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2024 Vol. 23 No. 12 Previous Issue   

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Review
Crop Science
Horticulture
Plant Protection
Animal Science · Veterinary Medicine
Agro-ecosystem & Environment
Agricultural Economics and Management
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Silage is an essential feed for ruminants, and its production process involves an anaerobic environment dominated by lactic acid bacteria. The micro-ecosystem in silage affects its quality. In the cover article, the authors discovered that pathogenic Klebsiella has the ability to produce α-tocopherol. Additionally, Klebsiella can coexist with the lactic acid-producing Lactobacillus plantarum. Further research revealed the mechanism of coexistence. Klebsiella supplies α-tocopherol to promote the proliferation of Lactobacillus plantarum, while its high residual capsular polysaccharides protect Klebsiella from being eliminated by the acid-producing Lactobacillus plantarum. This study provides a scientific basis for using Lactobacillus plantarum to enhance the hygienic quality and α-tocopherol content of rice straw silage. The cover photo provided by Dr. Qinhua Liu from Yunnan Agricultural University/Nanjing Agricultural University, China. For more details, please see pages 4186–4202.




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Review

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Trends in the global commercialization of genetically modified crops in 2023 Xingru Cheng, Haohui Li, Qiaoling Tang, Haiwen Zhang, Tao Liu, Youhua Wang 2024, 23(12): 3943-3952.  DOI: 10.1016/j.jia.2024.09.012 Abstract ( )   PDF in ScienceDirect   The commercialization of genetically modified (GM) crops has increased food production, improved crop quality, reduced pesticide use, promoted changes in agricultural production methods, and become an important new production strategy for dealing with insect pests and weeds while reducing the cultivated land area.  This article provides a comprehensive examination of the global distribution of GM crops in 2023.  It discusses the internal factors that are driving their adoption, such as the increasing number of GM crops and the growing variety of commodities.  This article also provides information support and application guidance for the new developments in global agricultural science and technology.

Crop Science

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Karyotype establishment and development of specific molecular markers of Aegilops geniculata Roth based on SLAF-seq  Yongfu Wang, Jianzhong Fan, Hong Zhang, Pingchuan Deng, Tingdong Li, Chunhuan Chen, Wanquan Ji, Yajuan Wang 2024, 23(12): 3953-3965.  DOI: 10.1016/j.jia.2023.09.014 Abstract ( )   PDF in ScienceDirect   The constant evolution of pathogens poses a threat to wheat resistance against diseases, endangering food security.  Developing resistant wheat varieties is the most practical approach for circumventing this problem.  As a close relative of wheat, Aegilops geniculata, particularly accession SY159, has evolved numerous beneficial traits that could be applied to improve wheat.  In this study, we established the karyotype of SY159 by fluorescence in situ hybridization (FISH) using the oligonucleotide probes Oligo-pTa535 and Oligo-pSc119.2 and a complete set of wheat–Ae. geniculata accession TA2899 addition lines as a reference.  Using specific-locus amplified fragment sequencing (SLAF-seq) technology, 400 specific markers were established for detecting the SY159 chromosomes with efficiencies reaching 81.5%.  The SY159-specific markers were used to classify the different homologous groups of SY159 against the wheat–Ae. geniculata addition lines.  We used these specific markers on the 7Mg chromosome after classification, and successfully confirmed their suitability for studying the different chromosomes of SY159.  This study provides a foundation for accelerating the application of SY159 in genetic breeding programs designed to improve wheat.

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Mapping and identification of QTLs for seed fatty acids in soybean (Glycine max L.) Yiwang Zhong, Xingang Li, Shasha Wang, Sansan Li, Yuhong Zeng, Yanbo Cheng, Qibin Ma, Yanyan Wang, Yuanting Pang, Hai Nian, Ke Wen 2024, 23(12): 3966-3982.  DOI: 10.1016/j.jia.2023.09.010 Abstract ( )   PDF in ScienceDirect   Soybean is one of the most important sources of vegetable oil.  The oil content and fatty acid ratio have attracted significant attention due to their impacts on the shelf-life of soybean oil products and consumer health.  In this study, a high-density genetic map derived from Guizao 1 and Brazil 13 was used to analyze the quantitative trait loci of palmitic acid (PA), stearic acid (SA), oleic acid (OA), linoleic acid (LA), linolenic acid (LNA), and oil content (OC).  A total of 54 stable QTLs were detected in the genetic map linkage analysis, which shared six bin intervals.  Among them, the bin interval on chromosome 13 (bin106–bin118 and bin123–bin125) was found to include stable QTLs in multiple environments that were linked to OA, LA, and LNA.  Eight differentially expressed genes (DEGs) within these QTL intervals were determined as candidate genes according to the combination of parental resequencing, bioinformatics and RNA sequencing data.  All these results are conducive to breeding soybean with the ideal fatty acid ratio for food, and provide the genetic basis for mining genes related to the fatty acid and oil content traits in soybean.

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Optimizing nitrogen management can improve stem lodging resistance and stabilize the grain yield of japonica rice in rice–crayfish coculture systems Qiang Xu, Jingyong Li, Hui Gao, Xinyi Yang, Zhi Dou, Xiaochun Yuan, Weiyan Gao, Hongcheng Zhang 2024, 23(12): 3983-3997.  DOI: 10.1016/j.jia.2024.02.002 Abstract ( )   PDF in ScienceDirect   Nitrogen (N) significantly affects rice yield and lodging resistance.  Previous studies have primarily investigated the impact of N management on rice lodging in conventional rice monoculture (RM); however, few studies have performed such investigations in rice–crayfish coculture (RC).  We hypothesized that RC would increase rice lodging risk and that optimizing N application practices would improve rice lodging resistance without affecting food security.  We conducted a two-factor (rice farming mode and N management practice) field experiment from 2021 to 2022 to test our hypothesis.  The rice farming modes included RM and RC, and the N management practices included no nitrogen fertilizer, conventional N application, and optimized N treatment.  The rice yield and lodging resistance characteristics, such as morphology, mechanical and chemical characteristics, anatomic structure, and gene expression levels, were analyzed and compared among the treatments.  Under the same N application practice, RC decreased the rice yield by 11.1–24.4% and increased the lodging index by 19.6–45.6% compared with the values yielded in RM.  In RC, optimized N application decreased the plant height, panicle neck node height, center of gravity height, bending stress, and lodging index by 4.0–4.8%, 5.2–7.8%, 0.5–4.5%, 5.5–10.5%, and 1.8–19.5%, respectively, compared with those in the conventional N application practice.  Furthermore, it increased the culm diameter, culm wall thickness, breaking strength, and non-structural and structural carbohydrate content by 0.8–4.9%, 2.2–53.1%, 13.5–19.2%, 2.2–24.7%, and 31.3–87.2%, respectively.  Optimized N application increased sclerenchymal and parenchymal tissue areas of the vascular bundle at the culm wall of the base second internode.  Furthermore, optimized N application upregulated genes involved in lignin and cellulose synthesis, thereby promoting lower internodes on the rice stem and enhancing lodging resistance.  Optimized N application in RC significantly reduced the lodging index by 1.8–19.5% and stabilized the rice yield (>8,570 kg ha–1 on average).  This study systematically analyzed and compared the differences in lodging characteristics between RM and RC.  The findings will aid in the development of more efficient practices for RC that will reduce N fertilizer application.

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A comparative study on the role of conventional, chemical, and nanopriming for better salt tolerance during seed germination of direct seeding rice Yixue Mu, Yusheng Li, Yicheng Zhang, Xiayu Guo, Shaokun Song, Zheng Huang, Lin Li, Qilin Ma, Mohammad Nauman Khan, Lixiao Nie 2024, 23(12): 3998-4017.  DOI: 10.1016/j.jia.2023.12.013 Abstract ( )   PDF in ScienceDirect   Salinity is one of the most significant risks to crop production and food security as it harms plant physiology and biochemistry.  The salt stress during the rice emergence stages severely hampers the seed germination and seedling growth of direct-seeded rice.  Recently, nanoparticles (NPs) have been reported to be effectively involved in many plant physiological processes, particularly under abiotic stresses.  To our knowledge, no comparative studies have been performed to study the efficiency of conventional, chemical, and seed nanopriming for better plant stress tolerance.  Therefore, we conducted growth chamber and field experiments with different salinity levels (0, 1.5, and 3‰), two rice varieties (CY1000 and LLY506), and different priming techniques such as hydropriming, chemical priming (ascorbic acid, salicylic acid, and γ-aminobutyric acid), and nanopriming (zinc oxide nanoparticles).  Salt stress inhibited rice seed germination, germination index, vigor index, and seedling growth.  Also, salt stress increased the over accumulation of reactive oxygen species (H2O2 and O2-·) and malondialdehyde (MDA) contents.  Furthermore, salt-stressed seedlings accumulated higher sodium (Na+) ions and significantly lower potassium (K+) ions.  Moreover, the findings of our study demonstrated that, among the different priming techniques, seed nanopriming with zinc oxide nanoparticles (NanoZnO) significantly contributed to rice salt tolerance.  ZnO nanopriming improved rice seed germination and seedling growth in the pot and field experiments under salt stress.  The possible mechanism behind ZnO nanopriming improved rice salt tolerance included higher contents of α-amylase, soluble sugar, and soluble protein and higher activities of antioxidant enzymes to sustain better seed germination and seedling growth.  Moreover, another mechanism of ZnO nanopriming induced rice salt tolerance was associated with better maintenance of K+ ions content.  Our research concluded that NanoZnO could promote plant salt tolerance and be adopted as a practical nanopriming technique, promoting global crop production in salt-affected agricultural lands.

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Effects of water and nitrogen rate on grain-filling characteristics under high-low seedbed cultivation in winter wheat Junming Liu, Zhuanyun Si, Shuang Li, Lifeng Wu, Yingying Zhang, Xiaolei Wu, Hui Cao, Yang Gao, Aiwang Duan 2024, 23(12): 4018-4031.  DOI: 10.1016/j.jia.2023.12.002 Abstract ( )   PDF in ScienceDirect   A high-efficiency mode of high-low seedbed cultivation (HLSC) has been listed as the main agricultural technology to increase land utilization ratio and grain yield in Shandong Province, China.  However, limited information is available on the optimized water and nitrogen management for yield formation, especially the grain-filling process, under HLSC mode.  A three-year field experiment with four nitrogen rates and three irrigation rates of HLSC was conducted to reveal the response of grain-filling parameters, grain weight percentage of spike weight (GPS), spike moisture content (SMC), and winter wheat yield to water and nitrogen rates.  The four nitrogen rates were N1 (360 kg ha–1 pure N), N2 (300 kg ha–1 pure N), N3 (240 kg ha–1 pure N), and N4 (180 kg ha–1 pure N), respectively, and the three irrigation quotas were W1 (120 mm), W2 (90 mm), and W3 (60 mm), respectively.  Results showed that the determinate growth function generally performed well in simulating the temporal dynamics of grain weight (0.989<R2<0.999, where R2 is the determination coefficient).  The occurrence time of maximum filling rate (Tmax) and active grain-filling period (AGP) increased with the increase in the water or nitrogen rate, whereas the average grain-filling rate (Gmean) had a decreasing trend.  The final 1,000-grain weight (FTGW) increased and then decreased with the increase in the nitrogen rates and increased with the increase in the irrigation rates.  The GPS and SMC had a highly significant quadratic polynomial relationship with grain weight and days after anthesis.  Nitrogen, irrigation, and year significantly affected the Tmax, AGP, Gmean, and FTGW.  Particularly, the AGP and FTGW were insignificantly different between high seedbed (HLSC-H) and low seedbed (HLSC-L) across the water and nitrogen levels.  Moreover, the moderate water and nitrogen supply was more beneficial for grain yield, as well as for spike number and grain number per hectare.  The principal component analysis indicated that combining 240–300 kg N ha–1 and 90–120 mm irrigation quota could improve grain-filling efficiency and yield for the HLSC-cultivated winter wheat.

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Mepiquat chloride increases the Cry1Ac protein content of Bt cotton under high temperature and drought stress by regulating carbon and amino acid metabolism Dian Jin, Yuting Liu, Zhenyu Liu, Yuyang Dai, Jianing Du, Run He, Tianfan Wu, Yuan Chen, Dehua Chen, Xiang Zhang 2024, 23(12): 4032-4045.  DOI: 10.1016/j.jia.2023.11.013 Abstract ( )   PDF in ScienceDirect   The effects of mepiquat chloride (DPC) on the Cry1Ac protein content in Bacillus thuringiensis (Bt) cotton boll shells under high temperature and drought stress were investigated to provide a theoretical reference for Bt cotton breeding and high-yield and -efficiency cotton cultivation.  This study was conducted using Bt cotton cultivar ‘Sikang 3’ during the 2020 and 2021 growing seasons at Yangzhou University Farm, Yangzhou, Jiangsu Province, China.  Potted cotton plants were exposed to high temperature and drought stress, and sprayed with either 20 mg L−1 DPC or water (CK).  Seven days after treatment, the Cry1Ac protein content, α-ketoglutarate content, pyruvic acid content, glutamate synthase activity, glutamic oxaloacetic transaminase activity, soluble protein content, and amino acid content were measured, and transcriptome sequencing was performed.  DESeq was used for differential gene analysis.  Under the DPC treatment, the Cry1Ac protein content increased by 4.7–11.9% compared to CK.  The α-ketoglutarate content, pyruvic acid content, glutamate synthase activity, glutamic oxaloacetic transaminase activity, soluble protein content, and amino acid content all increased.  Transcriptome analysis revealed 7,542 upregulated genes and 10,449 downregulated genes for DPC vs. CK.  Gene ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) analyses showed that the differentially expressed genes were mainly involved in biological processes, such as carbon and amino acid metabolism.  For example, genes encoding 6-phosphofructokinase, pyruvate kinase, glutamic pyruvate transaminase, pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase, glutamate synthase, 1-pyrroline-5-carboxylate dehydrogenase, glutamic oxaloacetic transaminase, amino-acid N-acetyltransferase, and acetylornithine deacetylase were all significantly upregulated.  The DPC treatment increased pyruvate, α-ketoglutarate, and oxaloacetate by increasing the operational rate of the glycolytic pathway of the citric acid cycle.  It also significantly upregulated the genes encoding glutamate synthase, pyrrolidine-5-carboxylic acid dehydrogenase, glutamate oxaloacetate transaminase, and N-acetylglutamate synthetase, while it downregulated the genes encoding glutamine synthetase.  Therefore, the synthesis of aspartic acid, glutamic acid, pyruvate, and arginine increased after treatment with DPC, and the Cry1Ac protein content was increased by regulating carbon and amino acid metabolism.

Horticulture

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Physiological and molecular mechanisms of cytokinin involvement in nitrate-mediated adventitious root formation in apples Muhammad Mobeen Tahir, Li Fan, Zhimin Liu, Humayun Raza, Usman Aziz, Asad Shehzaib, Shaohuan Li, Yinnan He, Yicen Lu, Xiaoying Ren, Dong Zhang, Jiangping Mao 2024, 23(12): 4046-4057.  DOI: 10.1016/j.jia.2024.07.045 Abstract ( )   PDF in ScienceDirect   Potassium nitrate (KNO3) promotes adventitious root (AR) formation in apple stem cuttings.  However, evidence for the possible involvement of cytokinin (CK) in KNO3-mediated AR formation in apples is still lacking.  In this study, we cultured GL-3 apple microshoots in different treatment combinations.  While the T1 (KNO3 9.4 mmol L–1+6-benzyl adenine (6-BA) 2.22 μmol L–1) and T3 (6-BA 2.22 μmol L–1) treatments completely inhibited AR formation, the control, T2 (KNO3 9.4 mmol L–1), and T4 (KNO3 9.4 mmol L–1+lovastatin (Lov) 1.24 μmol L–1) treatments developed ARs.  However, T4-treated microshoots developed fewer and shorter ARs, indicating that optimum CK synthesis is needed for normal AR growth.  This also suggests that these fewer and shorter ARs developed because of the presence of KNO3 in the same medium.  The anatomy of the stem basal part indicated that the inhibition of CK biosynthesis delayed AR primordia formation.  The endogenous levels of indole‐3‐acetic acid (IAA) and zeatin riboside (ZR) were higher in T2-treated microshoots, while the abscisic acid (ABA), gibberellic acid 3 (GA3), and brassinosteroid (BR) levels were higher in T4-treated microshoots.  The expression levels of MdNRT1.1 and MdNRT2.1 were higher in T2-treated microshoots at 3 and 8 days, while MdRR2 and MdCKX5 were higher at 8 and 16 days, respectively.  Furthermore, higher IAA levels increased MdWOX11 expression, which in turn increased MdLBD16 and MdLBD29 expression in response to T2.  The combined expression of these genes stimulated adventitious rooting by upregulating cell cycle-related genes (MdCYCD1;1 and MdCYCD3;1) in response to T2 treatment.  This study shows that specific genes and hormonal pathways contribute to KNO3-CK-mediated adventitious rooting in apples.

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Whole-genome characterization of CKX genes in Prunus persica and their role in bud dormancy and regrowth  Xuehui Zhao, Jianting Liu, Xiling Fu, Long Xiao, Qingjie Wang, Chaoran Wang, Zhizhang Chen, Jiakui Li, Changkun Lu, Hui Cao, Ling Li 2024, 23(12): 4058-4073.  DOI: 10.1016/j.jia.2024.09.002 Abstract ( )   PDF in ScienceDirect   Bud dormancy is a complex physiological process of perennial woody plants living in temperate regions, and it can be affected by various phytohormones.  Cytokinin oxidase/dehydrogenases (CKXs) are a group of enzymes essential for maintaining cytokinin homeostasis, yet a comprehensive analysis of these enzymes in peach remains lacking.  Here, a total of 51 CKX members from different species, including six from peach, eleven from apple, nine from poplar, seven from Arabidopsis, eight from strawberry, and ten from rice, were identified using the Simple HMM Search tool of TBtools and a BLASTP program and classified into four groups using phylogenetic analysis.  Conserved motif and gene structure analysis of these 51 CKX members showed that 10 conserved motifs were identified, and each CKX gene contained at least two introns.  Cis-element analysis of PpCKXs showed that all PpCKX genes have light-responsive elements and at least one hormone-responsive element.  The changed relative expression levels of six PpCKX genes in peach buds from endodormancy to bud-break were observed by qRT-PCR.  Among them, the expression trend of PpCKX6 was almost opposite that of PpEBB1, a positive bud-break regulator in woody plants, around the bud-break stage.  Y1H, EMSA, and dual-luciferase assays indicated that PpEBB1 negatively regulated PpCKX6 through direct binding to a GCC box-like element located in the promoter region of PpCKX6.  In addition, a transient assay showed that overexpression of PpCKX6 delayed the bud-break of peach.  These results indicate that the PpCKX genes play an essential role in the dormancy-regrowth process, and PpCKX6 may act downstream of PpEBB1 directly to regulate the bud-break process, which further improves the hormone-regulatory network of dormancy-regrowth of woody plants, and provides new insights for molecular breeding and genetic engineering of peach.

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Nitrogen application regulates antioxidant capacity and flavonoid metabolism, especially quercetin, in grape seedlings under salt stress Congcong Zhang, Han Wang, Guojie Nai, Lei Ma, Xu Lu, Haokai Yan, Meishuang Gong, Yuanyuan Li, Ying Lai, Zhihui Pu, Li Wei, Guiping Chen, Ping Sun, Baihong Chen, Shaoying Ma, Sheng Li 2024, 23(12): 4074-4092.  DOI: 10.1016/j.jia.2024.07.013 Abstract ( )   PDF in ScienceDirect   Salt stress is a typical abiotic stress in plants that causes slow growth, stunting, and reduced yield and fruit quality.  Fertilization is necessary to ensure proper crop growth.  However, the effect of fertilization on salt tolerance in grapevine is unclear.  In this study, we investigated the effect of nitrogen fertilizer (0.01 and 0.1 mol L–1 NH4NO3) application on the salt (200 mmol L–1 NaCl) tolerance of grapevine based on physiological indices, and transcriptomic and metabolomic analyses.  The results revealed that 0.01 mol L–1 NH4NO3 supplementation significantly reduced the accumulation of superoxide anion (O2–·), enhanced the activities of superoxide dismutase (SOD) and peroxidase (POD), and improved the levels of ascorbic acid (AsA) and glutathione (GSH) in grape leaves compared to salt treatment alone.  Specifically, joint transcriptome and metabolome analyses showed that the differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) were significantly enriched in the flavonoid biosynthesis pathway (ko00941) and the flavone and flavonol biosynthesis pathway (ko00944).  In particular, the relative content of quercetin (C00389) was markedly regulated by salt and nitrogen.  Further analysis revealed that exogenous foliar application of quercetin improved the SOD and POD activities, increased the AsA and GSH contents, and reduced the H2O2 and O2–· contents.  Meanwhile, 10 hub DEGs, which had high Pearson correlations (R2>0.9) with quercetin, were repressed by nitrogen.  In conclusion, all the results indicated that moderate nitrogen and quercetin application under salt stress enhanced the antioxidant system defense response, thus providing a new perspective for improving salt tolerance in grapes.

Plant Protection

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A potential hyphal fusion protein complex with an important role in development and virulence interacts with autophagy-related proteins in Fusarium pseudograminearum Linlin Chen, Yixuan Shan, Zaifang Dong, Yake Zhang, Mengya Peng, Hongxia Yuan, Yan Shi, Honglian Li, Xiaoping Xing 2024, 23(12): 4093-4106.  DOI: 10.1016/j.jia.2023.09.005 Abstract ( )   PDF in ScienceDirect   Hyphal fusion (anastomosis) is a common process serving many important functions at various developmental stages in the life cycle of ascomycetous fungi.  However, the biological roles and molecular mechanisms in plant pathogenic fungi were widely unknown.  In this study, a hyphal fusion protein FpHam-2 was screened from a T-DNA insertion mutant library of Fusarium pseudograminearum, and FpHam-2 interacts with another 2 hyphal fusion protein homologues FpHam-3 and FpHam-4.  Each of these 3 genes deletion mutant revealed in similar defective phenotypes compared with the WT and complemented strains, including reduction in growth rate, defects in hyphal fusion and conidiation, more sensitive for cell membrane, cell wall and oxidative stress responses, and decreased in virulence.  The yeast two-hybrid assay was used to identify that FpHam-2 interacts with 3 autophagy-related proteins, including FpAtg3, FpAtg28 and FpAtg33.  Furthermore, FpHam-2-deletion mutant showed decreased accumulation of autophagic bodies in hypha.  In conclusion, FpHam-2, FpHam-3 and FpHam-4 have an essential role for hyphal fusion and regulating the growth, conidiation and virulence in F. pseudograminearum.

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A novel chorismate mutase effector secreted from root-knot nematode Meloidogyne enterolobii manipulates plant immunity to promote parasitism Tuizi Feng, Yuan Chen, Zhourong Li, Ji Pei, Deliang Peng, Huan Peng, Haibo Long 2024, 23(12): 4107-4119.  DOI: 10.1016/j.jia.2023.11.039 Abstract ( )   PDF in ScienceDirect   Meloidogyne spp. is an economically important plant-parasitic nematode distributed worldwide.  To fight with host immune system for successful parasitism, plant parasitic nematodes secrete effectors to promote infection.  In this study, we identified one chorismate mutase (CM) effector from M. enterolobii, named Me-CM.  Spatial and temporal expression assays exhibited Me-cm is expressed in esophageal glands and up-regulated at parasitic-stage juveniles.  Me-CM affects the pathogenicity of M. enterolobii based on the reduced infection rate, number of galls, egg masses, eggs per mass and multiplication rate collected from RNA silencing experiments.  We showed that Me-CM localized in the cytoplasm and nucleus of plant cells and decreased the expression level of the marker gene PR1 of salicylic acid (SA) pathway.  Besides, constitutive expression of Me-cm in Arabidopsis thaliana significantly reduced salicylic acid concentration.  These results suggested that M. enterolobii may secrete effector Me-CM to fight with plant immune systems via regulating SA signaling pathway when interacting with host plants, ultimately facilitating parasitism.

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Functional characterization of sensory neuron membrane protein 1a involved in sex pheromone detection of Apolygus lucorum (Hemiptera: Miridae) Yan Li, Xingkui An, Shuang Shan, Xiaoqian Pang, Xiaohe Liu, Yang Sun, Adel Khashaveh, Yongjun Zhang 2024, 23(12): 4120-4135.  DOI: 10.1016/j.jia.2024.03.043 Abstract ( )   PDF in ScienceDirect   The mirid bug Apolygus lucorum (Hemiptera: Miridae) is a polyphagous pest that affects a wide range of host plants.  Its control remains challenging mainly due to its rapid reproduction, necessitating an understanding of sex pheromone communication.  The recognition of sex pheromones is vital for courtship and mating behaviors, and is mediated by various chemosensory-associated proteins.  Among these, sensory neuron membrane protein (SNMP), a CD36-related protein, is suggested to play crucial roles in detecting sex pheromones.  In this study, we employed transcriptomic and genomic data from A. lucorum and phylogenetic approaches, and identified four putative SNMP genes (AlucSNMP1a, AlucSNMP1b, AlucSNMP2a, and AlucSNMP2b) with full open reading frames.  Expression analysis revealed the ubiquitous presence of AlucSNMP transcripts in multiple tissues, with only AlucSNMP1a exhibiting male-biased expression in the antennae, suggesting its potential role in male chemosensation.  Functional analysis using the Xenopus oocyte expression system, coupled with two-electrode voltage clamp recording, demonstrated that the co-expression of AlucSNMP1a with specific pheromone receptors (PRs) and the Odorant receptor co-receptor (Orco) significantly enhanced electrophysiological responses to sex pheromones compared to the co-expression of PRs and Orco alone.  Moreover, the results indicated that the presence of AlucSNMP1a not only affected the responsiveness to sex pheromones but also influenced the kinetics (activation and inactivation) of the induced signals.  In contrast, the co-expression of AlucSNMP1b with AlucPR/Orco complexes had no impact on the inward currents induced by two pheromone compounds.  An examination of the selective pressures on SNMP1 genes across 20 species indicated strong purifying selection, implying potential functional conservation in various insects.  These findings highlight the crucial role of AlucSNMP1a in the response to sex pheromones.

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Vesicular transport-related genes in Diaphorina citri are involved in the process of Candidatus Liberibacter asiaticus infection Yingzhe Yuan, Tao Peng, Aijun Huang, Jun He, Chenyang Yuan, Tianyuan Liu, Long Yi, Xuejin Cui, Xuefeng Wang, Changyong Zhou 2024, 23(12): 4136-4146.  DOI: 10.1016/j.jia.2024.03.063 Abstract ( )   PDF in ScienceDirect   Asian citrus psyllid (ACP, Diaphorina citri) is the major vector of Candidatus Liberibacter asiaticus (CLas), which is a bacterial pathogen causing the devastating citrus Huanglongbing (HLB) disease.  Diaphorina citri is known to carry CLas in a persistent and propagative manner.  Some studies have suggested that CLas may use the vesicular structures of D. citri cells as its propagation organelles.  However, the mechanisms by which CLas enters the D. citri cells and how vesicle-mediated trafficking is involved remain unclear.  In this study, we monitored the titer change of CLas in D. citri nymphs during the process of CLas acquisition from feeding on infected citrus plants.  We found that the titer of CLas increased with the acquisition access period.  After infection, there was a significant upregulation in the expression of several vesicular transport-related genes in D. citri.  The titer of CLas was significantly reduced in the midgut and whole insect body when endocytosis and the endosome network in D. citri were inhibited.  Furthermore, silencing the D. citri clathrin-heavy chain gene also led to a reduction in the CLas titer in D. citri.  These results suggest that CLas infection upregulates the genes related to vesicular transport in D. citri, which facilitates the invasion of endocytosis-dependent pathogens.

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Neuropeptide signaling systems are involved in regulating thermal tolerance in the oriental fruit fly Yang Yang, Hongfei Li, Changhao Liang, Donghai He, Hang Zhao, Hongbo Jiang, Jinjun Wang 2024, 23(12): 4147-4160.  DOI: 10.1016/j.jia.2024.04.003 Abstract ( )   PDF in ScienceDirect   Neuropeptides and their receptors are involved in the regulation and coordination of various physiological processes in insects.  Although various neuropeptides have been identified previously, the corresponding receptors remain unknown in the oriental fruit fly, Bactrocera dorsalis, an important agricultural insect pest.  It is also unclear whether neuropeptide signaling systems are involved in regulating the thermal tolerance of this notorious pest.  Here, we systematically identified 44 putative neuropeptide receptor genes which encode 66 protein sequences, and analyzed the spatio-temporal expression patterns of the neuropeptide ligands and their receptor genes in B. dorsalis.  We also analyzed changes in their transcript accumulation in two thermo-tolerant populations (heat and cold) of B. dorsalis.  The results showed that numerous neuropeptides and receptors participate in responding to thermal stresses during acclimation.  In particular, the expression of short neuropeptide F (sNPF) was up-regulated in the heat-tolerant population of B. dorsalis.  Moreover, proteomic data showed that sNPF was up-regulated in both thermo-tolerant populations of B. dorsalis.  The functional verification based on CRISPR/Cas9 demonstrated that sNPF is involved in regulating the tolerance to thermal stresses.  The results of this study enrich our knowledge on the function of neuropeptide sNPF in insects.  Moreover, this study demonstrated the role of neuropeptide signaling systems in thermal adaptation, contributing to a better understanding of the rapid invasiveness of B. dorsalis around the world.

Animal Science · Veterinary Medicine

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Dietary manganese supplementation inhibits abdominal fat deposition possibly by regulating gene expression and enzyme activity involved in lipid metabolism in the abdominal fat of broilers Xiaoyan Cui, Ke Yang, Weiyun Zhang, Liyang Zhang, Ding Li, Wei Wu, Yun Hu, Tingting Li, Xugang Luo 2024, 23(12): 4161-4171.  DOI: 10.1016/j.jia.2023.08.004 Abstract ( )   PDF in ScienceDirect   Excessive abdominal fat deposition seriously restricts the production efficiency of broilers.  Several studies found that dietary supplemental manganese (Mn) could effectively reduce the abdominal fat deposition of broilers, but the underlying mechanisms remain unclear.  The present study aimed to investigate the effect of dietary supplementation with the inorganic or organic Mn on abdominal fat deposition, and enzyme activity and gene expression involved in lipid metabolism in the abdominal fat of male or female broilers.  A total of 420 1-d-old AA broilers (half males and half females) were randomly allotted by body weight and gender to 1 of 6 treatments with 10 replicates cages of 7 chicks per cage in a completely randomized design involving a 3 (dietary Mn addition)×2 (gender) factorial arrangement.  Male or female broilers were fed with the Mn-unsupplemented basal diets containing 17.52 mg Mn kg–1 (d 1–21) and 15.62 mg Mn kg–1 (d 22–42) by analysis or the basal diets supplemented with 110 mg Mn kg–1 (d 1–21) and 80 mg Mn kg–1 (d 22–42) as either the Mn sulfate or the Mn proteinate with moderate chelation strength (Mn-Prot M) for 42 d.  The results showed that the interaction between dietary Mn addition and gender had no impact (P>0.05) on any of the measured parameters; abdominal fat percentage of broilers was decreased (P<0.003) by Mn addition; Mn addition increased (P<0.004) adipose triglyceride lipase (ATGL) activity, while Mn-Prot M decreased (P<0.002) the fatty acid synthase (FAS) activity in the abdominal fat of broilers compared to the control; Mn addition decreased (P<0.009) diacylglycerol acyltransferase 2 (DGAT2) mRNA expression level and peroxisome proliferator-activated receptor γ (PPARγ) mRNA and protein expression levels, but up-regulated (P<0.05) the ATGL mRNA and protein expression levels in the abdominal fat of broilers.  It was concluded that dietary supplementation with Mn inhibited the abdominal fat deposition of broilers possibly via decreasing the expression of PPARγ and DGAT2 as well as increasing the expression and activity of ATGL in the abdominal fat of broilers, and Mn-Prot M was more effective in inhibiting the FAS acitivity.

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Transcriptome analysis reveals the genetic basis of crest cushion formation in duck Lan Huang, Qixin Guo, Yong Jiang, Zhixiu Wang, Guohong Chen, Guobin Chang, Hao Bai 2024, 23(12): 4172-4185.  DOI: 10.1016/j.jia.2023.09.025 Abstract ( )   PDF in ScienceDirect   The Chinese crested duck is a unique duck breed having a bulbous feather shape on its duck head.  However, the mechanisms involved in its formation and development are unclear.  In the present study, RNA sequencing analysis was performed on the crested tissues of 6 Chinese crested ducks and the scalp tissues of 6 cherry valley ducks (CVs) from 2 developmental stages.  This study identified 261 differentially expressed genes (DEGs), 122 upregulated and 139 downregulated, in the E28 stage and 361 DEGs, 154 upregulated and 207 downregulated in the D42 stage between CC and CV ducks.  The subsequent results of weighted gene co-expression network analysis (WGCNA) revealed that the turquoise and cyan modules were associated with the crest trait in the D42 stage, meanwhile, the green, brown, and pink modules were associated with the crest trait in the E28 stage.  Venn analysis of the DEGs and WGCNA showed that 145 and 45 genes are associated between the D42 and E28 stages, respectively.  The expression of WNT16, BMP2, SLC35F2, SLC6A15, APOBEC2, ABHD6, TNNC2, MYL1, and TNNI2 were verified by real-time quantitative PCR.  This study provides an approach to reveal the molecular mechanisms underlying the crested trait development.

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Klebsiella as an α-tocopherol source facilitating Lactobacillus plantarum fermentation in rice straw silage Cheng Zong, Lu Tang, Tao Shao, Yu Xiao, Zhongyong Huang, Wanqi Jiang, Jiugang Zhu, Zhihao Dong, Mao Li, Qinhua Liu 2024, 23(12): 4186-4202.  DOI: 10.1016/j.jia.2023.11.036 Abstract ( )   PDF in ScienceDirect   Silage serves as the indispensable diet of ruminants, the increasing mechanism of α-tocopherol during silage making is unclear.  Rice straw lacks chlorophyll after harvesting the grain, this can eliminate the impact of tocopherols formed by the breakdown of chlorophyll.  Here, we explored the α-tocopherol source, its influencing factor, and its relationship with dominant lactic acid bacteria in rice straw silage treated without or with different additives (sodium benzoate, Lactobacillus plantarum, cell wall degrading enzymes, the combination of L. plantarum and cell wall degrading enzymes) and vacuum times (5, 8, 11, and 14 s) after ensiling for 42 d.  We found that the pathogenic Klebsiella was traced as the source of increased α-tocopherol in rice straw silage.  The residue air in the silo, pH value, and additive variety had impacts on Klebsiella activity, which was strongly active at levels of residue air in the silo and pH that were high.  As an acidic niche creator, L. plantarum was more effective than sodium benzoate in restraining Klebsiella.  Despite having a low acidity tolerance, Klebsiella was still present in rice straw silage treated with L. plantarum.  The relationship between Klebsiella and L. plantarum was that Klebsiella could afford α-tocopherol to the multiplication of L. plantarum and residue capsular polysaccharide protected Klebsiella from escaping the extinction in rice straw silage.

Agro-ecosystem & Environment

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Assessment of CH4 flux and its influencing drivers in the rice–wheat agroecosystem of the Huai River Basin, China Xiaolan Yu, Fangmin Zhang, Yanqiu Fang, Xiaohan Zhao, Kaidi Zhang, Yanyu Lu 2024, 23(12): 4203-4215.  DOI: 10.1016/j.jia.2024.03.076 Abstract ( )   PDF in ScienceDirect   To understand the CH4 flux variations and their climatic drivers in the rice–wheat agroecosystem in the Huai River Basin of China, the CH4 flux was observed by using open-path eddy covariance at a typical rice–wheat rotation system in Anhui Province, China from November 2019 to October 2021.  The variations and their drivers were then analyzed with the Akaike information criterion method.  CH4 flux showed distinct diurnal variations with single peaks during 9:00–13:00 local time.  The highest peak was 2.15 µg m–2 s–1 which occurred at 11:00 in the vegetative growth stage in the rice growing season (RGS).  CH4 flux also showed significant seasonal variations.  The average CH4 flux in the vegetative growth stage in the RGS (193.8±74.2 mg m–2 d–1) was the highest among all growth stages.  The annual total CH4 flux in the non-rice growing season (3.2 g m–2) was relatively small compared to that in the RGS (23.9 g m–2).  CH4 flux increased significantly with increase in air temperature, soil temperature, and soil water content in both the RGS and the non-RGS, while it decreased significantly with increase in vapor pressure deficit in the RGS.  This study provided a comprehensive understanding of the CH4 flux and its drivers in the rice–wheat rotation agroecosystem in the Huai River Basin of China.  In addition, our findings will be helpful for the validation and adjustment of the CH4 models in this region.

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Sugarcane/soybean intercropping with reduced nitrogen addition enhances residue-derived labile soil organic carbon and microbial network complexity in the soil during straw decomposition Tantan Zhang, Yali Liu, Shiqiang Ge, Peng Peng, Hu Tang, Jianwu Wang 2024, 23(12): 4216-4236.  DOI: 10.1016/j.jia.2024.02.020 Abstract ( )   PDF in ScienceDirect   Sugarcane/soybean intercropping with reduced nitrogen addition is an important sustainable agricultural pattern that can alter soil ecological functions, thereby affecting straw decomposition in the soil.  However, the mechanisms underlying changes in soil organic carbon (SOC) composition and microbial communities during straw decomposition under long-term intercropping with reduced nitrogen addition remain unclear.  In this study, we conducted an in-situ microplot incubation experiment with 13C-labeled soybean straw residue addition in a two-factor (cropping pattern: sugarcane monoculture (MS) and sugarcane/soybean intercropping (SB); nitrogen addition levels: reduced nitrogen addition (N1) and conventional nitrogen addition (N2)) long-term experimental field plot.  The results showed that the SBN1 treatment significantly increased the residual particulate organic carbon (POC) and residual microbial biomass carbon (MBC) contents during straw decomposition, and the straw carbon in soil was mainly conserved as POC.  Straw addition changed the structure and reduced the diversity of the soil microbial community, but microbial diversity gradually recovered with decomposition time.  During straw decomposition, the intercropping pattern significantly increased the relative abundances of Firmicutes and Ascomycota.  In addition, straw addition reduced microbial network complexity in the sugarcane/soybean intercropping pattern but increased it in the sugarcane monoculture pattern.  Nevertheless, microbial network complexity remained higher in the SBN1 treatment than in the MSN1 treatment.  In general, the SBN1 treatment significantly increased the diversity of microbial communities and the relative abundance of microorganisms associated with organic matter decomposition, and the changes in microbial communities were mainly driven by the residual labile SOC fractions.  These findings suggest that more straw carbon can be sequestered in the soil under sugarcane/soybean intercropping with reduced nitrogen addition to maintain microbial diversity and contribute to the development of sustainable agriculture.

Agricultural Economics and Management

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Taxing sugar-sweetened beverages in China: By volume or sugar content?  A consumer welfare approach Tianchang Zhai, Jingjing Wang, Lei Li, Wei Si 2024, 23(12): 4237-4249.  DOI: 10.1016/j.jia.2024.10.006 Abstract ( )   PDF in ScienceDirect   Using scanner data on the consumption of packaged liquid beverages by Chinese urban households, we estimated the price elasticity of various beverages and compared the effects of volume-based versus sugar-content-based taxation strategies on consumer welfare.  Compared to the volume-based tax, the sugar-content-based beverage tax was predicted to cost less in compensating variation under the same amount of sugar reduced, indicating that taxation based on sugar content may be more efficient in preserving consumer welfare.  Further comparison across different socioeconomic groups reveals that, given current beverage consumption status in China, the efficiency advantage of the sugar-content-based taxation strategy is more pronounced than that of the volume-based taxation.  Our conclusions can provide insights for the food industry and the government to reduce the sugar content in beverages.

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Factors affecting farmers’ choice to adopt risk management strategies: The application of multivariate and multinomial probit models Jamal Shah, Majed Alharthi 2024, 23(12): 4250-4262.  DOI: 10.1016/j.jia.2024.10.004 Abstract ( )   PDF in ScienceDirect   This study investigates the factors that impact farmers’ adoption of risk management strategies (RMS) in Pakistan during times of uncertainty.  The study examines farmers’ adoption of RMS using both multinomial probit (MNP) and multivariate probit (MVP).  Data were collected from 382 farmers sampled from four districts in Khyber-Pakhtunkhwa (KP) province of Pakistan via a multistage sampling technique.  This study utilizes the MNP model, considering the assumption of Independence of Irrelevant Alternatives (IIA) and incorporating correlated error terms.  The objective is to understand farmers’ behavior in risky situations and determine if there is heterogeneity.  Results are compared with the MVP model to assess robustness and gain deeper understanding of farmers’ decision-making processes.  The research findings reveal that our results are robust, and farmers behave homogeneously in various RMS scenarios.  Farmers adopt RMS individually or in combination to mitigate the adverse effects of natural calamities on their livelihood.  The risk-averse farmers, who perceive weather-related risks as a threat, access credits and information, and have farms close to a river are more likely to adopt RMS, irrespective of the format of the strategies available.  Moreover, the predicted probabilities and correlation of the RMS and RM categories have strengthened our model estimation.  These findings provide insights into the behavior of farmers in adopting RMS which are helpful for policymakers and stakeholders in developing strategies to mitigate the impacts of natural calamities on farmers.

Letter

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Identification and characterization of a no-grain mutant (nog1) in foxtail millet Hui Zhang, Hui Zhi, Di Yuan, Hongkai Liang, Wei Zhang, Guanqing Jia, Xianmin Diao 2024, 23(12): 4263-4266.  DOI: 10.1016/j.jia.2024.06.016 Abstract ( )   PDF in ScienceDirect   Increasing crop grain yields is an urgent global priority due to population growth, shrinking arable land, and severe climate change in recent years (Tang et al. 2023).  Unraveling the process of panicle development is crucial for enhancing the grain yield of cereal crops.  In the development of rice panicles, the inflorescence meristem (IM) gives rise to two types of lateral branch meristems (BMs): primary branch meristem (pBM) and secondary branch meristem (sBM).  The pBM generates sBM and spikelet meristems (SMs), and the sBM further differentiates into more SMs (Zhang and Yuan 2014).  A spikelet is the basic unit of inflorescence in Poaceae plants.  It originates from the spikelet meristem (SM) that determines the number of spikelets per panicle and consequently impacts crop yield formation (Doebley et al. 2006).  The seed setting rate, determined by spikelet development, is another crucial trait intimately linked to grain yield (Zhuang et al. 2024).  In plants, developmental defects in spikelets are typically categorized as either male sterility (Notsu et al. 2002; Liu et al. 2007; Luo et al. 2013) or female sterility (Li et al. 2022).  However, no mutant with completely sterile male and female reproductive organs has yet been identified so far. Foxtail millet (Setaria italica (L.) Beauv.) is a stress-tolerant annual cereal crop species from the Poaceae family (Muthamilarasan and Prasad 2015; He et al. 2023; Liang et al. 2023).  In recent decades, foxtail millet has been developed as a new model crop for deciphering panicle development due to its diploidy and small genome, short growth cycle, and self-pollination character (Doust et al. 2009; Diao et al. 2017; He et al. 2021).  In foxtail millet, each spikelet produces one fertile floret and one sterile floret.  The sterile one was degenerated, and the fertile one was enclosed by lemma, palea, and two lodicules derived from the sterile one (Hussin et al. 2021; Zhang et al. 2021).  Several abnormal panicle mutants have been identified in both foxtail millet and its wild type, green foxtail (Setaria viridis).  In foxtail millet, the silp1 mutant showed an increase in the length and width of primary branches, in company with a decrease in the number of fertilized spikelets and seed setting rate in the panicle (Xiang et al. 2017).  The siaux1-1 mutant displays a sparsely branched panicle compared to the wild type (Tang et al. 2021).  The simads34 mutant shows increased panicle width and decreased panicle length and grain yield in foxtail millet (Hussin et al. 2021).  In green foxtail, mutations in the svaux1 gene result in reduced inflorescence branches and spikelet numbers and increased panicle length (Huang et al. 2017).  Approximately 17% of the panicles in the brl1 mutant produce additional flowers, bristles and/or spikelets within each spikelet compared to one flower per spikelet in the wild type (Yang et al. 2021).  The inflorescences of the Svfon2 mutant exhibit abnormal apices and panicle tips, which are divided into two or more parts (Zhu et al. 2021).  However, few studies on panicle sterility mutants have been reported in both Setaria italica and Setaria viridis, with only one male-sterile mutant, sinp1, having been identified (Zhang et al. 2021).  Thus, the understanding of panicle infertility in foxtail millet remains limited. In this trial, we identified a completely sterile mutant sinog1 (no grain 1) from the EMS-induced mutant library of Yugu 1.  The mutant exhibited a slender panicle and was completely sterile with no seed set (Fig. 1-A).  Dissection observations revealed that all the reproductive organs of sinog1 florets gradually turned brown and eventually failed at the heading stage, leaving only two lemmas in the florets of sinog1, while the wild type was able to form mature floral organs normally (Fig. 1-B).  Additionally, the spikelet of the sinog1 mutant was significantly narrower and longer compared to the wild type (Fig. 1-A and C).  Furthermore, the spikelet of sinog1 mutant exhibited a significant increase in the content of indoleacetic acid (IAA) ((44.20±0.96) ng g–1 FW), abscisic acid (ABA) ((91.27±1.77) ng g–1 FW), and gibberellic acid 4 (GA4) ((2.04±0.03) ng g–1 FW) compared to the wild type ((30.64±0.59) ng g–1 FW, (63.96±1.53) ng g–1 FW, and (1.88±0.07) ng g–1 FW), and a significant decrease in the content of brassinosteroid (BR) ((1.85±0.05) ng g–1 FW) compared to the wild type ((2.02±0.05) ng g–1 FW) (Fig. 1-D). To identify the candidate genes responsible for the complete sterility in sinog1, we constructed an F2 population with 263A as the female parent and the residual heterozygote individuals, including the mutation of sinog1 (Deng3-3) as the male parent.  The residual heterozygote individual Deng3-3 was derived from the BC3 population generated by multiple rounds of backcrosses with Yugu 1.  Genetic and segregation ratio analysis suggested that the complete sterile phenotype of sinog1 was caused by a single recessive gene (χ²=0.06<χ² (0.05,1)=3.84).  Combining BSA-seq (45× coverage) and linkage verification analysis of 343 recessive individuals (completely sterile plants), the causal gene of sinog1 was finally mapped to a 1.85 Mb interval from 32.44 to 34.29 Mb on chromosome 5.  A total of 203 genes with sequence variations were screened out within the 1.85 Mb genomic interval (Fig. 1-E). Subsequently, the transcriptomes of the wild type and mutant panicle at the heading stage were analyzed, identifying 37,986 genes.  Among these genes, 5,014 are differentially expressed genes (DEGs) between the wild type and sinog1 (log2FC≥1 and FDR≤0.01), consisting of 2,707 significantly up-regulated and 2,307 significantly down-regulated genes.  The KEGG pathways analysis revealed that pathways related to ABC transporters, photosynthesis, and diterpenoid biosynthesis were significantly enriched in DEGs (Fig. 1-F).  Among these pathways, ABC transporters have been confirmed to regulate floral organ formation and panicle growth by transporting phytohormones and heavy metals in plants (Do et al. 2018; Naaz et al. 2023).  For instance, the mutation of ABCG26 in Arabidopsis leads to severely reduced fertility (Choi et al. 2011).  In rice, the knock-down of OsABCB14 can decrease the concentration and polar transport rates of auxin, and iron concentrations are also increased in the mutant (Xu et al. 2014).  These results suggest that the mutation gene of sinog1 might be an essential regulator for the proper expression of ABC transporters involved in floral development in foxtail millet. Among the 203 genes located in the candidate interval on chromosome 5, we identified 28 DEGs by RNA-seq (Fig. 1-G), including seven genes (Seita.5G274600, Seita.5G274500, Seita.5G270700, Seita.5G274000, Seita.5G283000, Seita.5G282700, and Seita.5G272800) that were highly expressed in the wild type and 21 genes that were highly expressed in sinog1.  Of the 28 common genes, ten had mutations in the exon regions, including Seita.5G283500 (Val649Leu), Seita.5G283600 (Leu247Ser, Asn396Glu, and Ser667Arg), and Seita.5G282700 (insert 6 bp, Ala78Ser), which have been annotated as steroid/xenobiotic-transporting ATPase in foxtail millet and ABC transporter in rice and Arabidopsis thaliana.  Interestingly, these three ABC pathway-related candidate genes were also enriched in the KEGG pathway of DEGs in sinog1.  It is noteworthy that ABC transporters have been verified to contribute to auxin and ABA transport, response to heavy metals such as iron and aluminum, and regulation of stomatal characters, which are important for plant growth and development (Do et al. 2018; Naaz et al. 2023).  Therefore, we inferred that ABC transporters may be key genes influencing morphological mutations in sinog1. In conclusion, we have identified a novel panicle sterile phenotype in foxtail millet, the sinog1 mutant, which exhibits complete sterility with aborted reproductive organs.  The morphological analysis confirmed that sinog1 showed complete flower abortion at the heading stage, and the candidate pathways contributing to this completely sterile phenotype were identified using the combined BSA-seq and RNA-seq methods in foxtail millet.  This research provides a theoretical basis for understanding florets development in foxtail millet and other crops.

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Development and evaluation of a RT-RAA-combined CRISPR/Cas12a assay for the detection of African horse sickness virus Yingzhi Zhang, Lei Na, Kui Guo, Jinhui Wang, Zhe Hu, Cheng Du, Xuefeng Wang, Xiaojun Wang 2024, 23(12): 4267-4271.  DOI: 10.1016/j.jia.2024.08.012 Abstract ( )   PDF in ScienceDirect   African horse sickness (AHS) is an acute and fatal vector-borne infectious disease of equids, caused by the African horse sickness virus (AHSV).  The World Organization for Animal Health (WOAH) has classified AHS as a notifiable animal disease, and AHS has also been classified as a Class I animal infectious disease in China.  AHS is mainly found in Africa, the Middle East and the Arabian Peninsula.  China is currently recognized by the WOAH as an AHS-free zone.  However, in 2020, there were outbreaks of AHS in 2 countries neighboring China, Thailand and Malaysia (Bunpapong et al. 2021), which increases the risk of the introduction of AHS into China.  Therefore, in order to prevent the occurrence of AHS in China and to further monitor the spread of the disease, the development of rapid, accurate and cost-effective diagnostic methods for the detection of AHSV is essential.   AHSV is a segmented double-stranded RNA virus belonging to the genus Orbivirus in the family Reoviridae.  It is mainly transmitted by midges (Maurer et al. 2022), and is able to infect all members of the Equidae, including horses, mules, donkeys, and zebras.  AHSV infection causes severe morbidity and mortality (up to 90%) in horses, while mules, donkeys and zebras are less susceptible than horses to the disease (Barnard 1998).   The AHSV genome contains 10 double-stranded RNA segments, encoding 7 structural proteins (VP1–7) and 4 non-structural proteins (NS1–4).  AHSV is a complex non-enveloped virus with an icosahedral capsid comprising 3 distinct concentric protein layers.  VP2 and VP5 are the components of the outer capsid of the virion.  VP2 is the major determinant of AHSV serotype, and 9 serotypes (AHSV-1 to AHSV-9) have been identified according to the VP2 antigenicity; VP3 and VP7 are the components of the major inner capsid of the virion; VP1, VP4 and VP6 constitute the minor inner capsid of AHSV.   AHSV RNA segment 7 (vp7) is highly conserved among all AHSV serotypes and is the primary molecular diagnostic target of AHSV.  The VP7 protein encoded by this segment is the major antigen of AHSV and is commonly used as a serological diagnostic for AHSV.  Real-time RT-PCR targeting vp7 is capable of detecting all known types of AHSV and is recommended by the WOAH for the detection of this virus (Aguero et al. 2008; Guthrie et al. 2013; WOAH 2019).   Recently, molecular diagnostics for infectious diseases have been developed based on clustered regularly interspaced short palindromic repeats-associated Cas endonucleases (CRISPR/Cas) systems combined with isothermal amplification techniques (Chen J S et al. 2018; Gootenberg et al. 2018; Myhrvold et al. 2018).  Some Cas proteins with non-specific endonuclease activity, such as Cas12a, activate auxiliary (non-specific) cleavage of nearby single-stranded non-target nucleic acids upon recognition of the target.  By modifying a single-stranded nucleic acid with a fluorophore quencher, which fluoresces upon cleavage of the Cas12a and crRNA complex, this activity can be used to detect the presence of specific cleavage.  The CRISPR/Cas12-based detection system has certain advantages over traditional nucleic acid diagnostic methods (qPCR), including rapidity, simplicity, low cost, and low equipment requirements.  In this study, we developed a sensitive detection method for AHSV using the CRISPR/Cas12a system combined with reverse transcription-recombinase-assisted amplification (RT-RAA) (CRISPR/Cas12a-RT-RAA), which specifically targets the vp7 RNA of AHSV.   To generate a CRISPR/Cas12-based AHSV detection system, a Cas12a from the Lachnospiraceae bacterium, LbCas12a protein, was first expressed in an Escherichia coli system and purified with Strep-Tactin Sepharose resin (Appendix A).  A single-stranded DNA (ssDNA) reporter labeled with a fluorophore and a quencher at the 2 termini (5´-6-FAM-TTATT-BHQ-3´) was synthesized by Sangon Biotech (Shanghai, China).  Ten crRNAs were designed to target the conserved region of the vp7 sequence of all AHSV strains (Appendices B and C).  These crRNAs with a repeat sequence were prepared using in vitro transcription following a previously described method (Wang et al. 2023).  In order to screen for an optimal crRNA for the sensitive detection of AHSV, 10 crRNAs were individually tested using a 25 μL CRISPR/Cas12-based reaction volume containing 0.4 μmol L–1 LbCas12a, 0.4 μmol L–1 ssDNA reporter, 1.2 μmol L–1 crRNA, 109 copies μL–1 vp7 plasmid DNA (pMD18-T-vp7, containing the entire vp7 sequence) and 2.5 μL NEBuffer 2.1 (10×).  The reaction was performed at 37°C for 50 min on a qPCR thermal cycler (Applied Biosystems QuantStusio 5 Real-Time PCR System, USA) with fluorescence measurements taken every 30 s.  Fluorescence detection suggested that crRNA10 showed the highest efficiency in this reaction system (Fig. 1-A).  Therefore, crRNA10 was identified as the best option for the AHSV CRISPR/Cas12a detection platform and was used in the subsequent experiments. Recombinase-assisted amplification (RAA) is an isothermal amplification technique that has been widely used to detect microbial pathogens (Chen C et al. 2018; Wang et al. 2020; Xue et al. 2020).  Recent studies have increasingly integrated the RAA assay with the CRISPR-Cas system, which provides a second detection step for amplification products, increasing detection sensitivity and specificity, and enabling more convenient and intuitive determination of detection results (Li et al. 2023).  Six specific RAA primers specifically targeting vp7 were designed based on the flanking sequence of the crRNA10 region (Appendix D).  To screen for the optimal RAA primer pair for the sensitive detection of AHSV, a standard RAA reaction was performed with pMD18-T-vp7 (at a concentration of 109 copies μL–1) as a template, using the RAA Nucleic Acid Amplification Kit (Qitian, China) and following the manufacturer’s instructions.  Following RAA amplification at 37°C for 30 min, the products of the RAA amplification were used as substrates for the CRISPR/Cas12a system detection.  As shown in Fig. 1-B, the strongest fluorescence signals for the CRISPR/Cas12a-RAA assay were detected when the F3/R3 primer pair was used.  The results showed that the F3/R3 primer pair had the best amplification efficiency and was therefore selected for the establishment of the CRISPR/Cas12a-RAA detection platform and used for subsequent experiments. To evaluate the sensitivity of the CRISPR/Cas12a-RAA detection platform, we prepared vp7 RNA in vitro using the HiScribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs, USA), using vp7 PCR products and with T7 promoter sequences as templates.  A total of 1 μL of vp7 RNA at different concentrations was used as a template for the RT-RAA reaction using RT-RAA Nucleic Acid Amplification Kit (Qitian, Wuxi, China) for 30 min, and then 1 μL of RAA amplification product was extracted and used as a substrate for the CRISPR/Cas12a detection system and reacted for 30 min.  The CRISPR/Cas12a-RT-RAA assay was developed in this way.  As shown in Fig. 1-C, the detection limit of the CRISPR/Cas12a-RT-RAA assay was 10 copies of the vp7 mRNA molecule per reaction.  However, the detection limit of the real-time RT-PCR assay established by Aguero in 2008 was 100 copies of the vp7 RNA molecule per reaction (Aguero et al. 2008) (Fig. 1-D).  In 2015, WOAH organized the AHS reference laboratory to conduct a comparison of trials to evaluate different conventional detection methods, and confirmed that the real-time RT-PCR established by Aguero in 2008 was one of the best detection methods for diagnosing AHSV (WOAH 2019).  Our results suggest that the CRISPR/Cas12a-RT-RAA assay has higher sensitivity compared to the real-time RT-PCR assay. To test the specificity of the CRISPR/Cas12a-RT-RAA assay to AHSV, other equine viral and bacterial pathogens were tested, including equine infectious anemia virus (EIAV), equine influenza virus (EIV), equine arthritis virus (EAV), equine herpesvirus-1 (EHV-1), equine herpesvirus-4 (EHV-4), Streptococcus equi subspecies equi (S. equi), and Salmonella enterica subsp. enterica serovar Abortusequi (S. Abortusequi).  All of these pathogens were stored in our laboratory and RNA/DNA from these pathogens was prepared as previously described (Chen et al. 2022).  As shown in Fig. 1-E, no fluorescence was observed when these equine pathogens were tested using the CRISPR/Cas12a-RT-RAA assay, whereas significant fluorescence was observed when the vp7 mRNA was tested, indicating that this CRISPR/Cas12a-RT-RAA method is highly specific for the detection of AHSV. Due to the lack of AHSV-positive samples, we evaluated the performance of the CRISPR/Cas12a-RT-RAA assay in clinical practice by adding vp7 mRNAs to mRNAs extracted from equine blood or tissue samples as positive controls, and equine blood or equine tissue mRNAs without vp7 mRNAs as negative controls.  A total of 20 equine mRNA samples, including 5 equine blood cell mRNA samples (S1–5), 5 equine blood cell mRNA samples with vp7 mRNA (S6–10), 5 equine tissue (heart, liver, spleen, lung and kidney) mRNA (S11–15), and 5 equine tissue mRNA with vp7 mRNA (S16–20), were prepared and assessed using the CRISPR/Cas12a-RT-RAA assay.  As shown in Fig. 1-F, strong fluorescence signals were detected in all equine mRNA samples with vp7 mRNA, but not in any equine mRNA samples without vp7 mRNA.  This result demonstrates that the CRISPR/Cas12a-RT-RAA assay is able to detect vp7 mRNA efficiently in samples collected under complex conditions and can be used as a back-up technology for the early field detection of AHSV. In conclusion, we reported the development and validation of a CRISPR/Cas12a-combined RT-RAA-based detection assay for AHSV with high specificity, sensitivity and convenience.  This assay targets the vp7 mRNA, a highly conserved segment of the AHSV genome that has not been observed to cross-react with the nucleic acids of 7 common equine pathogens, including EIAV, EIV, EAV, EHV-1, EHV-4, S. equi, and S. Abortusequi.  Notably, this assay was 10 times more sensitive than real-time RT-PCR.  In addition, the signal generated by the assay would be directly visible to the naked eye under UV light without the need for special instrumentation.  Therefore, the CRISPR/Cas12a combination RT-RAA assay developed here has the potential to be used as an alternative to traditional real-time RT-PCR assays for the rapid diagnosis of AHSV infection.  We will continue to optimize and improve the assay and expect that it will allow the detection of AHSV in the field and improve the early warning and diagnosis of AHS.