Avian metapneumovirus (aMPV), a paramyxovirus, causes acute respiratory diseases in turkeys and swollen head syndrome in chickens.  This study established a reverse genetics system for aMPV subtype B LN16-A strain based on T7 RNA polymerase.  Full-length cDNA of the LN16-A strain was constructed by assembling 5 cDNA fragments between the T7 promoter and hepatitis delta virus ribozyme.  Transfection of this plasmid, along with the supporting plasmids encoding the N, P, M2-1, and L proteins of LN16-A into BSR-T7/5 cells, resulted in the recovery of aMPV subtype B.  To identify an effective insertion site, the enhanced green fluorescent protein (EGFP) gene was inserted into different sites of the LN16-A genome to generate recombinant LN16-As.  The results showed that the expression levels of EGFP at the site between the G and L genes of LN16-A were significantly higher than those at the other two sites (between the leader and N genes or replacing the SH gene).  To verify the availability of the site between G and L for foreign gene expression, the VP2 gene of very virulent infectious bursal disease virus (vvIBDV) was inserted into this site, and recombinant LN16-A (rLN16A-vvVP2) was successfully rescued.  Single immunization of specific-pathogen-free chickens with rLN16A-vvVP2 induced high levels of neutralizing antibodies and provided 100% protection against the virulent aMPV subtype B and vvIBDV.  Establishing a reverse genetics system here provides an important foundation for understanding aMPV pathogenesis and developing novel vector vaccines.

Ear differentiation, grain development and their interaction with factors in the growing environment, such as temperature, solar radiation and precipitation, greatly influence grain number and grain weight, and ultimately affect summer maize production.  In this study, field experiments involving different sowing dates were conducted over three years to evaluate the effects of temperature factors, average solar radiation and total precipitation on the growth process, ear differentiation, fertilization characteristics, grain filling and yield of summer maize varieties with different growth durations.  Four hybrids were evaluated in Huang-Huai-Hai Plain (HHHP), China from 2018 to 2020 with five different sowing dates.  The results showed that the grain yield formation of summer maize was strongly impacted by the environment from the silking (R1) to milking (R3) stage.  Average minimum temperature (AT_min) was the key environmental factor that determined yield.  Reductions in the length of the growing season (r=–0.556, P<0.01) and the total floret number on ear (R²=0.200, P<0.001) were found when AT_min was elevated from the emerging (VE) to R1 stage.  Both grain-filling rate (R²=0.520, P<0.001) and the floret abortion rate on ear (R²=0.437, P<0.001) showed quadratic relationships with AT_min from the R1 to physiological maturity (R6) stage, while the number of days after the R1 stage (r=–0.756, P<0.01) was negatively correlated with AT_min.  An increase in AT_min was beneficial for the promotion of yield when it did not exceeded a certain level (above 23°C during the R1–R3 stage and 20–21°C during the R1-R6 stage).  Enhanced solar radiation and precipitation during R1–R6 increased the grain-filling rate (R²=0.562, P<0.001 and R²=0.229, P<0.05, respectively).  Compared with short-season hybrids, full-season hybrids showed much greater suitability for a critical environment.  The coordinated regulation of AT_min, ear differentiation and grain development at the pre- and post-silking stages improved maize yield by increasing total floret number and grain-filling rate, and by reducing the floret abortion rate on ear. 

Genetic transformation has been an effective technology for improving the agronomic traits of maize.  However, it is highly reliant on the use of embryonic callus (EC) and shows a serious genotype dependence.  In this study, we performed genomic sequencing for 80 core maize germplasms and constructed a high-density genomic variation map using our newly developed pipeline (MQ2Gpipe).  Based on the induction rate of EC (REC), these inbred lines were categorized into three subpopulations.  The low-REC germplasms displayed more abundant genetic diversity than the high-REC germplasms.  By integrating a genome-wide selective signature screen and region-based association analysis, we revealed 95.23 Mb of selective regions and 43 REC-associated variants.  These variants had phenotypic variance explained values ranging between 21.46 and 49.46%.  In total, 103 candidate genes were identified within the linkage disequilibrium regions of these REC-associated loci.  These genes mainly participate in regulation of the cell cycle, regulation of cytokinesis, and other functions, among which MYB15 and EMB2745 were located within the previously reported QTL for EC induction.  Numerous leaf area-associated variants with large effects were closely linked to several REC-related loci, implying a potential synergistic selection of REC and leaf size during modern maize breeding.

Avian metapneumovirus (aMPV) is a highly contagious pathogen that causes acute upper respiratory tract diseases in chickens and turkeys, resulting in serious economic losses.  Subtype B aMPV has recently become the dominant epidemic strain in China.  We developed an attenuated aMPV subtype B strain by serial passaging in Vero cells and evaluated its safety and efficacy as a vaccine candidate.  The safety test showed that after the 30th passage, the LN16-A strain was fully attenuated, as clinical signs of infection and histological lesions were absent after inoculation.  The LN16-A strain did not revert to a virulent strain after five serial passages in chickens.  The genomic sequence of LN16-A differed from that of the parent wild-type LN16 (wtLN16) strain and had nine amino acid mutations.  In chickens, a single immunization with LN16-A induced robust humoral and cellular immune responses, including the abundant production of neutralizing antibodies, CD4⁺ T lymphocytes, and the Th1 (IFN-γ) and Th2 (IL-4 and IL-6)

cytokines.  We also confirmed that LN16-A provided 100% protection against subtype B aMPV and significantly reduced viral shedding and turbinate inflammation.  Our findings suggest that the LN16-A strain is a promising live attenuated vaccine candidate that can prevent infection with subtype B aMPV.

The footprints of water and nitrogen (WF and NF) provide a comprehensive overview of the type and quantity of water consumption and reactive nitrogen (Nr) loss in crop production.  In this study, a field experiment over two years (2019 and 2020) compared three integrated agronomic practice management (IAPM) systems: An improved management system (T2), a high-yield production system (T3), and an integrated soil–crop management system (ISCM) using a local smallholder farmer’s practice system (T1) as control, to investigate the responses of WF, Nr losses, water use efficiency (WUE), and nitrogen use efficiency (NUE) to IAPM.  The results showed that IAPM optimized water distribution and promoted water use by summer maize.  The evapotranspiration over the whole maize growth period of IAPM increased, but yield increased more, leading to a significant increase in WUE.  The WUE of the T2, T3, and ISCM treatments was significantly greater than in the T1 treatment, in 2019 and 2020 respectively, by 19.8–21.5, 31.8–40.6, and 34.4–44.6%.  The lowest WF was found in the ISCM treatment, which was 31.0% lower than that of the T1 treatment.  In addition, the ISCM treatment optimized soil total nitrogen (TN) distribution and significantly increased TN in the cultivated layer.  Excessive nitrogen fertilizer was applied in treatment T3, producing the highest maize yield, and resulting in the highest Nr losses.  In contrast, the ISCM treatment used a reduced nitrogen fertilizer rate, sacrificing grain yield partly, which reduced Nr losses and eventually led to a significant increase in nitrogen use efficiency and nitrogen recovery.  The Nr level in the ISCM treatment was 34.8% lower than in the T1 treatment while NUE was significantly higher than in the T1 treatment by 56.8–63.1% in 2019 and 2020, respectively.  Considering yield, WUE, NUE, WF, and NF together, ISCM should be used as a more sustainable and clean system for sustainable production of summer maize.

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.

The common vetch (Vicia sativa L.) is a self-pollinated annual forage legume that is widely distributed worldwide.  It has wide adaptability and high nutritional value and is commonly used as an important protein source for livestock feed.  However, pod shattering seriously limits the yield of common vetch.  To clarify the mechanism of pod shattering in common vetch, the pod walls of three shattering-resistant (SR) accessions (B65, B135, and B392) and three shattering-susceptible (SS) accessions (L33, L170, and L461) were selected for transcriptome sequencing.  A total of 17190 differentially expressed genes (DEGs) were identified in the pod wall of B135 and L461 common vetch at 5, 10, 15, 20, and 25 days after anthesis.  KEGG analysis showed that “phenylpropanoid biosynthesis” was the most significantly enriched pathway, and 40 structural genes associated with lignin biosynthesis were identified and differentially expressed in B135 and L461 common vetch.  We analysed the DEGs in the pod wall of three SR and three SS accessions at 15 days after anthesis, and most of the DEGs were consistent with the significant enrichment pathways identified in B135 and L461 common vetch.  The total lignin content of SR accessions was significantly lower than the SS accessions.  The present study lays a foundation for understanding the molecular regulatory mechanism of pod shattering related to lignin biosynthesis in common vetch and provides reference functional genes for breeders to further cultivate shattering-resistant common vetch varieties.

Persistent overcast rain was an essential limiting factor for summer maize production, of which immediate impact was the dual pressure of waterlogging and shading.  However, the mechanism of independent and combined effects of waterlogging and shading induced maize yield losses are rarely studied, especially at different growth stages.  Denghai 605 (DH605) was selected to be subjected shading, waterlogging, and their combined stress at the 3rd leaf stage (V3), the 6th leaf stage (V6), and tasseling stage (VT).  Results showed that shading, waterlogging and their combination significantly limited the expansion of leaf area, and decreased leaf net photosynthetic rate (P_n) and net assimilation rate (NAR), thus reducing the crop growth rate (CGR) and biomass accumulation.  At the same time, compared to control, the process of lignin synthesis was inhibited under stressed treatment, resulting in reduced stem mechanical strength and a poor development of the vascular system, of which change significantly reduced efficiency of assimilate remobilization to the ear and ultimately grain yield.  The most significant effects of waterlogging and combined stresses on yield were occurred at V3 stage, followed by the V6 and VT stages.  The most significant effects of shading were occurred at VT stage, followed by the V6 and V3 stages.  Moreover, the compound stress exacerbated the damage brought about by a single stress.  It is predicted that climate change will increase the frequency of abiotic stress assemblages, and the results of these findings provide some direction for further research on maize breeding in summer maize under continuous rainy conditions in the future.

Due to the breeding of dense-resistant and lodging-resistant varieties in maize production, dense planting has become an effective means for achieving high and stable yields, while excellent hybrids are a prerequisite for reasonable dense planting in maize production.  Nonetheless, the photosynthetic mechanism of improving plant density tolerance of maize hybrids released at different era in China remains unclear.  This study aims to investigate the 40-year breeding effort for enhanced photosynthetic trait at different densities, and elucidate the physiological and ecological mechanisms of improving the density tolerance of maize hybrids.  We conducted a 3-year study in 2019, 2020, and 2021.  From 1970 to 2009, a comparison was made between the eight major hybrids promoted in China, divided into four decades, under three planting densities (45,000 (D1), 67,500 (D2), and 90,000 (D3) plants ha⁻¹).  At high density, modern hybrids had more rational canopy structure and leaf photosynthetic performance compared with old hybrids and specific leaf nitrogen has decreased slightly.  Among all treatments, the modern hybrids (2000s) were able to maintain higher net photosynthetic rate and photosynthetic nitrogen utilization efficiency (PNUE) at D3 density, and therefore possessed the highest grain yield (GY), which was 118.47% higher than that of the old hybrids (1970s).  Leaf area duration after anthesis, total chlorophyll content, photosynthesis key enzyme activities, and maximum efficiency of PSII photochemistry were all positively correlated with GY, with PNUE was more significantly correlated with GY indeed and is a key indicator for maize hybrids optimization.  Based on these results, breeders should continue to conduct hybrid selections under adverse and high-density conditions, focusing on the optimization of population structure and the continuous improvement of photosynthetic capacity, searching for the optimal leaf nitrogen-content status, so as to select and breed high-yielding and density-tolerance hybrids, which resulted in a sustained increase in maize GY.

Characterizing the N uptake and utilization of different maize hybrids is essential for optimizing N application and increasing the profits from maize production.  Research trials were conducted with controlled-release urea (CRU) as a base fertilizer (TC) and urea split application in one (T1), two (T2), and three (T3) stages to evaluate the effects on N uptake, NUE, and yield using the ¹⁵N tracer technique between two maize hybrids; DH518 (an mid-early-maturing hybrid) and DH605 (a late-maturing hybrid).  According to the results, compared with urea, CRU as a base fertilizer and urea split application in two and three stages significantly increased grain yield and NUE while reducing environmental N loss.  Compared with T1, the grain yields of the TC, T2, and T3 treatments were, respectively, increased by 11.1, 9.8, and 11.7% in DH518 and by 16.4, 15.7, and 22.9% in DH605.  Regression analysis showed that the grain yield of DH518 displayed a bilinear trend of an initial rapid increase and then a slow increase with the increase in post-anthesis N accumulation, total N accumulation, N recovery efficiency, and N nutrition index (NNI).  By contrast, DH605 consistently showed a linear regression relationship with a rapid increase.  The crop recovery N efficiency (CRN) values in the T3 treatment for urea applied at the sowing stage and topdressing at the V9 stage in DH518 were 60.0 and 62.4% higher than under topdressing at the VT stage, respectively, while the CRN values of urea topdressing at the V9 and VT stages in DH605 were 37.7 and 37.1% higher than when applied at the sowing stage, respectively.  The higher pre-anthesis N demand and shorter growth period of DH518 maintained the N supply–demand balance, resulting in NNI (NNI≥0.988) falling within the range of slow yield increase under the T2 and TC treatments, while the N status of DH605 plants only reached optimal levels in the T3 treatment.  Therefore, a split three-stage application of urea or applying CRU as a base fertilizer and topdressing with urea in the later growth stages is recommended for mid-late-maturing hybrids to obtain an optimal yield.  In addition, for mid-early-maturing hybrids, applying CRU or reducing the number of times of split application, e.g., a split two-stage application, can ensure an adequate N supply in the later growth stages and increase production and thus profits.

Effects of maize straw return and N fertilizer application on soil quality and crop yield have been extensively researched.  However, the effects of different amounts of maize straw returned to the field with different nitrogen application rates on the soil-crop system quality, abundance of functional N cycle microorganisms, N₂O emissions and crop N nutrition status of crops remain incompletely explored.  Objective of this study was to assess the effects of different summer maize straw return rates and N application rates on: i) soil quality and crop productivity; ii) the community of N cycle-functional microorganisms and N₂O emission, and iii) crop N status.  Results indicated that crop yields increased by 7.62 to 12.69% at 210 kg ha^-1 of N application for full straw return (SN) and half return (1/2SN) compared to the no-return treatment (CK).  No significant difference was recorded in yield between the full straw return reduced by 15% (178.5 kg N ha^-1) of N fertilizer (S-15%N) and SN.  Surface soil layer (0-20 cm) showed significantly higher levels of soil organic matter (SOM), the community of N-cycling functional microorganisms, crop N nutrition status and N uptake efficiency in SN, 1/2SN, and S-15%N as compared to other treatments.  S-15%N and 1/2SN reduced cumulative N₂O emission fluxes by 19.11 and 5.51%, respectively, compared to SN.  Furthermore, the nitrogen nutrient index (NNI) of 1/2SN, S-15%N was closer to the critical N requirement than SN.  In summary, the decision schemes for optimal straw return and N application (1/2SN and S-15%N) based on SOM, NNI, cumulative N₂O emission fluxes and yield can be applied to the annual production of winter wheat and summer maize in China as compared to SN.

Research on the yield-enhancing mechanisms of maize through ‘smart’ plant morphology under dense planting conditions is a critical focus in modern agriculture.  However, the issue of yield stability in dense-planted maize, particularly regarding lodging resistance, remains insufficiently examined in the academic literature.  A three-year field experiment was conducted using three hybrids (XD20, DH618 and DH605) and three plant density treatments (6.0×10⁴, 7.5×10⁴, and 9.0×10⁴ plants ha^-1) to investigate the effects of planting density on lodging resistance and yield of summer maize hybrids with different plant morphologies.  According to the results, increasing planting density significantly boosted the yield of DH605, while the yields of XD20 and DH618 exhibited an initial increase followed by stabilization.  Compared to the low-density (L) treatment, the height parameters and center of gravity of summer maize under the high-density (H) treatment were significantly elevated.  This was accompanied by a pronounced reduction in light transmittance within the bottom and ear layers, a decrease in the mechanical strength of basal internodes, and an increased risk of lodging, particularly for the XD20 hybrid.  DH605 improved mechanical strength by enhancing the light distribution within the ear and bottom layers, and by optimizing basal internode characteristics.  Ultimately, the grain yield under the DH605-H treatment increased by 10.68 to 34.11% relative to XD20-H, with a concurrent reduction in lodging rates ranging from 72.66 to 92.29%.  Cellulose content within basal internodes and the total area of vascular bundles in the outer layer were key factors, explaining 61.70% of mechanical strength variance.  Therefore, high planting density significantly increased yield but also lodging susceptibility.  Optimizing plant morphology improved canopy light distribution, dry matter composition and anatomical structure of basal internodes, enhancing lodging resistance and grain yield in densely planted maize. 

High-density planting can better utilize the yield potential of modern varieties.  However, under traditional row spacing conditions, increasing planting density brings about poor light distribution and limited yield improvement, highlighting the need for further exploration of optimal row spacing in relation to planting density.  To assess the effect of delaying leaf senescence in the lower canopy by changing row spacing on the photosynthetic performance of the canopy and its regulatory impact on yield.  A two-year field trial (2019-2020) was conducted on Zhengdan 958 for this study. Four treatments were set up: LR60 (6.75 plants m^-2, 60 cm row spacing, conventional planting); HR60, HR80, and HR100 (8.25 plants m^-2, with row spacings of 60, 80, and 100 cm, respectively).  Quantitative analysis was conducted on canopy structure, population photosynthesis, and grain yield.  Maize canopy leaf area index (LAI), photosynthetically active radiation (PAR), canopy apparent photosynthesis (CAP), biomass distribution, yield were measured.  The results showed that the high-density treatments significantly increased the yield compared to LR60.  Among the high-density treatments, HR80 exhibited an average yield increase of 8.47% compared to HR60 over two years.  This was primarily attributed to HR80 enhancing the utilization of photosynthetically active radiation in the lower canopy after silking, delaying the decrease of LAI in the layers below the ear, and increasing CAP, resulting in a significant increase in biomass.  HR80 increased yield by an average of 8.17% over HR100, due to significant increase in RUE during the grain-filling period.  Furthermore, HR80 showed a significant increase in source-sink ratio compared to both HR60 and HR100, as well as an increase in ¹³C-photosynthetic products partitioning to the grains, and a significant increase in kernel number.  Thus, row spacing configuration should be adapted to the planting density for optimal yield.  Specifically, appropriate row spacing can optimize the population structure, enhancing light distribution within the middle and lower canopy layers, and improving the canopy apparent photosynthesis and light utilization, which will support higher yields in maize.

Enhancing soil organic carbon (SOC) stocks is a key aspect of modern agriculture, but whether this can be achieved by incorporating legume green manure crops in cereal production to substitute synthetic N fertilizers is unknown. This study used a six-year (2017-2022) field study to explore the impacts of intercropping green manure with maize and reducing nitrogen fertilization on SOC stocks, while specifically focusing on the relationship between aggregate composition and carbon sequestration. Maize intercropped with common vetch (M/V), maize intercropped with rapeseed (M/R), and sole maize (M), were each tested at conventional (N2, 360 kg ha^-1) and reduced (N1, 270 kg ha^-1, 25% reduced) N application rates. Soil was sampled in 2020, 2021, and 2022. Compared with sole maize, intercropping with green manure (M/V and M/R) significantly increased SOC stocks which compensated for any negative effect due to the 25% reduction in N application. Based on 3-year averages, intercropping with M/V and M/R increased the SOC content compared to sole maize (M) by 12.1 and 9.1%, respectively, with intercropping further mitigating the negative impact of reduced nitrogen application. There was no significant difference between M/V and M/R. The SOC content at N1 was reduced by 9.3-10.5% compared to that at N2 in sole maize, but the differences in SOC stocks between N1 and N2 were not significant in the intercropping patterns (M/V and M/R). The intercropped M/V and M/R showed 20.9 and 16.3% higher SOC contents compared to sole maize at N1, with no differences at N2. Intercropping green manure led to a 5.3% greater SOC in the 0-20 cm depth soil in 2022 compared to that in 2020, due to the cumulative effect of two years of green manure intercropping. Intercropping green manure (M/V and M/R) increased the proportion of macroaggregates (>0.25 mm) and aggregate stability while reducing the proportion of microaggregates compared to sole maize under the N1 application. Structural equation modeling indicated that cropping patterns and nitrogen application levels mainly affect SOC indirectly by regulating the composition of macroaggregates and aggregate organic carbon (AOC). Correlation analysis further revealed that the composition of macroaggregates is significantly and positively correlated with the SOC content (R²=0.64). In addition, intercropping green manure can maintain high crop yields by increasing SOC under reduced chemical nitrogen application. The results of this study show that intercropping green manure with grain crops can be a viable measure for increasing SOC sinks and maize productivity by optimizing the aggregate composition with reduced N application in the Oasis Irrigation Area.