and the mean weight diameter (MWD) of aggregates in the GMRS treatment was greater (by 18.9 and 3.41%, respectively) than in the RS treatment, while the proportion of silt+clay particles (<0.053 mm) was lower (by 14.4%). The concentration of SOC in microaggregates (0.053–0.25 mm) and silt+clay particles was higher in the GMRS treatment than in GM and RS treatments individually. The concentration and proportion of free light organic carbon (fLOC) in aggregates of various particle sizes and bulk soil was greater in the GMRS treatment than the RS treatment, whereas the concentration and proportion of mineral-associated organic carbon in small macroaggregates, microaggregates, and bulk was lower in the GMRS treatment than in the RS treatment. The proportion of intra-aggregate particulate organic carbon (iPOC) was greater in the GMRS treatment than in GM treatment. The GMRS treatment had strong positive effects on iPOC in small macroaggregates, suggesting that SOC was transferred from fLOC to iPOC. In conclusion, co-utilizing green manure and rice straw cultivated the SOC pool by increasing the concentration of fLOC and improved soil carbon stability by promoting the sequestration of organic carbon in iPOC as a form of physical protection.
    Inversion tillage with straw incorporation affects the patterns of soil microbial co-occurrence and multi-nutrient cycling in a Hapli-Udic Cambisol
    CHEN Xu, HAN Xiao-zeng, WANG Xiao-hui, GUO Zhen-xi, YAN Jun, LU Xin-chun, ZOU Wen-xiu
    2023, 22(5): 1546-1559.  DOI: 10.1016/j.jia.2022.12.011
    Abstract ( )   PDF in ScienceDirect  
    Inversion tillage with straw amendment is widely applied in northeastern China, and it can substantially increase the storage of carbon and improve multiple subsoil functions. Soil microorganisms are believed to be the key to this process, but research into their role in subsoil amelioration is limited. Therefore, a field experiment was conducted in 2018 in a region in northeastern China with Hapli-Udic Cambisol using four treatments: conventional tillage (CT, tillage to a depth of 15 cm with no straw incorporation), straw incorporation with conventional tillage (SCT, tillage to a depth of 15 cm), inversion tillage (IT, tillage to a depth of 35 cm) and straw incorporation with inversion tillage (SIT, tillage to a depth of 35 cm). The soils were managed by inversion to a depth of 15 or 35 cm every year after harvest. The results indicated that SIT improved soil multi-nutrient cycling variables and increased the availability of key nutrients such as soil organic carbon, total nitrogen, available nitrogen, available phosphorus and available potassium in both the topsoil and subsoil. In contrast to CT and SCT, SIT created a looser microbial network structure but with highly centralized clusters by reducing the topological properties of average connectivity and node number, and by increasing the average path length and the modularity. A Random Forest analysis found that the average path length and the clustering coefficient were the main determinants of soil multi-nutrient cycling. These findings suggested that SIT can be an effective option for improving soil multi-nutrient cycling and the structure of microbial networks, and they provide crucial information about the microbial strategies that drive the decomposition of straw in Hapli-Udic Cambisol.
    Effects of planting patterns plastic film mulching on soil temperature, moisture, functional bacteria and yield of winter wheat in the Loess Plateau of China
    ZHAO Xiao-dong, QIN Xiao-rui, LI Ting-liang, CAO Han-bing, XIE Ying-he
    2023, 22(5): 1560-1573.  DOI: 10.1016/j.jia.2023.02.026
    Abstract ( )   PDF in ScienceDirect  

    The yield of winter wheat is hindered by drought and low temperature in the Loess Plateau of China.  Two common mulching methods to conserve soil moisture, ridge furrows with plastic film mulching (RP) and flat soil surfaces with plastic film mulching (FP) are helpful for wheat production.  Our previous study indicated that FP could improve wheat yield more effectively than RP, but the reason remains unclear.  The effect of mulching method on functional bacteria also needs to be further studied.  In this study, winter wheat was employed to evaluate the impacts of mulching method on soil temperature, moisture content, microorganisms and grain yield.  The results showed that FP had a warming effect when the soil temperature was low and a cooling effect when the temperature was too high.  However, the ability to regulate soil temperature in the RP method was unstable and varied with year.  The lowest negative accumulated soil temperature was found in the FP treatment, which was 20–89 and 43–99% lower than that of the RP and flat sowing with non-film mulching control (NP) treatments, respectively.  Deep soil moisture was better transferred to topsoil for wheat growth in the FP and RP treatments than the NP treatment, which made the topsoil moisture in the two treatments (especially FP) more sufficient than that in the NP treatment during the early growing stage of wheat.  However, due to the limited water resources in the study area, there was almost no difference between treatments in topsoil water storage during the later stage.  The wheat yield in the FP treatment was significantly higher, by 12–16 and 23–56%, respectively, than in the RP and NP treatments.  Significant positive correlations were observed among the negative accumulated soil temperature, spike number and wheat yield.  The Chao1 and Shannon indices in the RP treatment were 17 and 3.9% higher than those in the NP treatment, respectively.  However, according to network relationship analysis, the interspecific relationships of bacteria were weakened in the RP treatment.  Phosphorus solubilizing, ammonification and nitrification bacteria were more active in the RP than in the FP treatment, and microbes with nitrate reduction ability and plant pathogens were inhibited in the RP treatment, which improved nutrient availability and habitat for wheat.

    Food Science
    Elucidation of the structure, antioxidant, and interfacial properties of flaxseed proteins tailored by microwave treatment
    YU Xiao, DUAN Zi-qiang, QIN Xiao-peng, ZHU Ying-ying, HUANG Feng-hong, PENG Deng-feng, BAI Yan-hong, DENG Qian-chun
    2023, 22(5): 1574-1589.  DOI: 10.1016/j.jia.2023.04.021
    Abstract ( )   PDF in ScienceDirect  
    The microwave treatment is commonly applied to flaxseed to release nutrients, inactivate enzymes, remove cyanogens, and intensify flavors. The current study aimed to explore the influences of microwave exposure on the antioxidant and interfacial properties of flaxseed protein isolates (FPI), focusing on the altering composition and molecular structure. The results showed that after microwave exposure (700 W, 1–5 min), more compact assembly of storage proteins and subsequent permeation by membrane fragments of oil bodies occurred for cold-pressing flaxseed flours. Moreover, the particle sizes of FPI was progressively reduced with the decrement ranged from 37.84 to 60.66% , whereas the zeta potential values initially decreased and then substantially recovered during 1–5 min of microwave exposure. The conformation unfolding, chain cross-linking, and depolymerization were sequentially induced for FPI based on the analysis of fluorescence emission spectra, secondary structure, and protein subunit profiles, thereby affecting the dispersion or aggregation properties between albumin and globulin fractions in FPI. Microwave exposure retained specific phenolic acids and superior antioxidant activities of FPI. The inferior gas–water interface absorption and the loose/porous assembly structure were observed for the foams prepared by FPI, concurrent with obviously shrinking foaming properties upon microwave exposure. Improving oil–water interface activities of FPI produced the emulsion droplets with descending sizes and dense interface coating, which were then mildly destabilized due to the lipid leakage and weakened rheological behavior with microwave exposure extended to 5 min. Our findings elucidated that microwave treatment could tailor the application functionality of protein fractions in flaxseed based on their structural remodeling.
    The effects of maltodextrin/starch in soy protein isolate–wheat gluten on the thermal stability of high-moisture extrudates
    XIE Si-han, WANG Zhao-jun, HE Zhi-yong, ZENG Mao-mao, QIN Fang, Benu ADHIKARI, CHEN Jie
    2023, 22(5): 1590-1602.  DOI: 10.1016/j.jia.2023.04.013
    Abstract ( )   PDF in ScienceDirect  
    This study aimed to investigate the interaction between maltodextrin/starch of different molecular weight distributions and soy protein isolate (SPI)–wheat gluten (WG) matrix during high-moisture extrusion.  Two maltodextrins (dextrose equivalent (DE): 10 and 20) and wheat starch were extruded with SPI–WG blend in a system of 65, 70, and 75% moisture to investigate their effects on texture and thermal stability.  Incorporating 5% maltodextrin (DE10) in the SPI–WG matrix improved the fiber structure and thermal stability.  When wheat starch was thoroughly gelatinized during subsequent sterilization, the fiber structure and thermal stability were also improved.  It was found that the plasticization caused by small-molecular weight saccharides and enhanced phase separation caused by large-molecular weight saccharides changed the melting temperature of blends and significantly improved the texture and thermal stability of extrudates.
    Developing a duplex ARMS-qPCR method to differentiate genotype I and II African swine fever viruses based on their B646L genes
    DING Lei-lei, REN Tao, HUANG Lian-yu, Weldu TESFAGABER, ZHU Yuan-mao, LI Fang, SUN En-cheng, BU Zhi-gao, ZHAO Dong-ming
    2023, 22(5): 1603-1607.  DOI: 10.1016/j.jia.2023.02.035
    Abstract ( )   PDF in ScienceDirect  

    African swine fever (ASF), caused by the African swine fever virus (ASFV), is an acute, hemorrhagic, and contagious disease of domestic pigs and wild boars.  The disease is notifiable and listed by the World Organization for Animal Health (WOAH) (Wang N et al. 2019).  The outcomes of ASF infection can be peracute, acute, subacute, and chronic, depending on the virulence of ASFVs.  According to the report of WOAH (https://www.woah.org/app/uploads/2022/12/asf-report24.pdf), from January 2020 to December 2022, ASF led to more than  2 million pig losses.  Currently, ASFV persists continuously in more than 23 countries and poses a serious threat to the global swine industry.  ASF invaded China on 3 August, 2018, caused by genotype II virulent Georgia-07-like ASFVs (Wen et al. 2019; Zhao et al. 2019; Wang et al. 2020; Wang L et al. 2022).  An experimental study showed that Georgia-07-like ASFV HLJ/18 isolated in China is highly lethal and efficiently transmissible in domestic pigs (Zhao et al. 2019; Jiang et al. 2021).  During the past four years, genotype II Georgia-07-like ASFVs dominantly spread in China.  However, the low virulent genotype II and I ASFVs have been successively reported in China in 2020 and 2021, respectively (Sun et al. 2021a, b; Shi et al. 2022).  Compared with the high virulent genotype II HLJ/18 strain, the low virulent genotype I and II ASFVs had lower virulence and high transmissibility in pigs and induced persistent and chronic infection showing irregular virus shedding at low levels (Sun et al. 2021a, b; Tsegay et al. 2022; Wang P et al. 2022).  Notably, when different genotype I and genotype II viruses infect the same pig in the field, a novel virus may be generated through viral genome recombination, which brings new problems and challenges for the prevention and control of ASF in China.  Thus, a diagnostic method that differentiates genotype I and II ASFVs with high sensitivity and stability is urgently needed and will be helpful for the prevention and control of ASF in China.  

    ASFVs have been divided into at least 24 genotypes based on the C-terminus of the B646L gene with 478 nt (Bastos et al. 2003).  B646L gene is one of the most used target genes for ASF diagnosis, which is also the target gene for the WOAH recommended PCR and fluorescent quantitative PCR assays (Agüero et al. 2003; King et al. 2003).  Sanger sequencing of targeted amplification of the B646L genes is the main genotyping approach for ASFVs.  Recently, Li et al. (2022) developed the duplex real-time PCR assay based on the ASFV E296R gene, and Cao et al. (2022) established the TaqMAN-MGB probe assay based on the N-terminal sequences of the B646L gene (Cao et al. 2022; Li et al. 2022), which could distinguish genotype I and II ASFVs with detection limits of 10 copies.  However, the target genes or regions in their methods were out of ASFV genotyping regions.  

    Single nucleotide polymorphism (SNP) is a single base change at a specific position in the genome of different individuals and can be used as a genotyping marker for the detection of different individual genotypes (Gut 2001).  The amplification refractory mutation system (ARMS), also named Allele-specific PCR (AS-PCR), relies on the extension of primer only when its 3´ end has a perfect complement to the template (Wang M et al. 2019).  ARMS-qPCR technology has been developed and widely used in SNP detection and genotyping (Ochsenreither et al. 2010; Shi et al. 2013; Wang M et al. 2019).  Compared with other assays for SNP detection and genotyping, ARMS-qPCR has the advantage of low-cost, simple operation, high sensitivity, and rapid and real-time detection.

    Here, 126 complete or partial B646L genes of ASFVs, including 78 genotype I and 48 genotype II viruses, were obtained from the GenBank database, and their information is shown in Appendix A.  After analyzing these genes by the MegAlign Software (DNAStar), there were 4 SNPs in the C-terminus of the B646L gene, differentiating genotype I viruses from genotype II viruses (Fig. 1-A).  Two SNPs at sites 1 656 and 1 710 were used to design primers and probes for differential detection of genotype I and II ASFVs (Fig. 1-A).  As previously described (Huang et al. 1992; Liu et al. 2012), primers (I F, II F and R) and probes (probe 1 and probe 2) were designed with the targeted gene sequences using Primer 5 Software (Fig. 1-B; Appendix B).  The duplex ARMS-qPCR reaction system volume was 25 μL: 12.5 μL of 2× HyperProbe Mixture (GENFINE), 0.5 μL of I F, II F and R primers (10 μmol L–1), 0.5 μL of probe 1 and probe 2 (10 μmol L–1), 5 μL of template DNA, and 5 μL of ddH2O.  The duplex ARMS-qPCR was performed by using the Bio-Rad CFX96 Touch Real-Time PCR Detection System with the following reaction conditions: 95°C for 30 s, followed by 40 cycles of 95°C for 10 s, and 60°C for 30 s.  Fluorescence signal was detected at the end of each cycle of extension step.  For the positive sample of genotype I ASFV, FAM and Cy5 fluorophores could be detected; however, for the positive sample of genotype II ASFV, only FAM fluorophore could be detected (Fig. 1-B).  

    The standard curve test revealed that for the standard plasmids of genotype I ASFV, the slopes were –3.3825 for Cy5 and –3.1906 for FAM; the correlation coefficient R2 was 0.999 for Cy5 and 0.998 for FAM; the amplification efficiency was 97.53% for Cy5 and 100.06% for FAM, respectively (Fig. 1-C); for the standard plasmids of genotype II ASFV, the slope was –3.2983 for FAM, the correlation coefficient R2 was 0.992 for FAM, the amplification efficiency was 100.01% for FAM, whereas Cy5 fluorophore could not be detected (Fig. 1-C).  In addition, the sensitivity of the duplex ARMS-qPCR was 10 copies per reaction for both genotype I and II ASFVs (Fig. 1-D).  Thus, these results indicated that the duplex ARMS-qPCR assay has high efficiency and sensitivity.  

    We then evaluated the specificity of the duplex ARMS-qPCR.  The nucleic acids of 7 other swine viruses, including PRRSV, CSFV, PRV, PCV2, PEDV, TGEV, and PoRV, were used as templates.  There were 3 amplification curves obtained for genotype I ASFV (FAM and Cy5 signals) and II ASFV (FAM signal), whereas no amplification curve was recorded for the nucleic acids of PRRSV, CSFV, PRV, PCV2, PEDV, TGEV, and PoRV, as well as genotype II ASFV (Cy5 signal) and ddH2O (Fig. 1-E).  The results demonstrated that the duplex ARMS-qPCR has a good specificity without cross-reactivity with other swine viruses.

    The results of the stable detection limit test showed that for the standard plasmids of genotype I ASFV, all 12 replicates were tested positive at the dilution of 10 copies, while 7/12 replicates were tested positive at the dilution of 5 copies (Fig. 1-F); for the standard plasmids of genotype II ASFV, all 12 replicates were tested positive at the dilution of 10 copies, while 6/12 replicates were tested positive at the dilution of 1 copy (Fig. 1-F).  Thus, the stable detection limit of the duplex ARMS-qPCR was 10 copies per reaction for both genotype I and II ASFVs (Fig. 1-F).

    We further assessed the repeatability and reproducibility of the duplex ARMS-qPCR.  The assay tested the standard plasmids of 3 concentrations (106, 104, and 102 copies).  For the standard plasmids of genotype I ASFV, the intra- and inter-assay variation of Ct value for the duplex ARMS-qPCR ranged from 0.07 to 0.93% and 1.2 to 2.17% in FAM fluorescence channel and from 0.38 to 1.02% and 0.85 to 1.27% in Cy5 fluorescence channel, respectively (Table 1).  For the standard plasmids of genotype II ASFV, the intra- and inter-assay variation of Ct value for the duplex ARMS-qPCR ranged from 0.27 to 0.61% and 0.77 to 1.07% in FAM fluorescence channel (Table 1).  These findings suggested that the duplex ARMS-qPCR assay has satisfactory repeatability and reproducibility.

    Finally, we evaluated the duplex ARMS-qPCR compared with WOAH-qPCR.  A total of 40 samples were detected using both assays, including blood, oral and rectal swabs, tissues, and cell cultures from pigs or PAMs infected by genotype I and II ASFVs.  Animal studies have evaluated the virulence and transmissibility of genotype I ASFV SD/DY-I/21 and genotype II virus HLJ/18 (Zhao et al. 2019; Sun et al. 2021a), respectively.  The results showed that 36 samples, including 18 of genotype I ASFV and 18 of genotype II ASFV were detected to be positive and differentiated by the duplex ARMS-qPCR, which were consistent with the results of the WOAH-qPCR (Appendix C).  

    In summary, we developed a duplex ARMS-qPCR assay based on ASFV genotyping region of B646L gene, which can effectively differentiate genotype I and II ASFVs.  The assay had high sensitivity and specificity and exhibited good results in detecting samples, including blood, oral and rectal swabs, tissues, and cell culture.  Whether our method could be used for differentiating other genotypes of ASFVs is needed for further evalution.  However, just genotype I and II ASFVs are spreading outside Africa.  Thus, our method will provide an additional epidemiological investigation tool to implement effective ASFV control and prevention.