H9N2 avian influenza virus (AIV) has widely circulated in poultry worldwide and sporadic infections in humans and mammals.  During our surveillance of chicken from 2019 to 2021 in Shandong Province, China, we isolated 11 H9N2 AIVs.  Phylogenetic analyses showed that the eight gene segments of the 11 isolates were closely related to several sublineages of Eurasian lineage: BJ/94-like clades (HA and NA genes), G1-like clades (PB2 and M genes), and SH/F/98-like clades (PB1, PA, NP and NS genes).  The isolates showed mutation sites that preferentially bind to human-like receptors (HA) and mammalian fitness sites (PB2, PB1 and PA), as well as mutations in antigen and drug resistance sites.  Moreover, studies with mice revealed four isolates with varying levels of pathogenicity.  The average antibody titer of the H9N2 AIVs was 8.60 log2.  Based on our results, the epidemiological surveillance of H9N2 AIVs should be strengthened.

Osteoblasts are considered as a major factor contributing to bone development and mineralization, however, few studies have been done to establish and evaluate the primary cultured tibial osteoblast model of broiler chicks.  Therefore, in the present study, two experiments were conducted to establish and evaluate the primary cultured tibial osteoblast model of broiler chicks.  In experiment 1, osteoblasts were isolated from the tibia of one-day-old Arbor Acre male broiler chicks using the explant method and identified through the cell morphology, alkaline phosphatase (ALP) and alizarin red staining.  Experiment 2 was carried out to evaluate the vitality and mineralization of primary cultured tibial osteoblasts of broilers on days 4, 8, 12, 16, 20, 24, 28 and 32 after incubation, respectively.  The results from experiment 1 demonstrated that primary cultured tibial osteoblasts of broilers showed a spindle-shaped, triangular or polygonal morphology.  More than 95% of the cells were stained blue-black after ALP staining, and mineralized nodules were formed after 4 days of continuous incubation.  in experiment 2, lactate dehydrogenase (LDH) activity stayed at a relatively stabilized level although incubation time affected (P=0.0012) it during the whole culture period.  Additionally, incubation time affected (P≤0.0001) the number and proportion of the area of mineralized nodules.  They increased linearly and quadratically (P<0.04) with the increase of incubation time, and remained at a stabilized level from 24 to 32 days of incubation.  The estimates of the optimal incubation time were 17 and 26 days based on the best fitted broken-line or quadratic models (P<0.0001) of the number and proportion of the area of mineralized nodules, respectively.  These results indicate that the primary cultured tibial osteoblast model of broilers has been established successfully by the explant method, and it showed typical osteoblast morphology and characteristics of ALP activity and mineralization, and could maintain a relatively stabilized vitality from 4 to 32 days of incubation; and the optimal incubation time of primary tibial osteoblasts was 17 to 26 days.  Therefore, it could be used to further study the underlying mechanisms of bone development and mineralization of broiler chicks.

Soil depth is critical for eco-hydrological modeling, carbon storage calculation and land evaluation.  However, its spatial variation is poorly understood and rarely mapped.  With a limited number of sparse samples, how to predict soil depth in a large area of complex landscapes is still an issue.  This study constructed an ensemble machine learning model, i.e., quantile regression forest, to quantify the relationship between soil depth and environmental conditions.  The model was then combined with a rich set of environmental covariates to predict spatial variation of soil depth and straightforwardly estimate the associated predictive uncertainty in the 140 000 km² Heihe River basin of northwestern China.  A total of 275 soil depth observation points and 26 covariates were used.  The results showed a model predictive accuracy with coefficient of determination (R²) of 0.587 and root mean square error (RMSE) of 2.98 cm (square root scale), i.e., almost 60% of soil depth variation explained.  The resulting soil depth map clearly exhibited regional patterns as well as local details.  Relatively deep soils occurred in low lying landscape positions such as valley bottoms and plains while shallow soils occurred in high and steep landscape positions such as hillslopes, ridges and terraces.  The oases had much deeper soils than outside semi-desert areas, the middle of an alluvial plain had deeper soils than its margins, and the middle of a lacustrine plain had shallower soils than its margins.  Large predictive uncertainty mainly occurred in areas with a lack of soil survey points.  Both pedogenic and geomorphic processes contributed to the shaping of soil depth pattern of this basin but the latter was dominant.  This findings may be applicable to other similar basins in cold and arid regions around the world.

Auxin response factors (ARFs) play key roles throughout the whole process of plant growth and development, and mediate auxin response gene transcription by directly binding with auxin response elements (AuxREs).  However, their functions in abiotic stresses are largely limited, especially in apples.  Here, the auxin response factor gene MdARF2 (HF41569) was cloned from apple cultivar ‘Royal Gala’ (Malus×domestica Borkh.).  Phylogenetic analysis showed that ARF2 proteins are highly conserved among different species and MdARF2 is the closest relative to PpARF2 of Prunus persica, but they differ at the DNA level.  MdARF2 contains three typical conserved domains including the B3 DNA-binding domain, Auxin_resp domain and AUX_IAA domain.  The subcellular localization demonstrated that MdARF2 is localized in the nucleus.  The three-dimensional structure prediction of the proteins showed that MdARF2 is highly similar with AtARF2, and they contain helices, folds, and random coils.  The promoter of MdARF2 contains cis-acting elements which respond to various stresses, as well as environmental and hormonal signals.  Expression analysis showed that MdARF2 is widely expressed in all tissues of apple, with the highest expression of MdARF2 in root.  Functional analysis with a series of MdARF2 transgenic apple calli indicated that MdARF2 can reduce the sensitivity to ABA signaling and enhance salt tolerance in apple.  In summary, the results of this research provide a new basis for studying the regulation of abiotic stresses by ARFs.

Monoclonal antibodies (mAbs) are widely used in virus research and disease diagnosis.  The nucleoprotein (NP) of influenza A virus (IAV) plays important roles in multiple stages of the virus life cycle.  Therefore, generating conserved mAbs against NP and characterizing their properties will provide useful tools for IAV research.  In this study, two mAbs against the NP protein, 10E9 and 3F3, were generated with recombinant truncated NP proteins (NP-1 and NP-2) as immunogens.  The heavy-chain subclass of both 10E9 and 3F3 was determined to be IgG2α, and the light-chain type was κ.  Truncation and site-specific mutation analyses showed that the epitopes of mAbs 10E9 and 3F3 were located in the N terminal 84–89 amino acids and the C terminal 320–324 amino acids of the NP protein, respectively.  We found that mAbs 10E9 and 3F3 reacted well with the NP protein of H1–H15 subtypes of IAV.  Both 10E9 and 3F3 can be used in immunoprecipitation assay, and 10E9 was also successfully applied in confocal microscopy.  Furthermore, we found that the 10E9-recognized ₈₄SAGKDP₈₉ epitope and 3F3-recognized ₃₂₀ENPAH₃₂₄ epitope were highly conserved in NP among all avian and human IAVs.  Thus, the two mAbs we developed could be used as powerful tools in the development of diagnostic methods of IAV, and also surely promote the basic research in understanding the replication mechanisms of IAV.

Trichomes are specialized structures developed from epidermal cells and can protect plants against biotic and abiotic stresses.  Trichomes cover carrots during the generative phase.  However, the morphology of the carrot trichomes and candidate genes controlling the formation of trichomes are still unclear.  This study found that carrot trichomes were non-glandular and unbranched hairs distributed on the stem, leaf, petiole, pedicel, and seed of carrot.  Resequencing analysis of a trichome mutant with sparse and short trichomes (sst) and a wild type (wt) with long and dense trichomes on carrot stems was conducted.  A total of 15 396 genes containing nonsynonymous mutations in sst were obtained, including 42 trichome-related genes.  We also analyzed the transcriptome of the trichomes on secondary branches when these secondary branches were 10 cm long between wt and sst and obtained 6 576 differentially expressed genes (DEGs), including 24 trichome-related genes.  qRT-PCR validation exhibited three significantly up-regulated DEGs, 20 significantly down-regulated, and one with no difference.  We considered both the resequencing and transcriptome sequencing analyses and found that 12 trichome-related genes that were grouped into five transcription factor families containing nonsynonymous mutations and significantly down-regulated in sst.  Therefore, these genes are potentially promising candidate genes whose nonsynonymous mutations and down-regulation may result in scarce and short trichomes mutation on carrot stems in sst.

Rabbit hepatitis E virus (HEV) has been reported for years and is thought to have the potential for zoonotic transmission from rabbits to humans.  As reported, HEV genotype 3 (gt3) is the most prevalent form of HEV in rabbits.  To determine the prevalence of HEV in commercial rabbit livers, 176 liver samples were collected from an abattoir in Hebei Province, China.  Three (1.7%) samples tested positive for RNA of HEV-ORF2 (open reading frames-2).  Sequence analysis showed that the three isolates shared high identities with each other (94.08–98.85%).  Further analysis showed that all the rabbit strains clustered together in the branch of HEV gt3.  Further study by immunohistochemistry (IHC) assays showed that 131 (74.4%) liver samples were positive for HEV ORF2 protein.  Pathological changes including cell degeneration, inflammatory cell infiltration and bile duct epithelial cell hyperplasia were observed under microscopy.  These findings indicated the presence of HEV in commercial livers of rabbits.  Additional studies should be conducted to investigate the infectivity of rabbit HEV (rHEV) and the potential risks of zoonotic transmission of rHEV from rabbits to human beings.

Bleeding canker, a devastating disease of pear trees (Pyrus pyrifolia L.), was first reported in the 1970s in Jiangsu, China and more recently in other provinces in China.  Trees infected with bleeding canker pathogen, Dickeya fangzhongdai, develop cankers on the trunks and branches, and a rust-colored mixture of bacterial ooze and tree sap could be seen all over the trunks and branches.  In this study, we provided detail descriptions of the symptoms and epidemiology of bleeding canker disease.  Based on pathogenic and phenotypic characterizations, we identified the causal agent of bleeding canker of pear as D. fangzhongdai.  Dickeya fangzhongdai strains isolated from pear were also pathogenic on Solanum tuberosum, Brassica pekinensis, Lycopersicon esculentum, and Phalaenopsis aphrodite based on artificial inoculation, and the pathogen were more virulent on potato than that of D. solani strain.  This study provides new information about this disease and bleeding canker disease of pear.

This study aimed to compare the efficiencies of clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9-mediated gene knock-ins with zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) in bovine and dairy goat fetal fibroblasts.  To test the knock-in efficiency, a set of ZFNs and CRISPR/Cas9 plasmids were designed to edit the bovine myostatin (MSTN) gene at exon 2, while a set of TALENs and CRISPR/Cas9 plasmids were designed for editing the dairy goat β-casein gene at exon 2.  Donor plasmids utilizing the ZFNs, TALENs, and CRISPR/Cas9 cutting sites were constructed in the GFP-PGK-NeoR plasmid background, including a 5´ and 3´ homologous arm flanking the genes humanized Fat-1 (hFat-1) or enhanced green fluorescent protein (eGFP).  Subsequently, the ZFNs, TALENs, or CRISPR/Cas9 and the hFat-1 or eGFP plasmids were co-transfected by electroporation into bovine and dairy goat fetal fibroblasts.  After G418 (Geneticin) selection, single cells were obtained by mouth pipetting, flow cytometry or a cell shove.  The gene knock-in events were screened by PCR across the homologous arms.  The results showed that in bovine fetal fibrobalsts, the efficiencies of ZFNs-mediated eGFP and hFat-1 gene knock-ins were 13.68 and 0%, respectively.  The efficiencies of CRISPR/Cas9-mediated eGFP and hFat-1 gene knock-ins were 77.02 and 79.01%, respectively.  The eGFP gene knock-in efficiency using CRISPR/Cas9 was about 5.6 times higher than when using the ZFNs gene editing system.  Additionally, the hFat-1 gene knock-in was only obtained when using the CRISPR/Cas9 system.  The difference of knock-in efficiencies between the ZFNs and CRISPR/Cas9 systems were extremely significant (P<0.01).  In the dairy goat fetal fibroblasts, the efficiencies of TALENs-mediated eGFP and hFat-1 gene knock-ins were 32.35 and 26.47%, respectively.  The efficiencies of eGFP and hFat-1 gene knock-ins using CRISPR/Cas9 were 70.37 and 74.29%, respectively.  The knock-in efficiencies difference between the TALENs and CRISPR/Cas9 systems were extremely significant (P<0.01).  This study demonstrated that CRISPR/Cas9 was more efficient at gene knock-ins in domesticated animal cells than ZFNs and TALENs.  The CRISPR/Cas9 technology offers a new era of precise gene editing in domesticated animal cell lines. 

The effects of antibacterial compounds produced by Saccharomyces cerevisiae in Koumiss on pathogenic Escherichia coli O₈ and its cell surface characteristics were investigated.  S. cerevisiae isolated from Koumiss produced antibacterial compounds which were active against pathogenic E. coli O₈ as determined by the Oxford cup method.  The aqueous phases from S. cerevisiae at pH=2.0 (S2) and pH=8.0 (S8) were extracted and tested, respectively.  The organic acids of S2 and S8 were determined by high performance liquid chromatography (HPLC), and the concentrations of killer toxins were determined by enhanced bicinchoninic acid (BCA) Protein Assay Kit.  The minimum inhibition concentration (MIC) and the minimum bactericidal concentration (MBC) of S2 and S8 on E. coli O₈ were determined by the broth microdilution method.  The effects of S2 and S8 on the growth curve of E. coli O₈ were determined by turbidimetry, and the hydrophobicities of E. coli O₈ cell surface were determined using the microbial adhesion to solvents method, the permeation of E. coli O₈ cell membrane were determined by the o-nitrophenyl-β-D-galactoside (ONPG) method.  Aqueous phases at pH 2.0 and 8.0 had larger inhibition zones and then S2 and S8 were obtained by freeze-drying.  The main component in S2 was citric acid and it was propanoic acid in S8.  Other organic acids and killer toxins were also present.  Both the MICs of S2 and S8 on E. coli O₈ were 0.025 g mL^–1, the MBCs were 0.100 and 0.200 g mL^–1, respectively.  The normal growth curve of E. coli O₈ was S-shaped, however, it changed after addition of S2 and S8.  E. coli O₈ was the basic character, and had a relatively hydrophilic surface.  The hydrophobicity of E. coli O₈ cell surface and the permeation of E. coli O₈ cell membrane were increased after adding S2 and S8.  The present study showed that S2 and S8 inhibit the growth of pathogenic E. coli O₈ and influence its cell surface characteristics.

Adverse selection is an operating risk of crop insurance.  Based on survey data on crop insurance collected by questionnaires in Inner Mongolia, China, the paper uses non-parametric analysis and econometric models to estimate the relationship between conditions for crop production and farmers’ insurance decision in order to test the existence of farmers’ adverse selection.  The results show farmers’ adverse selection does exist, but settling a claim by negotiation and premium subsidy from governments at all levels can defuse farmers’ adverse selection under the current system of crop insurance.  Risk regionalization, heterogeneous insurance contract and product innovation may decrease adverse selection to some extent.

Infection by hepatitis B virus (HBV) results in acute and chronic liver damages in humans. Liver products of broilers as a primary food consumed in our daily life have a close connection with public health. The prevalence of the virus in livers and serum of broilers is of great significance, owning to the potential transmission between chickens and humans. Liver tissues and serum samples were tested to investigate the prevalence of hepatitis B virus infection in slaughtered broilers, for expression of HBV antigens and antibodies. The distribution and positive rate of hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg) and hepatitis B e antigen (HBeAg) in liver samples were examined using immunohistochemistry. HBsAg was mainly located in the cytoplasm of hepatocytes with a positivity of 81.61% whereas HBeAg and HBcAg were primarily located in the nucleus of hepatocytes with a positivity of 40.13 and 49.10%, respectively. Enzyme-linked immunosorbent assay (ELISA) analysis of serum for HBV serological markers demonstrated a high prevalence of hepatiits B surface antibody (HBsAb, 54.91%) and hepatitis B core antibody (HBcAb, 27.68%), whereas HBeAb, HBsAg and HBeAg were rarely detectable. Classic hepatitis pathological changes, including swollen hepatocytes, focal parenchymal necrosis, lymphocytic infiltration and hyperplasia of fibrous connective tissues were observed using histopathological analysis. Some of the liver samples were found positive for HBV DNA using nested PCR. Sequence comparison confirmed that all sequences shared 97.5–99.3% identity with human HBV strains. These results demonstrated the existence of HBV in livers and serums of broilers. Animals or animal products contaminated with HBV could raise an important public health concern over food safety and zoonotic risk.

In this study, 14 wheat cultivars with contrasting yield and N use efficiency (NUE) were used to investigate the agronomic and NUE-related traits, and the N assimilation-associated enzyme activities under low and high N conditions. Under deficient-N, the cultivars with high N uptake efficiency (UpE) and high N utilization efficiency (UtE) exhibited higher plant biomass, yields, and N contents than those with medium and low NUEs. The high UpE cultivars accumulated more N than other NUE type cultivars. Under sufficient-N, the tested cultivars showed similar patterns in biomass, yield, and N content to those under deficient-N, but the varietal variations in above traits were smaller. In addition, the high UpE cultivars displayed much more of root biomass and larger of root length, surface area, and volume than other NUE type cultivars, indicating that the root morphological traits under N deprivation are closely associated with the plant biomass through its improvement of the N acquisition. The high UtE cultivars showed higher activities of nitrate reductase (NR), nitrite reductase (NIR), and glutamine synthetase (GS) at stages of seediling, heading and filling than other NUE type cultivars under both low and high N conditions. Moreover, the high UpE and UtE cultivars also displayed higher photosynthetic rate under deficient-N than the medium and low NUE cultivars. Together, our results indicated that the tested wheat cultivars possess dramatically genetic variations in biomass, yield, and NUE. The root morphological traits and the N assimilation enzymatic acitivities play critical roles in regulating N accumulation and internal N translocation under the N-starvation stress, respectively. They can be used as morphological and biochemical references for evaluation of UpE and UtE in wheat.

Mycotoxin contamination in agro-food systems has been a serious concern globally during the last few decades. Mycotoxins are toxic secondary metabolites produced by fungi when they grow in agro-food products and feedstuff. Several detection techniques have been developed in recent years to detect mycotoxins in the food and feed effectively. HPLC based techniques are very common in usage in the laboratories for the testing of mycotoxins. In recent years, immuno-based assays is widely used and have been reported at large due to its sensitivity and limited detection time. Immuno assay-based kits were developed effectively to be used in the fields and in storage systems to detect the mycotoxin levels. Microarray-based immunoassays developed in the recent years could simultaneously detect aflatoxin, ochratoxin, and zearalenone with the higher sensitivity. Aptamer-based assays could target the detection of ochratoxin and aflatoxins and fumonisins at high specificity in food products. In recent years, several assays reported for the simultaneous multiple detection of different mycotoxin was based on HPLC and LC-MS/MS. There is a need for the use of these advanced technologies in the commercial scale.

The gene expression patterns of antioxidative enzymes in cucumber (Cucumis sativus L.) fruits at four different maturity stages, immature (3-8 d after anthesis (DAA), mature (9-16 DAA), breaker (17-22 DAA), and yellow (35-40 DAA), were determined before and after cold storage at 2°C for 9 d and after subsequent rewarming at 20°C for 2 d. The electrolyte leakage and malondialdehyde content in cucumber fruits were increased after cold storage and subsequent rewarming. Increased expressions of peroxidase, ascorbate peroxidase (APX), and monodehydroascorbate reductase after cold storage played an important role in cucumber fruits to cope with chilling injury. The elevated cyt-superoxide dismutase, catalase, APX and dehydroascorbate reductase after subsequent rewarming in cucumber fruits facilitated the recovery from chilling stress. The highest expression levels of all the seven antioxidative enzyme genes in yellow fruits might be responsible for the enhanced chilling tolerance. Cucumber fruits at earlier developmental stages was more susceptible to chilling stress than those at later stages. The relative higher gene expressions of antioxidative enzymes genes at earlier developmental stages may be the responses to the sever oxidative stress caused by chilling injury.

14-3-3 proteins belong to a family of phosphoserine/threonine-binding modules and participate in a wide array of signal transduction and regulatory events. Our previous study demonstrated that Ta14-3-3 was significantly down-regulated in leaf and root tissues of hybrid wheat at the tillering stage. In this paper, three homoeologous Ta14-3-3 genes were cloned from common wheat (Triticum aestivum L., 2n=6x=42, AABBDD) and mapped on chromosomes 2A, 2B, and 2D, respectively. Transgenic Arabidopsis plants ectopically overexpressing Ta14-3-3 displayed shorter primary roots, delayed flowering and retarded growth rates, indicating that Ta14-3-3 acted as a growth inhibitor in Arabidopsis. In wheat, Ta14-3-3 was down-regulated in roots and leaves of hybrids as compared to their parental lines. We proposed that Ta14-3-3 proteins might regulate growth vigor in hybrid wheat.

Classical swine fever caused by Classical swine fever virus (CSFV) is a serious problem for swine industries in developing countries, which successful control of the disease have been relying on vaccination. However, classical swine fever still occurs in some immunized swine herds for various reasons. In this study, we conducted animal experiments to examine the influence of single or mixed infection with Porcine parvo virus (PPV), Pseudorabies virus (PRV) and Porcine reproductive and respiratory syndrome virus (PRRSV) on the protective immunity induced by the Lapinized hog cholera virus (HCLV) vaccine and the pathogenicity of CSFV. In experiment 1, pigs were first inoculated with PPV, PRV or PRRSV, then immunized with HCLV, and finally challenged with a highly virulent CSFV Shimen strain. All of the pigs immunized with HCLV survived after the challenge, while all of the pigs in the non-immunized control group died after the challenge. The pigs in the group immunized with HCLV did not show any clinical symptoms of classical swine fever and were negative with CSFV after the challenge. The pigs infected with the non-CSFV before HCLV immunization did not display any clinical symptoms after the challenge with CSFV Shiman strain, but 11 of the 12 pigs were positive with CSFV. In experiment 2, pre-infections with PPV, PRV, and PRRSV were followed by inoculation with a low-virulence CSFV strain (CSFV 39), and then the pigs were challenged with the CSFV Shimen strain. Infections by either PPV, PRV or PRRSV did not enhance the virulence of CSFV-39, but pigs infected by a mixture of the 3 viruses developed clinical symptoms after inoculation with CSFV-39. The mixed infection also increased mortality caused by the challenge with the CSFV Shimen strain. Together, these results showed PPV, PRV and PRRSV infections in pigs can reduce the efficacy of the HCLV vaccine and enhance the pathogenicity of CSFV, which may partly explain the immunization failure against CSFV in some swine herds.

To make agricultural systems sustainable in terms of their greenness and efficiency, it is essential to optimize the tillage and fertilization practices.  To assess the effect of tilling and fertilization practices in the wheat-maize cropping systems, we carried out a three-year field experiment designed to quantify the carbon footprint (CF), and energy efficiency of the cropping systems in the North China Plain.  As the study parameters, we used four tillage practices (no tillage (NT), conventional tillage (CT), rotary tillage (RT) and subsoiling rotary tillage (SRT)), and two fertilizer regimes (inorganic fertilizer (IF), and hybrid fertilizer with organic and inorganic components (HF)).  Our results indicated that the most prominent energy inputs and greenhouse gas (GHG) emissions could be ascribed to the use of fertilizers and fuel consumption.  Assuming the same fertilization regime, ranking the tillage patterns with respect to the value of the crop yield, the profit, the CF, the energy use efficiency (EUE) or the energy productivity (EP) for either wheat or maize always gave the following result: SRT>RT>CT>NT.  For the same tillage, the energy consumption associated with HF was higher than IF, but its GHG emissions and CF were lower while the yield and profit were better.  In terms of the overall performance, tilling is more beneficial than NT, and reduced tillage (RT and SRT) are more beneficial than CT.  The fertilization regime with the best overall performance was HF.  Combining SRT with HF has significant potential for reducing CF and increasing EUE, improving the sustainability.  Adopting measures promoting these optimizations can help overcome the challenges posed by lack of food security, energy crises and ecological stress.