Toxoplasma gondii is an important zoonotic parasite which has over 200 genotypes worldwide.  N⁶-methyladenosine (m⁶A) methylation is a common epigenetic modification in messenger RNAs (mRNAs), and has been implicated in many aspects of mRNA biology.  However, little is known about the difference in m⁶A methylation among different genotypes of T. gondii.  In the present study, we employed methylated RNA immunoprecipitation sequencing (MeRIP-seq) technology to identify key genes exhibiting m⁶A methylation in the three major clonal lineages (Types I, II and III) of T. gondii tachyzoites.  A total of 7,650, 8,359 and 7,264 m⁶A peaks were identified in 5,211, 5,607 and 4,974 genes in tachyzoites of RH (Type I), ME49 (Type II) and VEG strain (Type III), respectively.  By comparing RH vs. ME49, RH vs. VEG, and ME49 vs. VEG, 735, 192 and 615 differentially methylated peaks (DMPs) were identified in 676, 168 and 553 genes, respectively.  A combined MeRIP-seq and RNA-seq analysis revealed 172, 41 and 153 differentially methylated genes (DMGs) at both the m⁶A methylation and transcriptional level.  Gene Ontology term enrichment analysis of the DMPs identified differences related to Golgi apparatus, plasma membrane, signal transduction, RNA processing and catalytic step 2 spliceosome.  KEGG pathway enrichment analysis showed that the DMGs are mainly involved in endocytosis, systemic lupus erythematosus and mTOR signaling pathway.  These findings reveal genotype-specific differences in m⁶A methylation, which provide new resources for further investigations of the role of m⁶A in the pathobiology of T. gondii.

Cold stress is an important factor that limits apple production.  In this study, we examined the tissue-cultured plantlets of apple rootstocks ‘M9T337’ and ‘60-160’, which are resistant and sensitive to cold stress, respectively.  The enriched pathways of differentially expressed genes (DEGs) and physiological changes in ‘M9T337’ and ‘60-160’ plantlets were clearly different after cold stress (1°C) treatment for 48 h, suggesting that they have differential responses to cold stress.  The differential expression of WRKY transcription factors in the two plantlets showed that MdWRKY40is and MdWRKY48 are potential regulators of cold tolerance.  When we overexpressed MdWRKY40is and MdWRKY48 in apple calli, the overexpression of MdWRKY48 had no significant effect on the callus, while MdWRKY40is overexpression promoted anthocyanin accumulation, increased callus cold tolerance, and promoted the expression of anthocyanin structural gene MdDFR and cold-signaling core gene MdCBF2.  Yeast one-hybrid screening and electrophoretic mobility shift assays showed that MdWRKY40is could only bind to the MdDFR promoter.  Yeast two-hybrid screening and bimolecular fluorescence complementation showed that MdWRKY40is interacts with the CBF2 inhibitor MdMYB15L through the leucine zipper (LZ).  When the LZ of MdWRMY40is was knocked out, MdWRKY40is overexpression in the callus did not affect MdCBF2 expression or callus cold tolerance, indicating that MdWRKY40is acts in the cold signaling pathway by interacting with MdMYB15L.  In summary, MdWRKY40is can directly bind to the MdDFR promoter in order to promote anthocyanin accumulation, and it can also interact with MdMYB15L to interfere with its inhibitory effect on MdCBF2, indirectly promoting MdCBF2 expression, and thereby improving cold tolerance.  These results provide a new perspective for the cold-resistance mechanism of apple rootstocks and a molecular basis for the screening of cold-resistant rootstocks.

Organic acids are one of the most important factors influencing fruit flavors. The predominant organic acid in most pear cultivars is malic acid, but the mechanism controlling its accumulation remains unclear. In this study, by comparing gene expression levels and organic acid content, we revealed that the expression of PbPH5, which encodes a P_3A-ATPase, is highly correlated with malic acid accumulation in different pear species, with correlation coefficients of 0.932^**, 0.656^*, 0.900^**, and 0.518^* (^*, P<0.05 or ^**, P<0.01) in Pyrus bretschneideri Rehd., P. communis Linn., P. pyrifolia Nakai., and P. ussuriensis Maxim., respectively. Moreover, the overexpression of PbPH5 in pear significantly increased the malic acid content. In contrast, silencing PbPH5 via RNA interference significantly decreased its transcript level and the pear fruit malic acid content. A subcellular localization analysis indicated that PbPH5 is located in the tonoplast. Additionally, a phylogenetic analysis proved that PbPH5 is a PH5 homolog gene that is clustered with Petunia hybrida, Malus domestica, and Citrus reticulata genes. Considered together, these findings suggest PbPH5 is a functionally conserved gene. Furthermore, the accumulation of malic acid in pear fruits is at least partly related to the changes in PbPH5 transcription levels.

Cell death is an important physiological phenomenon in life.  It can be programmed or unprogrammed.  Unprogrammed cell death is usually induced by abiotic or biotic stress.  Recent studies have shown that many proteins regulate both cell death and immunity in plants.  Here, we provide a review on the advances in plant immunity with cell death, especially the molecular regulation and underlying mechanisms of those proteins involved in both cell death and plant immunity.  In addition, we discuss potential approaches toward improving plant immunity without compromising plant growth.

Soil salinity and alkalinity can inhibit crop growth and reduce yield, and this has become a global environmental concern. Combined changes in nitrogen (N) application and hill density can improve rice yields in sodic saline–alkaline paddy fields and protect the environment. We investigated the interactive effects of N application rate and hill density on rice yield and N accumulation, translocation and utilization in two field experiments during 2018 and 2019 in sodic saline–alkaline paddy fields. Five N application rates (0 (control), 90, 120, 150, and 180 kg N ha⁻¹ (N0–N4), respectively) and three hill densities (achieved by altering the distance between hills, in rows spaced 30 cm apart: 16.5 cm (D1), 13.3 cm (D2) and 10 cm (D3)) were utilized in a split-plot design with three replicates. Nitrogen application rate and hill density significantly affected grain yield. The mathematical model of quadratic saturated D-optimal design showed that with an N application rate in the range of 0–180 kg N ha⁻¹, the highest yield was obtained at 142.61 kg N ha⁻¹ which matched with a planting density of 33.3×10⁴ ha⁻¹. Higher grain yield was mainly attributed to the increase in panicles m^–2. Nitrogen application rate and hill density significantly affected N accumulation in the aboveground parts of rice plants and showed a highly significant positive correlation with grain yield at maturity. From full heading to maturity, the average N loss rate of the aboveground parts of rice plants in N4 was 70.21% higher than that of N3. This is one of the reasons why the yield of N4 treatment is lower than that of the N3 treatment. Nitrogen accumulation rates in the aboveground parts under treatment N3 (150 kg N ha⁻¹) were 81.68 and 106.07% higher in 2018 and 2019, respectively, than those in the control. The N translocation and N translocation contribution rates increased with the increase in the N application rate and hill density, whereas N productivity of dry matter and grain first increased and then decreased with the increase in N application rate and hill density. Agronomic N-use efficiency decreased with an increase in N application rate, whereas hill density did not significantly affect it. Nitrogen productivity of dry matter and grain, and agronomic N-use efficiency, were negatively correlated with grain yield. Thus, rice yield in sodic saline–alkaline paddy fields can be improved by combined changes in the N application rate and hill density to promote aboveground N accumulation. Our study provides novel evidence regarding optimal N application rates and hill densities for sodic saline–alkaline rice paddies.

It is well known that application of 5-aminolevulinic acid (ALA) could promote the plant growth under abiotic stress in oilseed rape (Brassica napus L.).  However, the specifics of its physiological and ultrastructural regulation under herbicide stress conditions are poorly understood.  In the present study, alleviating role of ALA in B. napus was investigated under four levels of herbicide propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino) benzoate (ZJ0273) (0, 100, 200 and
500 mg L^–1) with or without 1 mg L^–1 ALA treated for 48 or 72 h.  Results showed that after 48 h of herbicide stress, the growth of rape seedlings was significantly inhibited with the successive increases of the ZJ0273 concentrations from 0 to 500 mg L^–1, but this inhibition was obviously alleviated by exogenous application of ALA.  However, when treatment time prolonged to 72 h, the recovery effects of ALA could not be evaluated due to the death of plants treated with the highest concentration of ZJ0273 (500 mg L^-1).  Further, the root oxidizability and activities of antioxidant enzymes (superoxide dismutase, peroxidase and ascorbate peroxidase) were dramatically enhanced by the application of 1 mg L^–1 ALA under herbicide stress.  Therefore, plants treated with ALA dynamically modulated their antioxidant defenses to reduce reactive oxygen species (ROS) accumulation and malondialdehyde (MDA) content induced by herbicide stress.  Additionally, exogenously applied ALA improved the ultrastructure’s of chloroplast, mitochondria and nucleus, and induced the production of stress proteins.  Our results suggest that ALA could be considered as a potential plant growth regulator for the improvement of herbicide tolerance through alleviation of the physiological and ultrastructural changes induced by the herbicide in crop production.

There are 41 members of the CCT (CO, CO-like, and TOC1) domain-containing gene family in rice, which are divided into three subfamilies: COL (CONSTANS-like), CMF (CCT motif family), and PRR (pseudoresponse regulator).  The first flowering gene to be isolated by map-based cloning, Heading date 1 (Hd1), which is the orthologue of CO in rice, belongs to COL.  The central regulator of plant development, Ghd7, belongs to CMF.  The major role in controlling rice distribution to high latitudes, Ghd7.1/PRR37, belongs to PRR.  Both of Hd1, Ghd7 and Ghd7.1 simultaneously control grain number, plant height, and the heading date.  To date, 13 CCT family genes from these three subfamilies have been shown to regulate flowering.  Some of them have pleiotropic effects on grain yield, plant height, and abiotic stresses, and others function as circadian oscillators.  There are two independent photoperiod flowering pathways that are mediated by GI-Hd1-Hd3a/RFT and GI-Ehd1-Hd3a/RFT in rice.  CCT family genes are involved in both pathways.  The latest study reveals that protein interaction between Hd1 and Ghd7 integrates the two pathways.  CCT family genes are rich in natural variation because rice cultivars have been subjected to natural and artificial selection for different day lengths in the process of domestication and improvement.  Alleles of several crucial CCT family genes such as Hd1, Ghd7, and Ghd7.1 exhibit geographic distribution patterns and are highly associated with yield potentials.  In addition, CCT family genes are probably involved in the responses to abiotic stress, which should be emphasized in future work.  In general, CCT family genes play important roles in regulating flowering, plant growth, and grain yield.  The functional identification and elucidation of the molecular mechanisms of CCT family genes would help construct a flowering regulatory network and maximize their contribution to rice production.

Traditionally, Chinese indigenous cattle is geographically widespread.  The present study analyzed based on genome-wide variants to evaluate the genetic background among 157 individuals from four representative indigenous cattle breeds of Hubei Province of China: Yiling yellow cattle (YL), Bashan cattle (BS), Wuling cattle (WL), Zaobei cattle (ZB), and 21 individuals of Qinchuan cattle (QC) from the nearby Shanxi Province of China.  Linkage disequilibrium (LD) analysis showed the LD of YL was the lowest (r2=0.32) when the distance between markers was approximately 2 kb.  Principle component analysis (PCA), and neighbor-joining (NJ)-tree revealed a separation of Yiling yellow cattle from other geographic nearby local cattle breeds.  In PCA plot, the YL and QC groups were segregated as expected; moreover, YL individuals clustered  together more obviously.  In the NJ-tree, the YL group formed an independent branch and BS, WL, ZB groups were mixed.  We then used the FST statistic approach to reveal long-term selection sweep of YL and other 4 cattle breeds.  According to the selective sweep, we identified the unique pathways of YL, associated with production traits.  Based on the results, it can be proposed that YL has its unique genetic characteristics of excellence resource, and it is an indispensable cattle breed in China.   

Genomic selection has been demonstrated as a powerful technology to revolutionize animal breeding.  However, marker density and minor allele frequency can affect the predictive ability of genomic estimated breeding values (GEBVs).  To investigate the impact of marker density and minor allele frequency on predictive ability, we estimated GEBVs by constructing the different subsets of single nucleotide polymorphisms (SNPs) based on varying markers densities and minor allele frequency (MAF) for average daily gain (ADG), live weight (LW) and carcass weight (CW) in 1 059 Chinese Simmental beef cattle.  Two strategies were proposed for SNP selection to construct different marker densities: 1) select evenly-spaced SNPs (Strategy 1), and 2) select SNPs with large effects estimated from BayesB (Strategy 2).  Furthermore, predictive ability was assessed in terms of the correlation between predicted genomic values and corrected phenotypes from 10-fold cross-validation.  Predictive ability for ADG, LW and CW using autosomal SNPs were 0.13±0.002, 0.21±0.003 and 0.25±0.003, respectively.  In our study, the predictive ability increased dramatically as more SNPs were included in analysis until 200K for Strategy 1.  Under Strategy 2, we found the predictive ability slightly increased when marker densities increased from 5K to 20K, which indicated the predictive ability of 20K (3% of 770K) SNPs with large effects was equal to the predictive ability of using all SNPs.  For different MAF bins, we obtained the highest predictive ability for three traits with MAF bin 0.01–0.1.  Our result suggested that designing a low-density chip by selecting low frequency markers with large SNP effects sizes should be helpful for commercial application in Chinese Simmental cattle.

Both bolting and flowering times influence taproot and seed production in radish. FLOWERING LOCUS C (FLC) plays a key role in plant flowering by functioning as a repressor. Two genomic DNA sequences, a 3 046-bp from an early- and a 2 959-bp from a late-bolting radish line were isolated and named as RsFLC1 and RsFLC2, respectively, for they share approximately 87.03% sequence identity to the FLC cDNA sequences. The genomic DNA sequences, 1 466-bp and 1 744-bp, flanking the 5´-regions of RsFLC1 and RsFLC2, respectively, were characterized. Since both of them harbor the basic promoter elements, the TATA box and CAAT box, they were designated as PRsFLC1 and PRsFLC2. The transcription start site (TSS) was identified at 424 and 336 bp upstream of the start codon in PRsFLC1 and PRsFLC2, respectively. cis-regulatory elements including CGTCA (MeJA-responsive) and ABRE (abscisic acid-responsive) motifs were found in both promoters, while some cis-regulatory elements including TCA element and GARE-motif were present only in PRsFLC1. These sequence differences lead to the diversity of promoter core elements, which could partially result in the difference of bolting and flowering time in radish line NauDY13 (early-bolting) and Naulu127 (late-bolting). Furthermore, to investigate the activity of these promoters, a series of 5´-deletion fragment-GUS fusions were constructed and transformed into tobacco. GUS activity was detected in PRsFLC1-(1 to 4)-GUS-PS1aG-3 and PRsFLC2-(1 to 4)-GUS-PS1aG-3 transgenic tobacco leaf discs, and this activity progressively decreased from PRsFLC-1-GUS-PS1aG-3 to PRsFLC-5-GUS-PS1aG-3. Deletion analysis indicated that the cis-regulatory elements located at –395 bp to +1 bp may be critical for specifying RsFLC gene transcription.

Organophosphate insecticide residues on vegetable, fruit, tea and even grains are primary cause of food poisoning. Organophosphate compounds can cause irreversible inhibition of the activity of acetylcholinesterase and butyrylcholinesterase (BChE, EC 3.1.1.8), which are both candidates for rapid detection of organophosphate pesticides. To develop an easy-tohandle method for detecting organophosphate pesticides using BChE, BChE from human was optimized according to the codon usage bias of Pichia pastoris and successfully expressed in P. pastoris GS115. The codon-optimized cDNA shared 37.3% of the codon identity with the native one. However, the amino acid sequence was identical to that of the native human butyrylcholinesterase gene (hBChE) as published. The ratio of guanine and cytosine in four kinds of bases ((G+C) ratio) was simultaneously increased from 40 to 47%. The recombinant hBChE expression reached a total protein concentration of 292 mg mL–1 with an activity of 14.7 U mL–1, which was purified 3.2×103-fold via nickel affinity chromatography with a yield of 68% and a specific activity of 8.1 U mg–1. Recombinant hBChE was optimally active at pH 7.4 and 50°C and exhibited high activity at a wide pH range (>60% activity at pH 4.0 to 8.0). Moreover, it had a good adaptability to high temperature (>60% activity at both 50 and 60°C up to 60 min) and good stability at 70°C. The enzyme can be activated by Li+, Co+, Zn2+ and ethylene diamine tetraacetic acid (EDTA), but inhibited by Mg2+, Mn2+, Fe2+, Ag+ and Ca2+. Na+ had little effect on its activity. The values of hBChE of the Michaelis constant (Km) and maximum reaction velocity (Vm) were 89.4 mmol L–1 and 1 721 mmol min–1 mg–1, respectively. The bimolecular rate constants (Ki) of the hBChE to four pesticides were similar with that of electric eel AChE (EeAChE) and higher than that of horse BChE (HoBChE). All values of the half maximal inhibitory concentration of a substance (IC50) for hBChE were lower than those for HoBChE, but most IC50 for hBChE were lower than those for EeAChE except dichlorvos. The applicability of the hBChE was further verified by successful detection of organophosphate insecticide residues in six kinds of vegetable samples. Thus, hBChE heterologously over-expressed by P. pastoris would provide a sufficient material for development of a rapid detection method of organophosphate on spot and produce the organophosphate detection kit.

    Quorum sensing (QS) is a type of microbe-microbe communication system that is widespread among the microbial world, particularly among microorganisms that are symbiotic with plants and animals. Thereby, the cell-cell signalling is likely to occur in an anaerobic rumen environment, which is a complex microbial ecosystem. In this study, using six ruminally fistulated Liuyang black goats as experimental animals, we aimed to detect the activity of quorum sensing autoinducers (AI) both in vivo and in vitro and to clone the luxS gene that encoded autoinducer-2 (AI-2) synthase of microbial samples that were collected from the rumen of goats. Neutral detergent fiber (NDF) and soluble starch were the two types of substrates that were used for in vitro fermentation. The fermented fluid samples were collected at 0, 2, 4, 6, 8, 12, 24, 36, and 48 h of incubation. The acyl-homoserine lactones (AHLs) activity was determined using gas chromatography-mass spectrometer (GC-MS) analysis. However, none of the rumen fluid extracts that were collected from the goat rumen showed the same or similar fragmentation pattern to AHLs standards. Meanwhile, the AI-2 activity, assayed using a Vibrio harveyi BB170 bioassay, was negative in all samples that were collected from the goat rumen and from in vitro fermentation fluids. Our results indicated that the activities of AHLs and AI-2 were not detected in the ruminal contents from six goats and in ruminal fluids obtained from in vitro fermentation at different sampling time-points. However, the homologues of luxS in Prevotella ruminicola were cloned from in vivo and in vitro ruminal fluids. We concluded that AHLs and AI-2 could not be detected in in vivo and in vitro ruminal fluids of goats using the current detection techniques under current dietary conditions. However, the microbes that inhabited the goat rumen had the potential ability to secrete AI-2 signaling molecules and to communicate with each other via AI-2-mediated QS because of the presence of luxS.

The objective of the present investigation was to estimate the prevalence of Toxoplasma gondii infection and co-infection with porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV) and porcine circovirus type 2 (PCV-2) in pigs in China. A total of 372 tissues or serum samples collected from pigs distributed in 9 provinces/ municipalities of China during the period from February 2011 to November 2012 were assayed for T. gondii antigens and antibodies using enzyme linked immunosorbent assay (ELISA) technique, while the PCR was designed for the detection of the PRRSV, CSFV and PCV-2, respectively. The total positive rate of T. gondii, PRSSV, CSFV and PCV-2 was 9.14% (34/372), 50.00% (186/372), 37.10% (138/372) and 3.23% (12/372), respectively. Among the 34 T. gondii positive samples, 26 samples were simultaneously infected with T. gondii and viruses, while the remaining eight samples were infected with T. gondii alone. In addition, the co-infection rate of T. gondii with PRSSV, T. gondii with PRSSV and CSFV, T. gondii with PRSSV and PCV-2, T. gondii with CSFV and PCV-2, T. gondii with PRSSV, CSFV and PCV-2 was 1.61% (6/372), 4.03% (15/372), 0.27% (1/372), 0.27% (1/372) and 0.81% (3/372), respectively. The results of the present survey revealed that PRRSV and CSFV were the common pathogens co-existing with porcine toxoplasmosis in China, and both of them could increase the chances of T. gondii infection in pig. This is the first report of T. gondii co-infections with viruses in pigs. It is very important to understand the interactions of parasite and virus, and can be used as reference data for the control and prevention of co-infections of T. gondii and viruses in pigs.

In this study, four strains of Toxoplasma gondii with the same genetic type (Type I) originated from chicken, human, cat and swine were used to compare the immune responses in resistant chicken host to investigate the relationships between the parasite origins and the pathogenicity in certain host. A total of 300, 10-day-old chickens were allocated randomly into five groups which named JS (from chicken), CAT (from cat), CN (from swine), RH (from human) and a negative control group (–Ve) with 60 birds in each group. Tachyzoites of four different T. gondii strains (JS, CAT, CN and RH) were inoculated intraperitoneally with the dose of 1×107 in the four designed groups, respectively. The negative control (–Ve) group was mockly inoculated with phosphate-buffered saline (PBS) alone. Blood and spleen samples were obtained on the day of inoculation (day 0) and at days 4, 11, 25, 39 and 53 post-infection to screen the immunopathological changes. The results demonstrated some different immune characters of T. gondii infected chickens with that of mice or swine previous reported. These differences included up-regulation of major histocompatibility complex class II (MHC II) molecules in the early stage of infection, early peak expressions of interleukin (IL)-12 (IL-12) and -10 (IL-10) and long keep of IL-17. These might partially contribute to the resistance of chicken to T. gondii infection. Comparisons to chickens infected with strains from human, cat and swine, chickens infected with strain from chicken showed significant high levels of CD4+ and CD8+ T cells, interferon gamma (IFN-γ), IL-12 and IL-10. It suggested that the strain from chicken had different ability to stimulate cellular immunity in chicken.

The bacterial brown spot disease (BBS), caused primarily by Pseudomonas syringae pv. syringae van Hall (Pss), reduces plant vigor, yield and quality in maize. To reveal the nature of the defense mechanisms and identify genes involved in the effective host resistance, the dynamic changes of defense transcriptome triggered by the infection of Pss were investigated and compared between two maize near-isogenic lines (NILs). We found that Pss infection resulted in a sophisticated transcriptional reprogramming of several biological processes and the resistant NIL employed much faster defense responses than the susceptible NIL. Numerous genes encoding essential components of plant basal resistance would be able to be activated in the susceptible NIL, such as PEN1, PEN2, PEN3, and EDR1, however, in a basic manner, such resistance might not be sufficient for suppressing Pss pathogenesis. In addition, the expressions of a large number of PTI-, ETI-, PR-, and WRKY-related genes were pronouncedly activated in the resistant NIL, suggesting that maize employ a multitude of defense pathways to defend Pss infection. Six R-gene homologs were identified to have significantly higher expression levels in the resistant NIL at early time point, indicating that a robust surveillance system (gene-to-gene model) might operate in maize during Pss attacks, and these homolog genes are likely to be potential candidate resistance genes involved in BBS disease resistance. Furthermore, a holistic group of novel pathogen-responsive genes were defined, providing the repertoire of candidate genes for further functional characterization and identification of their regulation patterns during pathogen infection.

Eight compounds were isolated from the fermentation cultures of rice sheath blight pathogen Rhizoctonia solani Kühn. They were identified as ergosterol (1), 6β-hydroxysitostenone (2), sitostenone (3), m-hydroxyphenylacetic acid (4), methyl m-hydroxyphenylacetate (5), m-hydroxymethylphenyl pentanoate (6), (Z)-3-methylpent-2-en-1,5-dioic acid (7) and 3-methoxyfuran-2-carboxylic acid (8) by means of physicochemical and spectroscopic analysis. Among them, 2, 3, 5–8 were isolated from R. solani for the first time. All the compounds were evaluated for their biological activities. 4–6 and 8 showed their inhibitory activities on the radical and germ elongation of rice seeds. 1, 4 and 7 showed moderate antibacterial activity to some bacteria. 4, 7 and 8 exhibited weak inhibitory activities on spore germination of Magnaporthe oryzae. 8 showed moderate antioxidant activity with the 1,1-diphenyl-2-picryhydrazyl (DPPH) and β-carotene-linoleic acid assays. This is the first time to reveal compounds 5, 6 and 8 from rice sheath blight pathogen R. solani to have in vitro phytotoxic activity.

Myrosinase is a defense-related enzyme and is capable of hydrolyzing glucosinolates into a variety of compounds, some of which are toxic to pathogens and herbivores. Many studies revealed that a number of important vegetables or oil crops contain the myrosinase-glucosinolate system. However, the related promoter and genomic DNA sequences as well as expression profiles of myrosinase gene remain largely unexplored in radish (Raphanus sativus). In this study, the 2 798 bp genomic DNA sequence, designated as RsMyr2, was isolated and analyzed in radish. The RsMyr2 consisting of 12 exons and 11 introns reflected the common gene structure of myrosinases. Using the genomic DNA walking approach, the 5´-flanking region upstream of RsMyr2 with length of 1 711 bp was successfully isolated. PLACE and PlantCARE analyses revealed that this upstream region could be the promoter of RsMyr2, which contained several basic cis-regulatory elements including TATA-box, CAAT-box and regulatory motifs responsive to defense and stresses. Furthermore, recombinant pET-RsMyr2 protein separated by SDS-PAGE was identified as myrosinase with mass spectrometry. Real-time PCR analysis showed differential expression profiles of RsMyr2 in leaf, stem and root at different developmental stages (e.g., higher expression in leaf at cotyledon stage and lower in flesh root at mature stage). Additionally, the RsMyr2 gene exhibited up-regulated expression when treated with abscisic acid (ABA), methyl jasmonate (MeJA) and hydrogen peroxide (H2O2), whereas it was down-regulated by wounding (WO) treatment. The findings indicated that the expression of RsMyr2 gene was differentially regulated by these stress treatments. These results could provide new insight into elucidating the molecular characterization and biological function of myrosinase in radish.

Downy mildew (DM), caused by the fungus Peronospora parasitica, is a destructive disease of radish (Raphanus sativus L.) worldwide. Host resistance has been considered as an attractive and environmentally friendly approach to control the disease. However, the genetic mechanisms of resistance in radish to the pathogen remain unknown. To determine the inheritance of resistance to DM, F1, F2 and BC1F1 populations derived from reciprocal crosses between a resistant line NAU-dhp08 and a susceptible line NAU-qtbjq-06 were evaluated for their responses to DM at seedling stage. All F1 hybrid plants showed high resistance to DM and maternal effect was not detected. The segregation for resistant to susceptible individuals statistically fitted a 3:1 ratio in two F2 populations (F2(SR) and F2(RS)), and 1:1 ratio in two BC1F1 populations, indicating that resistance to DM at seedling stage in radish was controlled by a single dominant locus designated as RsDmR. A total of 1 972 primer pairs (1036 SRAP, 628 RAPD, 126 RGA, 110 EST-SSR and 72 ISSR) were screened, and 36 were polymorphic between the resistant and susceptible bulks, and consequently used for genotyping individuals in the F2 population. Three markers (Em9/ga24370, NAUISSR826700 and Me7/em10400) linked to the RsDmR locus within a 10.0 cM distance were identified using bulked segregant analysis (BSA). The SRAP marker Em9/ga24370 was the most tightly linked one with a distance of 2.3 cM to RsDmR. These markers tightly linked to the RsDmR locus would facilitate marker-assisted selection and resistance gene pyramiding in radish breeding programs.

The soil organic matter and nutrients are fundamental for the sustainability of pear production, but little is known about the spatial distribution of soil organic matter and nutrients in a pear orchard. With the soil of the pear (cv. Dangshansu on P.betulifolia Bunge. rootstock) orchard under clean and sod cultivation models as test materials, the experiment was conducted to evaluate spatial variability of soil organic matter (SOM), total nitrogen (STN), total phosphorus (STP), total potassium (STK), available nitrogen (SAN), and available potassium (SAK) in and between rows at different soil depths (0-60 cm). The SOM, STN, STP, STK, SAN and SAK of the different soil layers under the two tillage models were different in the vertical direction. The SOM, STN, STP and SAN in the 0-20 cm soil layer were higher than those in the 20-40 and 40- 60 cm soil layers. The STK of 40-60 cm soil layer was higher than that in the 0-20 and 20-40 cm soil layers. The STK increased with the depth of soil in the vertical direction in the clean cultivated pear orchard. Variability of the SOM, STN, STP, STK, SAN and SAK of sample sites in between rows of the same soil layer was found in the pear orchard soil in the horizontal direction under clean and sod cultivation management systems, except that STK of all sites did not show the difference in identical soil layers in the pear orchard under clean cultivation. The sod cultivation model improved the SOM, STN, and STK in the 0-20 cm soil layer in the pear orchard, and the three components increased by 12.8, 12.7 and 7.3% compared to clean cultivation, respectively. The results can be applicable to plan collection of orchard soil samples, assess orchard soil quality, and improve orchard soil management practices.

One strain of Toxoplasma gondii was successfully isolated from chickens in China by bioassay in mice. Antibodies and circulating antigens of T. gondii were assayed by the ELISA kits in 100 free range chickens from a rural area surrounding Funing, China. Fifty-three chickens were antibody-positive and 21 chickens were antigen positive. Hearts, brains, spleens, lungs, livers, and kidneys of 21 antibody or antigen-positive chickens were bioassayed in mice. One strain of T. gondii was isolated from 1 of 21 (4.76%) chickens. The isolated T. gondii killed all of the inoculated mice. Genotyping of this isolate using polymorphisms at the loci 5´-SAG2, 3´-SAG2, SAG3, cB21-4, L358, BTUB, and GRA6 revealed that it was Type I. These indicated that it was virulent for mice. This is the first report of isolation of T. gondii from chickens in China.

Regrowth traits of alfalfa (Medicago sativa L.) in spring are closely related to its fall dormancy before winter. In order todetermine the relationship between fall dormancy (FD) grade and hormone variation pattern and provide academic referencesfor the variety improvement and production of alfalfa, the variations of gibberellins (GA3), indole-3-acetic acid (IAA), andabscisic acid (ABA) in alfalfa roots during regrowth period in spring were examined by high performance liquidchromatography (HPLC). The study involved seven alfalfa cultivars that belonged to four fall dormant grades, i.e., 2, 4, 6,and 8. The results showed that the differences in spring regrowth among the alfalfa cultivars were partially associatedwith their root hormone levels. The alfalfa cultivars that belonged to the same dormancy grades presented similarvariation trends in endogenous hormone content in their roots during the spring regrowth stage. At the early regrowthstage, cultivars with a higher dormant grade had a higher GA3 concent and a lower ABA content in their roots than thecultivars with a lower dormant grade; and IAA content in roots of non- and semi-fall dormancy cultivars was higher thanthat of fall dormancy cultivars. During the whole period of spring regrowth, the root ABA content of fall dormancy alfalfacultivar is significantly higher than those of semi- and non-fall dormancy cultivars. GA3 contents in the roots of allcultivars under study showed a double-peak dynamic curve; root IAA contents of the studied cultivars presented adownward trend. But the trend did not significantly differed among the different fall dormant cultivars. The higher GA3content and lower ABA content in root of non-fall dormancy alfalfa lead to its earlier regrowth. Regrowth time and rate ofalfalfa can be regulated by exogenous GA3 or ABA at the early regrowth stage to meet producing requirement.

The application of imidacloprid insecticides for the management of forage grass pests, such as Therioaphis trifolii, is a widely adopted practice; however, the transgenerational impacts of sublethal doses of imidacloprid on T. trifolii remain largely unexplored. This research initially employed toxicity assessments to identify sublethal concentrations of imidacloprid in T. trifolii. Following this, a series of experiments—including life table analysis, transcriptome sequencing, evaluations of feeding efficiency, and assessments of immune capacity—were conducted to elucidate the diverse effects on the F₂ generation of T. trifolii resulting from exposure to sublethal concentrations of imidacloprid in the F₀ and F₁ generations. Toxicity testing experiments revealed that exposure to imidacloprid at concentrations of 0.1 mg/L and 0.5 mg/L did not significantly impact the survival and reproductive rates of a single generation of T. trifolii. When both F₀ and F₁ generations T. trifolii were subjected to a concentration of 0.5 mg/L imidacloprid, a notable decline in the intrinsic growth rate and reproductive capacity of F₂ generation T. trifolii was observed in comparison to the control treatment group. Honeydew secretion during feeding decreased by 36.7% within a 72-hour period. Fatty acid content decreased by 30.5%. And there was a significant impairment of hemolymph immune response to exogenous bacterial and fungal challenges. This study represented the inaugural investigation into the transgenerational impacts of sublethal concentrations of imidacloprid on T. trifolii. The findings of our study indicate that sublethal concentrations of imidacloprid cross generational inhibit the reproductive capacity, feeding efficiency, and immune function of T. trifolii. This research offers important insights for enhancing the application strategies of imidacloprid within the framework of integrated pest management for forage crops.

The long-term overuse of insecticides has accelerated the evolutionary development of insect resistance. In this process, carboxylesterases as pivotal enzymes in detoxification metabolism, play a critical role in the formation of pest resistance, with their enhanced activity and altered expression levels being closely associated with the development of resistance mechanisms. In this study, the VmCarEs-6 gene was screened and cloned based on the transcriptomic data of Therioaphis trifolii under reverse stress conditions. The aim was to investigate the role of this gene in the sensitivity of T. trifolii to chemical pesticides through RNA interference and inhibitor treatments. Indoor bioassay results demonstrated that exposure to LC₅₀ concentrations of lambda-cyhalothrin (LCT), isoprocarb (IPC), phoxim (PHX), and imidacloprid (IMI) significantly upregulated the expression of the VmCarEs-6 gene in T. trifolii. Following RNAi-mediated silencing of VmCarEs-6 using star polycation (SPc)-encapsulated double-stranded RNA, the mortality rates of aphids treated with the four insecticides increased by 35.6, 23.4, 31.1, and 23.3%, respectively, compared tothe control group. Additionally, the carboxylesterase inhibitor TPP exhibited a synergistic effect when combined with the aforementioned insecticides, with synergistic ratios increasing by 1.54, 1.28, 1.24, and 1.17, respectively, consistent with the RNAi results. Field trials further validated the indoor findings, showing that on the 5^th day after application, the control efficacy of LCT+TPP, IPC+TPP, PHX+TPP, and IMI+TPP combinations improved by 35.6, 21.5, 46.0, and 70.1%, respectively, compared to the use of chemical pesticides alone.The functional inhibition of the VmCarEs-6 gene in T. trifolii through RNAi and TPP treatment significantly impaired the pest's detoxification metabolism, thereby enhancing its sensitivity to chemical pesticides. This study provides a critical theoretical foundation for elucidating the mechanisms of resistance in piercing-sucking pests and developing targeted pest control products.