Single-time fertilization (STF) with controlled release blended fertilizer (CRBF) improves grain yield and nitrogen use efficiency (NUE) in rice production.  However, the impact of soil nitrogen (N) distribution and root growth on rice yield and NUE under STF with CRBF remains unclear.  Here, a two-year field experiment investigated the effects of two fertilizer types (normal urea (U) and CRBF) and two single-time fertilization methods (broadcast and side-deep fertilization) on the soil N distribution, plant N uptake, root characteristics, grain yield, and NUE.  The results showed that CRBF under STF increased the averages of plant dry matter accumulation, N uptake, grain yield, nitrogen recovery efficiency (NRE), and nitrogen agronomic efficiency (NAE) by 8.29, 21.85, 10.57, 79.28, and 74.8% compared to the other treatments, respectively.  Side-deep fertilization with CRBF further increased NUE by 12.78% compared to broadcast.  Moreover, CRBF under STF increased the leaf SPAD value and glutamine synthetase (GS)/glutamine oxoglutarate aminotransferase (GOGAT) activity by 5.93 and 25.58%, respectively.  CRBF under STF increased the soil inorganic N concentration and showed a “rising early and stabilizing later” pattern.  In addition, CRBF under STF improved rice root growth and increased the averages of root biomass, total root number, root average diameter, total root length, total root surface area, and total root volume by 28.30, 28.56, 18.64, 13.38, 35.26, and 37.06%, respectively, at the tillering and heading stages.  Partial least squares path modeling indicated that CRBF under STF increased the soil inorganic N concentration which improved root morphology, thereby increasing N uptake and improving the rice yield and NUE.  Taken together, our findings show that CRBF with single-time fertilization is the preferred N fertilizer strategy for achieving high yield and efficiency in rice, and that side-deep fertilization is the optimal fertilization method.

Peanut (Arachis hypogaea L.) is an important oil and edible protein crop.  Its fatty acid composition not only influences the quality of peanut oil but also impacts flavor, shelf life, and consumer health.  Peanut oil is comprised of approximately 80% oleic acid (C18:1) and linoleic acid (C18:2), 10% palmitic acid (C16:0), and the remaining 10% includes stearic acid (C18:0), arachidic acid (C20:0), gadoleic acid (C20:1), behenic acid (C22:0), and lignoceric acid (C24:0).  To unravel the genetic foundation of fatty acid content and delve into QTL localization, high-density SNP microarrays were used to genotype the RIL population of ‘SunOleic 97R’ × ‘NC94022’.  A genetic linkage map was constructed with 3,141 SNP markers, covering a total genetic distance of 3,051.81 cM.  Sixty quantitative trait loci (QTLs) associated with fatty acids were distributed in 11 linkage groups, with phenotypic variance explained (PVE) ranging from 1.37 to 44.92%.  Notably, the QTLs qFAT_A05.1 and qFAT_A08.1 are multiple-effect loci contributing to various fatty acid compositions.  Moreover, 15 haplotypes for the QTLs qFAT_A05.1 and qFAT_A08.1 were identified through genotyping 178 peanut germplasms.  Haplotype analysis in a natural population confirmed the close relationship of the QTLs with the contents of oil, oleic acid, lignoceric acid, palmitic acid and behenic acid.  This study serves as a valuable reference for selecting improved peanut genotypes with superior oil quality and desirable fatty acid composition.

Cadmium (Cd) contamination in wheat farmland is increasing at an alarming rate, posing threats to food security and public health.  Breeding and utilizing wheat varieties characterized by low Cd accumulation levels constitute an effective strategy in the battle against wheat Cd contamination.  The adoption of molecular marker-assisted approaches can greatly expedite the selection and enhancement of wheat varieties with low Cd accumulation.  Nonetheless, research concerning the genes associated with wheat cadmium accumulation remains scarce.  In this study, a high-density 660K SNP array was employed for conducting a genome-wide association study (GWAS) on the grain Cd concentration (GCdC), bioconcentration factor (BCF) and translocation factor (TF) in 175 wheat germplasms.  The findings revealed 401 significant SNPs identified across three diverse environments.  Linkage disequilibrium analysis revealed 30 core quantitative trait loci (QTLs) capable of reliably modulating wheat Cd accumulation phenotypes.  Through gene annotation, transcriptomics, and gene molecular features, four candidate genes (TraesCS7B02G000200, TraesCS4A02G035900, TraesCS4A02G040900, and TraesCS5D02G564000) were identified as potential constituents in the biological process of wheat Cd accumulation.  Furthermore, six wheat germplasms exhibiting low grain Cd accumulation were isolated, and two kompetitive allele specific PCR (KASP) markers conducive to breeding selection were developed.  These findings provide valuable genetic resources for cultivating wheat with low Cd accumulation and establish a foundation for understanding the molecular mechanisms underlying low Cd accumulation in wheat.  The candidate genes and KASP markers elucidated in this research have potential for effective use in genetic enhancement and marker-assisted selection in the breeding of wheat with low Cd accumulation.

Probiotics are considered to exert beneficial effects in humans and animals by modulating the structure and metabolic functions of the gut microbiota.  Therefore, the identification of functional probiotics and in-depth exploration of the characteristics and applicability of probiotics are of paramount significance for the development of more effective probiotic products and the formulation of personalized probiotic treatment strategies in the fields of human health and livestock farming.  However, due to current limitations in sequencing technologies and considering that microbial communities may encompass closely related lineages, rendering metagenome assembly complex, the generation of complete metagenome-assembled genomes (cMAGs) is hindered.  This limitation constrains our comprehensive resolution at the probiotic strain level.  In this review, we summarized the effects of probiotics on gut microbiota balance and host health from a functional perspective.  The technical methods of functional probiotics identification were summarized from the technical point of view.  Furthermore, we introduced methods for microbial metagenome assembly to elucidate the associated progress and advantages and disadvantages of these approaches.  Finally, we highlight more advanced metagenomic assembly techniques that may help us assemble high-fidelity intestinal metagenomes, providing powerful tools for the identification of functional probiotics.

Intervention strategies to control non-point source nitrogen (N) and phosphorus (P) pollution in agriculture are expensive and there is a trade-off between engineering cost and treatment effectiveness.  Implementing strategies often result in unsatisfactory outcomes and massive engineering costs when managing diffusive pollution in agricultural catchments.  To address this issue, this paper proposes a robust, handy, catchment N&P decision support system (CNPDSS), an Android-based smartphone system integrated with a web-based geographic information system (GIS).  The CNPDSS aims to provide artificial intelligence-driven decisions that minimize N&P loadings and engineering costs for mitigating pollution in agricultural catchments.  It consists of four components: a general user interface (GUI), GIS, N&P pollution modeling (NPPM), and a DSS.  The CNPDSS simplifies the GUI and integrates GIS modules to create a user-friendly interface, enabling non-professional users to operate the system easily through intuitive actions.  The NPPM uses straightforward empirical models to predict N&P loadings, enhancing efficiency by avoiding excessive parameters.  Taking into account the N&P movement pathway in the catchment, the DSS incorporates three control measures: source reduction in farmland (before migration stage), process retention by ecological ditch (midway transport stage), and down-end purification by constructed wetland (waterbody discharge stage), to formulate a comprehensive ternary controlling strategy.  To optimize the cost-effectiveness of any proposed N&P control strategies for sub-catchments, a differential evolution algorithm (DEA) is employed in CNPDSS to carry out a dual-objective decision-making optimization computation.  In this study, the CNPDSS is applied to a case study in an agricultural catchment in Central China to develop the most cost-effective ternary N&P control strategies that ensure the catchment water quality within Criterion III of the Chinese Surface Water Quality Standard GB3838-2002 is met (total N concentration≤1.0 mg L⁻¹ and total P concentration≤0.2 mg L⁻¹).  Our results demonstrate that the CNPDSS is feasible and also possesses an adaptive design and flexible architecture to enable its generalization and extension to support strong hands-on applications in other catchments.

Thinopyrum ponticum (2n=10×=70), a wild relative of common wheat (Triticum aestivum L.), is considered an invaluable genetic resource for wheat improvement due to its abundance of genes conferring resistance to biotic and abiotic stresses.  This study focused on the CH97 line, derived from the BC₁F₇ progeny of a cross between wheat cv. 7182 and Th. ponticum.  Cytological evidence showed that CH97 has 42 chromosomes, forming 21 bivalents at meiotic metaphase I, with the bivalents subsequently separating and moving to opposite poles during meiotic anaphase I.  Through a combination of fluorescence in situ hybridization (FISH), genomic in situ hybridization (GISH), multicolor GISH (mc-GISH), and liquid array analysis, it was determined that CH97 comprises 40 wheat chromosomes and two alien chromosomes from the E^e genome of Th. ponticum, featuring the absence of a pair of 5D chromosomes and variations in 1B, 6B, and 7B chromosomes.  These findings confirm that CH97 is a stable wheat-Th. ponticum 5E (5D) alien disomic substitution line.  Inoculation experiments revealed that CH97 exhibits high resistance to wheat powdery mildew and stripe rust throughout the growth period, in contrast to the highly susceptible common wheat parent 7182.  Compared to 7182, CH97 displayed improvements in thousand-kernel weight and kernel length.  Additionally, utilizing specific-locus amplified fragment sequencing (SLAF-seq) technology, chromosome 5E-specific molecular markers were developed and validated, achieving a 33.3% success rate, facilitating marker-assisted selection for disease resistance in wheat.  Overall, the CH97 substitution line, with its resistance to diseases and improved agronomic traits, represents valuable new germplasm for wheat chromosome engineering and breeding.

Apple fruit firmness is a crucial index for measuring the internal quality of apples, which influences palatability, storage capacity and transportability.  The primary cause of reduced firmness during fruit development is the hydrolysis of cell wall polysaccharides.  Xyloglucan endotransglycosylase/hydrolase (XTH) is a key enzyme involved in the depolymerization of cell wall polysaccharides, but the mechanism of its involvement in the formation of fruit firmness remains unclear.  Here, we identified the gene MdXTH2 by integrating metabolomic and transcriptomic data, and analyzed its function and molecular mechanism in the formation of apple fruit firmness.  The results showed downward trends in both fruit firmness and cell wall components throughout fruit development.  The contents of cell wall material, cellulose, and hemicellulose in various apple varieties exhibited significant positive correlations with firmness, with total correlation coefficients of 0.862, 0.884, and 0.891, respectively.  Overexpression of MdXTH2 significantly increased fruit firmness in apple and tomato, inhibited fruit ripening, and significantly suppressed the growth of calli.  The upstream transcription factor MdNAC72 of the MdXTH2 gene can promote the expression of fruit ripening-related genes.  Furthermore, dual-luciferase, yeast one-hybrid, and electrophoretic mobility shift assay (EMSA) demonstrated that MdNAC72 down-regulates the transcription of MdXTH2 by binding to its promoter.  In summary, the results of this study provide a strategy for examining fruit quality regulation and a theoretical basis for breeding apple varieties with moderate firmness through genetic improvement.

The inappropriate use of cephalosporins lead to the occurrence and global spread of bacteria resistant to these antimicrobials.  In this study, we isolated four Escherichia albertii strains from broilers in eastern China.  The antimicrobial susceptibility and genomic characterization of these E. albertii isolates were determined.  Our results revealed that these four E. albertii isolates exhibited resistance to tetracyclines, chloramphenicol, β-lactams, aminoglycosides, polymyxin B, sulfonamides, quinolones, and other antimicrobials.  Among them, EA04 isolate was multidrug resistant and harbored extended-spectrum β-lactamases (ESBL) genes bla_CTX-M and bla_TEM.  Whole genome sequencing and core-genome multilocus sequence typing (cgMLST) based on all ST4638 E. albertii for EA04 inferred highly probable epidemiological links between selected human isolates.  Additionally, the ESBL genes bla_TEM-141 and bla_CTX-M-55 were coexistent in an approximately 75 kb IncFII plasmid pEA04.2 in EA04.  Comparative analysis indicated that genes bla_TEM-141 and bla_CTX-M-55 were located in IS15-bla_CTX-M-55-wbuC-bla_TEM-141-IS26 region, which similar structures were identified in various bacteria.  Furthermore, the plasmid pEA04.2 could be transferable to E. coli EC600 and lead to the resistance to third-generation cephalosporins.  These results suggested that chicken potentially serve as a reservoir for multidrug resistant E. albertii, which increases the risk of horizontal transfer of antimicrobial resistance between humans, animals and environment.

Cattle carcass traits are economically important in the beef industry.  In the present study, we identified 184 significant genes and 822 alternative genes for 7 carcass traits using genome-wide association studies (GWAS) in 1,566 Huaxi beef cattle.  We then identified 5,860 unique cis-genes and 734 trans-genes in 227 longissimus dorsi muscle (LDM) samples to better understand the genetic regulation of gene expression.  Our integration study of the GWAS and cis-eQTL analysis detected 13 variants regulating 12 identical genes, in which one variant was also detected in fine-mapping analysis.  Moreover, using a transcriptome-wide association study (TWAS), we identified 4 genes (TTC30B, HMGA1, PRKD3 and FXN) that were significantly related to carcass chest depth (CCD), carcass length (CL), carcass weight (CW) and dressing percentage (DP).  This study identified variants and genes that may be useful for understanding the molecular mechanism of carcass traits in beef cattle.

Malate dehydrogenase (MDH) is a widely expressed enzyme that plays a key role in plant growth, development, and stress responses.  However, information on MDH genes in the soybean genome is limited.  Seventeen members of the soybean MDH family were identified by genome-wide analysis, and the genes were analyzed for the presence of conserved protein motifs.  The genes were divided into five clusters according to their phylogenetic relationships.  The intracellular localizations of six GmMDHs were determined by confocal microscopy of Arabidopsis mesophyll protoplasts.  Transcripts of GmMDHs were significantly increased by abiotic stress (drought, salt, and alkalinity) and hormone treatments, as shown by an analysis of cis-regulatory elements and quantitative real-time polymerase chain reaction (qRT-PCR).  The GmMDHs displayed unique expression patterns in various soybean tissues.  Notably, the expression levels of a chloroplast isoform (GmMDH2) were unusually high under salt stress, presumably indicating a critical role in soybean responses to salinity.  Expression of GmMDH2 in Escherichia coli showed that the recombinant enzyme has nicotinamide adenine dinucleotide phosphate (NADP)-dependent MDH activity.  The redox states of the NADP (reduced form) (NADPH) pool and antioxidant activities were shown to be modulated by GmMDH2 gene overexpression, which in turn reduced reactive oxygen species (ROS) formation in transgenic soybean, significantly enhancing the salt stress resistance.  Gene-based association analysis showed that variations in GmMDH2 were strongly linked to seedling salt tolerance.  A polymorphism potentially associated with salt tolerance was discovered in the promoter region of GmMDH2.  These findings not only improve our understanding of the stress response mechanism by identifying and characterizing the MDH gene family throughout the soybean genome but they also identified a potential candidate gene for the future enhancement of salt tolerance in soybean.  

The wheat above-ground biomass (AGB) is an important index that shows the life activity of vegetation, which is of great significance for wheat growth monitoring and yield prediction.  Traditional biomass estimation methods specifically include sample surveys and harvesting statistics.  Although these methods have high estimation accuracy, they are time-consuming, destructive, and difficult to implement to monitor the biomass at a large scale.  The main objective of this study is to optimize the traditional remote sensing methods to estimate the wheat AGB based on improved convolutional features (CFs).  Low-cost unmanned aerial vehicles (UAV) were used as the main data acquisition equipment.  This study acquired RGB and multi-spectral (MS) image data of the wheat population canopy for two wheat varieties and five key growth stages.  Then, field measurements were conducted to obtain the actual wheat biomass data for validation.  Based on the remote sensing indices (RSIs), structural features (SFs), and convolutional features (CFs), this study proposed a new feature named AUR-50 (Multi-source combination based on convolutional feature optimization) to estimate the wheat AGB.  The results show that AUR-50 could more accurately estimate the wheat AGB than RSIs and SFs, and the average R² exceeded 0.77.  AUR-50_MS had the highest estimation accuracy (R² of 0.88) in the overwintering period.  In addition, AUR-50 reduced the effect of the vegetation index saturation on the biomass estimation accuracy by adding CFs, where the highest R² was 0.69 at the flowering stage.  The results of this study provide an effective method to evaluate the AGB in wheat with high throughput and a research reference for the phenotypic parameters of other crops.

Caspases, which play key roles in cell apoptosis, undergo alternative splicing to form different splicing variants that can regulate the apoptotic process.  Lepidopteran insect caspases undergo alternative splicing, although the functions of their splicing variants are still unclear.  The Spodoptera exigua caspase-5 (SeCaspase-5) gene was cloned and found to produce four different splicing variants with different gene sequences and protein functional domains, which were named SeCaspase-5a, SeCaspase-5b, SeCaspase-5c and SeCaspase-5d.  Overexpression of these variants in S. exigua cells (Se-3) showed that SeCaspase-5a had a proapoptotic function, whereas SeCaspase-5b, SeCaspase-5c and SeCaspase-5d did not.  Semi-qPCR analysis revealed that the expression of the SeCaspase-5 variants significantly differed during Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection.  Furthermore, the SeCaspase-5 variants were constructed into the AcMNPV bacmid and transfected into Se-3 cells, which revealed that SeCaspase-5a promoted cell apoptosis and reduced virus production, whereas SeCaspase-5b, SeCaspase-5c and SeCaspase-5d did not promote cell apoptosis but instead increased virus production.  Moreover, an analysis of the interactions between the SeCaspase-5 variants revealed that SeCaspase-5a directly interacted with SeCaspase-5b, SeCaspase-5c and SeCaspase-5d.  Coexpression of these variants in Se-3 cells also revealed that SeCaspase-5b, SeCaspase-5c and SeCaspase-5d inhibited the proapoptotic function of SeCaspase-5a, resulting in a reduction in the percentage of apoptotic cells by about 20%.  These results indicate that SeCaspase-5 undergoes alternative splicing and is involved in regulating the apoptosis induced by baculovirus infection.  These findings increase our understanding of the functions of lepidopteran insect caspases and provide new insights into the mechanism of host-cell apoptosis induced by baculoviruses.

The eutrophication of rivers and lakes is becoming increasingly common, primarily because of pollution from agricultural non-point sources.  We investigated the effects of optimized water and fertilizer treatments on agricultural non-point source pollution in the Nansi Lake basin.  The water heat carbon nitrogen simulator model (WHCNS model) was used to analyze water and nitrogen transport in wheat fields in Nansi Lake basin.  Four water and fertilizer treatments were set up: conventional fertilization and irrigation (CK), reduced controlled-release fertilizer and conventional irrigation (F2W1), an equal amount of controlled-release fertilizer and reduced irrigation (F1W2), and reduced controlled-release fertilizer and reduced irrigation (F2W2).  The results indicated that the replacement of conventional fertilizers with controlled-release fertilizers, combined with reduced irrigation, led to reduced nitrogen loss.  Compared with those of the CK, the cumulative nitrogen leaching and ammonia volatilization of F2W1 were reduced by 8.90 and 41.67%, respectively; under F1W2, the same parameters were reduced by 12.50 and 15.99%, respectively.  Compared with the other treatments, F2W2 significantly reduced nitrogen loss while producing a stable yield.  Compared with those of the CK, ammonia volatilization and nitrogen loss due to leaching were reduced by 29.17 and 27.13%, respectively, water and nitrogen use efficiencies increased by 11.38 and 17.80%, respectively.  F2W2 showed the best performance among the treatments, considering water and fertilizer management.  Our findings highlight the effectiveness of optimizing water and fertilizer application in improving the water and nitrogen use efficiency of wheat, which is of great significance for mitigating nitrogen loss from farmland in the Nansi Lake basin.

The optimized management of crop fertilization is very important for improving crop yield and reducing the consumption of chemical fertilizers. Critical nutrient values can be used for evaluating the nutritional status of a crop, and they reflect the nutrient concentrations above which the plant is sufficiently supplied for achieving the maximum potential yield. Based on on-farm surveys of 504 farmers and 60 field experimental sites in the drylands of China, we proposed a recommended fertilization method to determine nitrogen (N), phosphorus (P), and potassium (K) fertilizer input rates for wheat production, and then validated the method by a field experiment at 66 different sites in northern China. The results showed that wheat grain yield varied from 1.1 to 9.2 t ha⁻¹, averaging 4.6 t ha⁻¹, and it had a quadratic relationship with the topsoil (0−20 cm) nitrate N and soil available P contents at harvest. However, yield was not correlated with the inputs of N, P, and K fertilizers. Based on the relationship (exponential decay model) between 95–105% of the relative yield and topsoil nitrate N, available P, and available K contents at wheat harvest from 60 field experiments, the topsoil critical nutrient values were determined as 34.6, 15.6, and 150 mg kg⁻¹ for soil nitrate N, available P, and available K, respectively. Then, based on five groups of relative yield (>125%, 115–125%, 105–115%, 95–105%, and <95%) and the model, the five groups of topsoil critical nutrient levels and fertilization coefficients (Fc) were determined. Finally, we proposed a new method for calculating the recommended fertilizer input rate as: Fr=Gy×Nr×Fc, where Fr is the recommended fertilizer (N/P/K) input rate; Gy is the potential grain yield; Nr is the N(N_rN), P(N_rP), and K(N_rK) nutrient requirements for wheat to produce 1,000 kg of grain; and Fc is a coefficient for N(N_c)/P(P_c)/K(K_c) fertilizer. A 2-year validated experiment confirmed that the new method reduced N fertilizer input by 17.5% (38.5 kg N ha⁻¹) and P fertilizer input by 43.5% (57.5 kg P₂O₅ ha⁻¹) in northern China and did not reduce the wheat yield. This outcome can significantly increase the farmers’ benefits (by 7.58%, or 139 US$ ha⁻¹).  Therefore, this new recommended fertilization method can be used as a tool to guide N, P, and K fertilizer application rates for dryland wheat production.

Water is the key factor limiting dryland wheat grain yield.  Mulching affects crop yield and yield components by affecting soil moisture.  Further research is needed to determine the relationships between yield components and soil moisture with yield, and to identify the most important factor affecting grain yield under various mulching measures.  A long-term 9-year field experiment in the Loess Plateau of Northwest China was carried out with three treatments: no mulch (CK), plastic mulch (M_P) and straw mulch (M_S).  Yield factors and soil moisture were measured, and the relationships between them were explored by correlation analysis, structural equation modeling and significance analysis.  The results showed that compared with CK, the average grain yields of M_P and M_S increased by 13.0 and 10.6%, respectively.  The average annual grain yield of the M_P treatment was 134 kg ha^–1 higher than the M_S treatment.  There were no significant differences in yield components among the three treatments (P<0.05).  Soil water storage of the M_S treatment was greater than the M_P treatment, although the differences were not statistically significant.  Soil water storage during the summer fallow period (SWSSF) and soil water storage before sowing (SWSS) of M_S were significantly higher than in CK, which increased by 38.5 and 13.6%, respectively.  The relationship between M_P and CK was not statistically significant for SWSSF, but the SWSS in M_P was significantly higher than in CK.  In terms of soil water storage after harvest (SWSH) and water consumption in the growth period (ET), there were no significant differences among the three treatments.  Based on the three analysis methods, we found that spike number and ET were positively correlated with grain yield.  However, the relative importance of spike number to yield was the greatest in the M_P and M_S treatments, while that of ET was the greatest in CK.  Sufficient SWSSF could indirectly increase spike number and ET in the three treatments.  Based on these results, mulch can improve yield and soil water storage.  The most important factor affecting the grain yield of dryland wheat was spike number under mulching, and ET with CK.  These findings may help us to understand the main factors influencing dryland wheat grain yield under mulching conditions compared to CK.

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.

Roots are vital for crop growth, development, yield and tolerance to various types of environmental stress.  Numerous genetic loci associated with soybean root morphological traits have been identified, but few genes associated with these traits have been identified.  In this study, seven quantitative trait loci (QTLs) containing stable SNPs significantly associated with the root dry weight in soybeans were identified through a genome-wide association study.  Among these QTLs, qRDW14-2 presented the greatest significance.  In qRDW14-2, the gene GmRGD14, encoding the lysophosphatidic acid acyltransferase LPAT4, was identified as a candidate.  GmRGD14, in block63, which contained the significant SNP S14_6521715, had the highest expression level in soybean roots, and its Arabidopsis homologous mutant lpat4 presented more lateral roots than did the control Col-0.  GmRGD14 was localized primarily to the cell membrane and endoplasmic reticulum.  The heterologous overexpression of GmRGD14 in Arabidopsis significantly increased the lateral root number, which was similar to the phenotype of atlpat4.  Furthermore, overexpression of GmRGD14 resulted in a greater total root length, root tip number, root surface area and root volume in the hairy roots of transgenic soybean plants than in those of WT soybean plants, whereas knockdown of the gene via RNA interference in soybean hairy roots resulted in the opposite phenotype.  GmRGD14, which is highly genetically variable in wild soybean, has been gradually utilized during soybean domestication.  Overall, this study revealed that GmRGD14 is a new key gene that plays a role in root growth, providing a new genetic target for breeding elite soybean varieties with strong root systems.

Avian pathogenic Escherichia coli (APEC) could cause colibacillosis, which is economically devastating to poultry industries worldwide. The bacterial membrane is critical to its environment adaptability and virulence. The inner membrane protein TolA maintains membrane integrity, but the roles of which in fitness and pathogenesis of APEC are not completely understood. Thus, the tolA gene mutant and complemented strains of APEC were constructed and characterized. We found that mutant strain ΔtolA was damaged in inner and outer membranes, and showed altered morphology, impaired flagella production, reduced motility, increased outer membrane vesicles (OMVs) production, decreased resistance to antibiotics and environmental stress. Deletion of tolA gene resulted in a significant decrease in biofilm formation and interbacterial competition, due to the downregulated expression of biofilm-associated genes and type VI secretion system (T6SS) genes, respectively. In addition, the mutant strain exhibited diminished serum bactericidal resistance, reduced cell infection capacity, decreased intracellular survival, consequently, leading to attenuated bacterial survival and virulence in mice. Compared with the wild-type and complemented strains, mutant strain induced less expression of inflammatory cytokine interleukin 1 beta (IL-1β) in HD-11 macrophages, consistent with the pathological damage in mice. In conclusion, inner membrane protein TolA contributed to the antibiotic resistance, environment adaptability, biofilm formation and virulence of APEC.

Wheat (Triticum aestivum L.) is a vital staple crop globally, with its grain microelement content playing a crucial role in human nutrition and health. In this study, the concentrations of eight essential microelements (micronutrients and toxic elements): iron (Fe), manganese (Mn), copper (Cu), zinc (Zn), selenium (Se), chromium (Cr), cadmium (Cd), and arsenic (As), were quantified in 272 wheat varieties using inductively coupled plasma mass spectrometry (ICP-MS) under three different environments. A genome-wide association study (GWAS) was conducted using 176,357 molecular markers, comprising 163,223 single-nucleotide polymorphisms (SNPs) and 13,134 insertion-deletion (InDels) variants, identified through RNA sequencing. A total of 196 significant markers associated with microelement content traits were identified across 21 chromosomes in various environments. Of these, 14 significant markers consistently appeared across environments, forming 13 QTLs and linking to 45 candidate genes. Among these, 29 genes were homologs of known genes in Arabidopsis and rice, while 16 were novel candidates. Haplotype analysis indicated significant phenotypic variation in microelement accumulation, with TraesCS6A02G204300^Hap2 notably enhancing iron content. This study provides valuable insights into the genetic architecture of microelement accumulation in wheat grains and introduces novel genetic resources for breeding wheat varieties aimed at improving micronutrient content and ensuring food safety.

Fusarium head blight (FHB), primarily caused by Fusarium graminearum, is a globally destructive fungal disease that not only reduces cereal crop yields but also threatens food safety due to mycotoxin contamination. In this study, we systematically characterized histone acetyltransferases in F. graminearum and revealed the overlapping functions between FgSas3 and FgRtt109. The double deletion mutant Fgsas3 Fgrtt109 showed severely impaired vegetative growth, conidiation, mycotoxin production, as well as complete loss of sexual reproduction and pathogenicity. Furthermore, integrated transcriptome and metabolome analyses revealed that this double mutant had significant dysregulation in carbohydrate metabolism, particularly in the disaccharides to monosaccharides conversion. This metabolic shift was evidenced by the reduced disaccharide concentrations, accumulated monosaccharide and their derivatives, and enhanced growth on disaccharide-supplemented medium in the Fgsas3 Fgrtt109 double mutant. Taken together, our results demonstrate that FgSas3 and FgRtt109 synergistically regulate carbohydrate metabolism, which in turn modulates fungal development, and plant infection.

Rift Valley fever virus (RVFV) is a highly virulent zoonotic pathogen posing substantial risks to global public health and livestock industries. Classified by the WHO as a priority pathogen with high pandemic potential, RVFV underscores the critical need for fundamental research to accelerate the development of vaccines and antiviral agents. In this study, we engineered a replication-defective RVFV system that preserves the capacity for host cell infection and a single round of genomic replication. Targeted deletion of the envelope glycoprotein genes Gn and Gc, together with the non-structural protein NSm resulted in a replication-incompetent virus capable of producing infectious particles only in trans-complementing cell lines engineered to express the missing components. This system enables safe experimentation under BSL-2 containment. By incorporating a biotin acceptor peptide (AP) tag into the viral L protein and leveraging biotin-streptavidin bridging for quantum dot conjugation, we developed a highly specific, protein-level labeling platform for single-virus tracking of RVFV. This advanced methodology permits real-time visualization of the viral life cycle from the point of cellular entry. Using this system, we have obtained the first live-cell imaging evidence that RVFV undergoes microtubule-dependent transport via endocytic vesicles during infection. Our findings provide unprecedented insight into the dynamic post-entry trafficking of RVFV and establish a versatile and safe strategy applicable to the study of other high-containment pathogens.

Fusarium head blight (FHB) and crown rot (FCR) threaten global wheat production, demanding sustainable biocontrol solutions. We isolated 50 indigenous Clonostachys strains (C. chloroleuca, C. rosea, C. rogersoniana) from Chinese agroecosystems and identified key biocontrol traits. Critically, sporulation rate—not mycelial growth—correlates with Fusarium suppression efficacy, revolutionizing agent selection criteria. Five elite strains completely prevented perithecia formation on crop residues. Microscopy revealed direct mycoparasitism through perithecial wall adhesion and enzymatic destruction of asci/ascospores. Transcriptomics of parasitized perithecia showed Fusarium stress responses including ABC transporter induction, membrane remodeling, and DNA repair activation, confirming membrane damage and genotoxic stress. Strain Cc878 exhibited dual-mode protection: suppressing residue-borne inoculum while establishing root endophytism via seed treatment. This protected against multiple soilborne diseases (FCR, common root rot, take-all) without yield penalties and primed systemic immunity through MAPK/phenylpropanoid pathways. Genome analysis revealed extensive secretomes, CAZymes, and secondary metabolite clusters underpinning biocontrol mechanisms. This integrated strategy combining inoculum reduction with immunity priming provides a sustainable alternative to chemical fungicides for managing devastating Fusarium diseases in wheat production systems.

Ammonia-oxidizing microorganisms (AOMs) mediate a pivotal yet poorly understood step in agricultural nitrogen cycling under long-term fertilization. To identify the key microbial drivers of ammonia oxidation, two DNA stable isotope probing (DNA-SIP) experiments were conducted on soils under long-term fertilization regimes. Through integrated DNA-SIP and targeted inhibition approaches (C₂H₂, simvastatin, C₈H₁₄), we intended to resolve functional partitioning among ammonia-oxidizing archaea (AOA), bacteria (AOB), and comammox Nitrospira in soils subjected to multi-decadal fertilization regimes. SIP revealed the exclusive functional dominance of AOB (primarily Nitrosospira cluster 3a), which drove over 85% of autotrophic nitrification in fertilized soils. In contrast, AOA activity was significant only in non-fertilized (CK) and mineral N-only (N) soils. Notably, comammox Nitrospira showed negligible functional engagement, with no labeled DNA detected. The inhibitor 1-octyne (C₈H₁₄) broadly suppressed both AOB (by 61.9–88.9%) and, unexpectedly, AOA (by up to 42.7%), challenging its specificity. Simvastatin preferentially inhibited comammox Nitrospira clade B. High-throughput sequencing confirmed Nitrososphaera (AOA) and Nitrosospira 3a as keystone nitrifiers, with no active comammox phylotypes detected. These findings challenge assumptions of comammox metabolic prominence in agroecosystems, demonstrating that long-term fertilization restructures nitrification networks through species sorting driven by high ammonium availability, leading to the competitive dominance of AOB. Our work establishes AOB as primary nitrogen cycle engineers in intensively managed soils, providing a molecular blueprint for precision nitrogen management to mitigate environmental impacts.

Weak seedling vigor of machine-transplanted rice during the recovery stage often limits basal-tillering nitrogen (N) uptake and yield, particularly under the split urea application with the increasing N. In this experiment, the effect of tandem long-mat seedlings (TLMS) transplanted with seedling fertilizer (SF) on yield and N use efficiency (NUE) was studied. Three-season field experiments at two sites consisting of TSF (transplanting with 7.0 kg ha^-1 SF) and T (transplanting without SF) based on five different dosages of basal-tillering N were conducted to comprehensively study the effect of SF in field. The results show that SF released rapidly after transplanting and significantly increased dry matter accumulation, N uptake and their rates during tillering stage. Consequently, TSF showed enhanced growth with early emerging tillers, significantly higher proportion of effective tillers and panicle numbers, and 7.8% higher yield than T. Consistently, basal tillering and total NUE (13.8%) of TSF was significantly higher than that of T. Notably, in some growing seasons, even with a 37.5 kg ha^-1 reduction in basal-tillering N, TSF still achieved a comparable dry weight, N uptake and yield to that of T, supporting the quantitative significance of seedling fertilizer in TLMS. Further quantification through regression analysis of yield and dosage verified that 7.0 kg ha^-1 N supplied via SF was equally effective as 24.1 kg/ha basal-tillering N, in terms of yield response. Overall, TLMS transplanting with SF is an effective strategy to enhance the early growth vigor, improve yield and NUE, and reduce basa-tillering N input in machine transplanted rice. This study successfully integrates the soilless nursery establishment with SF within mechanical rice-transplanting system and quantitatively demonstrating its contribution to post-transplantation performance of rice seedlings.

Wheat is one of the most important food crops in the world, and grain number per spike (GNS) is one of the most important factors affecting wheat grain yield. Therefore, identifying genes controlling GNS is important for wheat production. However, wheat is an allohexaploid species and has a highly complex genome, and the isolation of wheat genes is challenging. In this study, we identified a candidate gene, TraesCS4B02G047100 (TaRLK-4B), associated with GNS using both quantitative trait locus (QTL) mapping and genome-wide association study (GWAS) based on RNA-Seq. We obtained three homozygous mutant lines, bb-1, bb-2 and bb-3, using the CRISPR/Cas9 gene editing system. Compared with Fielder (wild type, WT), the three mutant lines exhibited significant reductions in GNS, total spikelets per spike (TSS) and spike length (SL). These results indicated that TaRLK-4B positively regulates GNS and its related traits TSS and SL. The TaRLK-4B gene contains two exons and one intron and belongs to the largest subfamily of LRR-RLKs. We performed RNA-Seq analysis using spikes from the WT and bb-2 mutant line at the tillering stage. A total of 1,193 differentially expressed genes (DEGs) were identified, including previously cloned TaSPL17 homologous genes in the three subgenomes and their orthologous gene OsIPA1 (OsSPL14) in rice. Combining RNA-Seq data of TaRLK-4B and DAP-Seq data of the TaSPL17-7D, we identified 136 overlapping genes which are likely downstream targets of TaSPL17-7D. Therefore, we hypothesized that TaRLK-4B represents a novel putative component of the TaSPL17-centered regulatory network. In addition, haplotype analysis revealed that Haplotype 2 (Hap2) is a favorable haplotype for increasing GNS, but the thousand-grain weight (TGW) was not significantly different between Haplotype 1 (Hap1) and Hap2.