In the face of agricultural labor shortages, reducing labor and costs in rice production while meeting demand or increasing yield is crucial for sustainable agricultural development.  Using crop straw boards and raising seedlings at a high-density can reduce labor demand and enhance rice yield.  This study investigated the effects of seeding density and transplanting age on tillering patterns, panicle formation rates, and yield to determine the optimal cultivation practices for maximizing rice yield.  Two-year field experiments were conducted in Sihong County, Jiangsu Province, China, using the japonica rice variety Nanjing 5718.  Five seeding densities (150–350 g/tray) and four transplanting ages (10–25 days) were evaluated to assess their impacts on tillering patterns, panicle formation rates, and yield.  Innovative crop straw boards were employed to enhance planting efficiency and reduce dependence on soil for raising seedlings.  This approach also lessened tillage layer destruction, promoting sustainable practices.  The results indicated that increasing seeding density significantly altered tillering and panicle formation patterns by reducing the occurrence and panicle formation rates of lower-position tillers.  Although the occurrence of middle- and high-position tillers increased, the overall number of panicles per hill decreased, especially at higher densities, negatively affecting yield.  Reducing the transplanting age promoted the emergence and panicle formation of lower-position tillers, thus mitigating these negative effects.  Specifically, compared to traditional methods (150 g/tray, 20-day seedlings), the higher seeding density (300 g/tray) and reduced transplanting age (15-day seedlings) increased total panicle number by 3.79–4.73% and yield by 3.38–5.05%.  Combining higher seeding densities with reduced transplanting ages offers significant advantages over conventional practices by enhancing resource utilization and improving tillering efficiency.  These findings provide actionable recommendations for optimizing rice cultivation practices and contribute to sustainable agricultural development.

Vegetable soybean ((Glycine max (L.) Merr.), commonly referred to as edamame, holds significant agricultural importance in China as a legume vegetable harvested at the pod-filling stage (R6).  The visual appeal of vegetable soybeans is crucial for consumer preference and marketability, and it depends on factors such as pod length, pod width, and pod color.  This study cultivated 264 vegetable soybeans in Nanjing, Huai’an, and Nantong, Jiangsu Province, China to assess pod traits using PlantPhenoM, a system for pod phenotypic identification and analysis.  The results revealed a variability range of 8.64 to 30.00% in appearance quality traits among the vegetable soybeans.  Leveraging phenotypic data and employing a genome-wide association study (GWAS) identified 525 SNPs significantly linked to the appearance quality traits in different regions.  In addition, five candidate genes (Glyma.04G004700, Glyma.15G051600, Glyma.18G225700, Glyma.18G225900, and Glyma.18G272300) associated with target traits were identified, and KASP markers for S04_372771 (pod length), S18_51477324 (pod width), and S18_55553200 (pod color) were developed.  This study offers valuable insights for breeding superior vegetable soybean varieties and lays the groundwork for exploring candidate genes and molecular markers related to appearance and quality traits in vegetable soybeans.

Phosphorus (P) is an essential nutrient element that is critical for plant growth and ecosystem functionality. The soil P cycle plays multiple roles, such as sustaining plant growth and productivity, regulating nutrient balance within ecosystems, and enhancing ecosystem adaptability and resilience. This cycle is influenced by factors such as the restoration approach and microbial community dynamics. However, the extent to which the restoration approach alters the P cycle in karst ecosystems and the underlying microbial mechanisms remain poorly understood. The P-cycle multifunctionality index (P-cycle MFI) serves as a comprehensive indicator for evaluating soil P cycle function, and it provides insights into changes in the P cycle between different restoration approaches. To investigate the shifts in soil P-cycle MFI and microbial mechanisms between different restoration approaches, we analyzed soil available P (AP), total P (TP), microbial biomass P (MBP), and the activities of acid phosphatase (ACP) and alkaline phosphatase (ALP). These data were used to calculate the P-cycle MFI by averaging the Z-scores between two restoration approaches (artificial restoration of forest (AF) and natural restoration of forest (NF)) and a control (cropland, CP) at six subtropical karst ecosystem sites in China. We also determined the soil organic carbon (SOC), exchangeable calcium (Ca) and magnesium (Mg), pH, bulk density (BD), microbial biomass C (MBC), and microbial biomass nitrogen (MBN), as well as the community structure, relative abundance, diversity indices, and co-occurrence networks of phoD-harboring bacteria. The results showed that the community structure of phoD-harboring bacteria varied significantly among AF, NF, and CP and across different temperature gradients. These bacteria exhibited increasing complexity and tightness in co-occurrence networks from CP to AF and then to NF, along with the ACP and ALP activities, but not the TP and AP contents. The P-cycle MFI values were significantly higher in NF compared to AF and CP, and the variation was significantly explained by restoration approach, temperature, MBC, MBN, SOC, exchangeable Ca, BD, community structure of phoD-harboring bacteria, and exchangeable Mg. Furthermore, natural restoration had a more substantial impact on the P-cycle MFI than temperature by enhancing SOC, microbial biomass, the complexity and co-occurrence network tightness of the phoD-harboring bacterial community structure, and ACP and ALP activities, but it reduced soil BD. The rare genera of phoD-harboring bacteria significantly influenced the variation of soil P-cycle MFI compared to the dominant genera. This study highlights the importance of rare genera of phoD-harboring bacteria in driving soil P-cycle multifunctionality in karst ecosystems, with natural restoration being more effective than artificial methods for enhancing soil organic matter and microbial community complexity.

Understanding the spatial distributions and corresponding variation mechanisms of key soil nutrients in fragile karst ecosystems can assist in promoting sustainable development.  However, due to the implementation of ecological restoration initiatives such as land-use conversions, novel changes in the spatial characteristics of soil nutrients remain unknown.  To address this gap, we explored nutrient variations and the drivers of the variation in the 0–15 cm topsoil layer using a regional-scale sampling method in a typical karst area in northwest Guangxi Zhuang Autonomous Region, Southwest China.  Descriptive statistics, geostatistics, and spatial analysis were used to assess the soil nutrient variability.  The results indicated that soil organic carbon (SOC), total nitrogen (TN), total phosphorus (TP), and total potassium (TK) concentrations showed moderate variations, with coefficients of variance being 0.60, 0.60, 0.71, and 0.72, respectively.  Moreover, they demonstrated positive spatial autocorrelations, with global Moran’s indices being 0.68, 0.77, 0.64, and 0.68, respectively.  However, local Moran’s index values were low, indicating large spatial variations in soil nutrients.  The best-fitting semi-variogram models for SOC, TN, TP, and TK concentrations were spherical, Gaussian, exponential, and exponential, respectively.  According to the classification criteria of the Second National Soil Census in China, SOC and TN concentrations were relatively sufficient, with the proportions of rich and very rich levels being up to 90.9 and 96.0%, respectively.  TP concentration was in the medium-deficient level, with the areas of medium and deficient levels accounting for 33.7 and 30.1% of the total, respectively.  TK concentration was deficient, with the cumulative area of extremely deficient, very deficient, and deficient levels accounting for 87.6% of the total area.  Consequently, the terrestrial ecosystems in the study area were more vulnerable to soil P and K than soil N deficiencies.  Furthermore, variance partitioning analysis of the influencing factors showed that, except for the interactions, the single effect of other soil properties accounted more for soil nutrient variations than spatial and environmental variables.  These results will aid in the future management of terrestrial ecosystems.

Ogura cytoplasmic male sterility (Ogura CMS) was first identified in wild radish (Raphanus sativus) and resulted in complete pollen abortion.  However, the molecular mechanism of Ogura CMS in Chinese cabbage remains unclear.  A cytological analysis confirmed nuclear degradation during the late uninucleate stage of pollen development, which diminished by the tricellular stage.  Concurrently, tapetal cells exhibited abnormal enlargement and vacuolation starting from the tetrad stage.  Serious developmental defects were observed in the pollen wall.  During early pollen development, genes associated with cytochrome c and programmed cell death (PCD) were upregulated in the Ogura CMS line, while genes involved in pollen wall mitosis were downregulated.  Conversely, at the late stage of pollen development, peroxisome and autophagy-related genes in the Ogura CMS line were upregulated.  The mitochondrial orf138 gene mutation triggered the PCD process in tapetal cells, leading to their abnormal enlargement and the degradation of their contents, eventually resulting in vacuolation at the tricellular stage.  These tapetal defects hindered the provision of adequate sporopollenin and nutrients to the microspores, consequently leading to abnormal pollen wall development and abnormal mitosis in the microspores.  Ultimately, nuclear dispersion commenced during the late uninucleate stage, and autophagy occurred in the late stage of pollen development.  Consequently, the plant could not produce functional pollen, resulting in male sterility in Chinese cabbage.  Studies of Ogura CMS can promote the production and application of male sterile materials and enrich male sterile resources, which is of great significance for hybrid breeding.

Enhancing photosynthetic efficiency is a major goal for improving crop yields under agricultural field conditions and is associated with chloroplast biosynthesis and development.  In this study, we demonstrate that Golden2-like 1a (BnGLK1a) plays an important role in regulating chloroplast development and photosynthetic efficiency.  Overexpressing BnGLK1a resulted in significant increases in chlorophyll content, the number of thylakoid membrane layers and photosynthetic efficiency in Brassica napus, while knocking down BnGLK1a transcript levels through RNA interference (RNAi) had the opposite effects.  A yeast two-hybrid screen revealed that BnGLK1a interacts with the abscisic acid receptor PYRABACTIN RESISTANCE 1-LIKE 1–2 (BnPYL1–2) and CONSTITUTIVE PHOTOMORPHOGENIC 9 SIGNALOSOME 5A subunit (BnCSN5A), which play essential roles in regulating chloroplast development and photosynthesis.  Consistent with this, BnGLK1a-RNAi lines of B. napus display hypersensitivity to the abscisic acid (ABA) response.  Importantly, overexpression of BnGLK1a resulted in a 10% increase in thousand-seed weight, whereas seeds from BnGLK1a-RNAi lines were 16% lighter than wild type.  We propose that BnGLK1a could be a potential target in breeding for improving rapeseed productivity.  Our results not only provide insights into the mechanisms of BnGLK1a function, but also offer a potential approach for improving the productivity of Brassica species.

Pseudorabies (PR) is an acute infectious disease of pigs caused by the PR virus (PRV) and results in great economic losses to the pig industry worldwide.  PRV glycoprotein E (gE)-based enzyme-linked immunosorbent assay (ELISA) has been used to distinguish gE-deleted vaccine-immunized pigs from wild-type virus-infected pigs to eradicate PR in some countries.  Nanobody has the advantages of small size and easy genetic engineering and has been a promising diagnostic reagent.  However, there were few reports about developing nanobody-based ELISA for detecting anti-PRV-gE antibodies.  In the present study, the recombinant PRV-gE was expressed with a bacterial system and used to immunize the Bactrian camel.  Then, two nanobodies against PRV-gE were screened from the immunized camel by phage display technique.  Subsequently, two nanobody-HRP fusion proteins were expressed with HEK293T cells.  The PRV-gE-Nb36-HRP fusion protein was selected as the probe for developing the blocking ELISA (bELISA) to detect anti-PRV-gE antibodies.  Through optimizing the conditions of bELISA, the amount of coated antigen was 200 ng per well, and dilutions of the fusion protein and tested pig sera were separately 1:320 and 1:5.  The cut-off value of bELISA was 24.20%, and the sensitivity and specificity were 96.43 and 92.63%, respectively.  By detecting 233 clinical pig sera with the developed bELISA and a commercial kit, the results showed that the coincidence rate of two assays was 93.99%.  Additionallly, epitope mapping showed that PRV-gE-Nb36 recognized a conserved conformational epitope in different reference PRV strains.  Simple, great stability and low-cost nanobody-based bELISA for detecting anti-PRV-gE antibodies were developed.  The bELISA could be used for monitoring and eradicating PR.

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway play a pivotal role in innate immunity.  Among invertebrates, Domeless receptors serve as the key upstream regulators of this pathway.  In our study on Bactrocera dorsalis, we identified three cytokine receptors: BdDomeless1, BdDomeless2, and BdDomeless3.  Each receptor encompasses five fibronectin-type-III-like (FN III) extracellular domains and a transmembrane domain.  Furthermore, these receptors exhibit the increased responsiveness to diverse pathogenic challenges.  Notably, only BdDomeless3 is upregulated during symbiont-like viral infections.  Moreover, silencing BdDomeless3 enhanced the infectivity of Bactrocera dorsalis cripavirus (BdCV) and B. dorsalis picorna-like virus (BdPLV), underscoring BdDomeless3’s crucial role in antiviral defense of B. dorsalis.  Following the suppression of Domeless3 expression, six antimicrobial peptide genes displayed decreased expression, potentially correlating with the rise in viral infectivity.  To our knowledge, this is the first study identifying cytokine receptors associated with the JAK/STAT pathway in tephritid flies, shedding light on the immune mechanisms of B. dorsalis.

Straw return is a promising strategy for managing soil organic carbon (SOC) and improving yield stability.  However, the optimal straw return strategy for sustainable crop production in the wheat (Triticum aestivum L.)–cotton (Gossypium hirsutum L.) cropping system remains uncertain.  The objective of this study was to quantify the long-term (10 years) impact of carbon (C) input on SOC sequestration, soil aggregation and crop yields in a wheat–cotton cropping system in the Yangtze River Valley, China.  Five treatments were arranged with a single-factor randomized design as follows: no straw return (Control), return of wheat straw only (Wt), return of cotton straw only (Ct), return of 50% wheat and 50% cotton straw (Wh-Ch) and return of 100% wheat and 100% cotton straw (Wt-Ct).  In comparison to the Control, the SOC content increased by 8.4 to 20.2% under straw return.  A significant linear positive correlation between SOC sequestration and C input (1.42–7.19 Mg ha⁻¹ yr⁻¹) (P<0.05) was detected.  The percentages of aggregates of sizes >2 and 1–2 mm at the 0–20 cm soil depth were also significantly elevated under straw return, with the greatest increase of the aggregate stability in the Wt-Ct treatment (28.1%).  The average wheat yields increased by 12.4–36.0% and cotton yields increased by 29.4–73.7%, and significantly linear positive correlations were also detected between C input and the yields of wheat and cotton.  The average sustainable yield index (SYI) reached a maximum value of 0.69 when the C input was 7.08 Mg ha⁻¹ yr⁻¹, which was close to the maximum value (SYI of 0.69, C input of 7.19 Mg ha⁻¹ yr^–1) in the Wt-Ct treatment.  Overall, the return of both wheat and cotton straw was the best strategy for improving SOC sequestration, soil aggregation, yields and their sustainability in the wheat–cotton rotation system.

Increasing crop grain yields is an urgent global priority due to population growth, shrinking arable land, and severe climate change in recent years (Tang et al. 2023).  Unraveling the process of panicle development is crucial for enhancing the grain yield of cereal crops.  In the development of rice panicles, the inflorescence meristem (IM) gives rise to two types of lateral branch meristems (BMs): primary branch meristem (pBM) and secondary branch meristem (sBM).  The pBM generates sBM and spikelet meristems (SMs), and the sBM further differentiates into more SMs (Zhang and Yuan 2014).  A spikelet is the basic unit of inflorescence in Poaceae plants.  It originates from the spikelet meristem (SM) that determines the number of spikelets per panicle and consequently impacts crop yield formation (Doebley et al. 2006).  The seed setting rate, determined by spikelet development, is another crucial trait intimately linked to grain yield (Zhuang et al. 2024).  In plants, developmental defects in spikelets are typically categorized as either male sterility (Notsu et al. 2002; Liu et al. 2007; Luo et al. 2013) or female sterility (Li et al. 2022).  However, no mutant with completely sterile male and female reproductive organs has yet been identified so far.

Foxtail millet (Setaria italica (L.) Beauv.) is a stress-tolerant annual cereal crop species from the Poaceae family (Muthamilarasan and Prasad 2015; He et al. 2023; Liang et al. 2023).  In recent decades, foxtail millet has been developed as a new model crop for deciphering panicle development due to its diploidy and small genome, short growth cycle, and self-pollination character (Doust et al. 2009; Diao et al. 2017; He et al. 2021).  In foxtail millet, each spikelet produces one fertile floret and one sterile floret.  The sterile one was degenerated, and the fertile one was enclosed by lemma, palea, and two lodicules derived from the sterile one (Hussin et al. 2021; Zhang et al. 2021).  Several abnormal panicle mutants have been identified in both foxtail millet and its wild type, green foxtail (Setaria viridis).  In foxtail millet, the silp1 mutant showed an increase in the length and width of primary branches, in company with a decrease in the number of fertilized spikelets and seed setting rate in the panicle (Xiang et al. 2017).  The siaux1-1 mutant displays a sparsely branched panicle compared to the wild type (Tang et al. 2021).  The simads34 mutant shows increased panicle width and decreased panicle length and grain yield in foxtail millet (Hussin et al. 2021).  In green foxtail, mutations in the svaux1 gene result in reduced inflorescence branches and spikelet numbers and increased panicle length (Huang et al. 2017).  Approximately 17% of the panicles in the brl1 mutant produce additional flowers, bristles and/or spikelets within each spikelet compared to one flower per spikelet in the wild type (Yang et al. 2021).  The inflorescences of the Svfon2 mutant exhibit abnormal apices and panicle tips, which are divided into two or more parts (Zhu et al. 2021).  However, few studies on panicle sterility mutants have been reported in both Setaria italica and Setaria viridis, with only one male-sterile mutant, sinp1, having been identified (Zhang et al. 2021).  Thus, the understanding of panicle infertility in foxtail millet remains limited.

In this trial, we identified a completely sterile mutant sinog1 (no grain 1) from the EMS-induced mutant library of Yugu 1.  The mutant exhibited a slender panicle and was completely sterile with no seed set (Fig. 1-A).  Dissection observations revealed that all the reproductive organs of sinog1 florets gradually turned brown and eventually failed at the heading stage, leaving only two lemmas in the florets of sinog1, while the wild type was able to form mature floral organs normally (Fig. 1-B).  Additionally, the spikelet of the sinog1 mutant was significantly narrower and longer compared to the wild type (Fig. 1-A and C).  Furthermore, the spikelet of sinog1 mutant exhibited a significant increase in the content of indoleacetic acid (IAA) ((44.20±0.96) ng g^–1 FW), abscisic acid (ABA) ((91.27±1.77) ng g^–1 FW), and gibberellic acid 4 (GA₄) ((2.04±0.03) ng g^–1 FW) compared to the wild type ((30.64±0.59) ng g^–1 FW, (63.96±1.53) ng g^–1 FW, and (1.88±0.07) ng g^–1 FW), and a significant decrease in the content of brassinosteroid (BR) ((1.85±0.05) ng g^–1 FW) compared to the wild type ((2.02±0.05) ng g^–1 FW) (Fig. 1-D).

To identify the candidate genes responsible for the complete sterility in sinog1, we constructed an F₂ population with 263A as the female parent and the residual heterozygote individuals, including the mutation of sinog1 (Deng3-3) as the male parent.  The residual heterozygote individual Deng3-3 was derived from the BC₃ population generated by multiple rounds of backcrosses with Yugu 1.  Genetic and segregation ratio analysis suggested that the complete sterile phenotype of sinog1 was caused by a single recessive gene (χ²=0.06<χ² (0.05,1)=3.84).  Combining BSA-seq (45× coverage) and linkage verification analysis of 343 recessive individuals (completely sterile plants), the causal gene of sinog1 was finally mapped to a 1.85 Mb interval from 32.44 to 34.29 Mb on chromosome 5.  A total of 203 genes with sequence variations were screened out within the 1.85 Mb genomic interval (Fig. 1-E).

Subsequently, the transcriptomes of the wild type and mutant panicle at the heading stage were analyzed, identifying 37,986 genes.  Among these genes, 5,014 are differentially expressed genes (DEGs) between the wild type and sinog1 (log₂FC≥1 and FDR≤0.01), consisting of 2,707 significantly up-regulated and 2,307 significantly down-regulated genes.  The KEGG pathways analysis revealed that pathways related to ABC transporters, photosynthesis, and diterpenoid biosynthesis were significantly enriched in DEGs (Fig. 1-F).  Among these pathways, ABC transporters have been confirmed to regulate floral organ formation and panicle growth by transporting phytohormones and heavy metals in plants (Do et al. 2018; Naaz et al. 2023).  For instance, the mutation of ABCG26 in Arabidopsis leads to severely reduced fertility (Choi et al. 2011).  In rice, the knock-down of OsABCB14 can decrease the concentration and polar transport rates of auxin, and iron concentrations are also increased in the mutant (Xu et al. 2014).  These results suggest that the mutation gene of sinog1 might be an essential regulator for the proper expression of ABC transporters involved in floral development in foxtail millet.

Among the 203 genes located in the candidate interval on chromosome 5, we identified 28 DEGs by RNA-seq (Fig. 1-G), including seven genes (Seita.5G274600, Seita.5G274500, Seita.5G270700, Seita.5G274000, Seita.5G283000, Seita.5G282700, and Seita.5G272800) that were highly expressed in the wild type and 21 genes that were highly expressed in sinog1.  Of the 28 common genes, ten had mutations in the exon regions, including Seita.5G283500 (Val₆₄₉Leu), Seita.5G283600 (Leu₂₄₇Ser, Asn₃₉₆Glu, and Ser₆₆₇Arg), and Seita.5G282700 (insert 6 bp, Ala₇₈Ser), which have been annotated as steroid/xenobiotic-transporting ATPase in foxtail millet and ABC transporter in rice and Arabidopsis thaliana.  Interestingly, these three ABC pathway-related candidate genes were also enriched in the KEGG pathway of DEGs in sinog1.  It is noteworthy that ABC transporters have been verified to contribute to auxin and ABA transport, response to heavy metals such as iron and aluminum, and regulation of stomatal characters, which are important for plant growth and development (Do et al. 2018; Naaz et al. 2023).  Therefore, we inferred that ABC transporters may be key genes influencing morphological mutations in sinog1.

In conclusion, we have identified a novel panicle sterile phenotype in foxtail millet, the sinog1 mutant, which exhibits complete sterility with aborted reproductive organs.  The morphological analysis confirmed that sinog1 showed complete flower abortion at the heading stage, and the candidate pathways contributing to this completely sterile phenotype were identified using the combined BSA-seq and RNA-seq methods in foxtail millet.  This research provides a theoretical basis for understanding florets development in foxtail millet and other crops.

Rapeseed (Brassica napus L.) is a major oil crop worldwide that is vigorously promoted for cultivation in China.  Boron (B) is an essential micronutrient for plant growth and development.  However, agricultural soils in rapeseed planting areas often show either B deficiency or severe B deficiency.  Increasing the resistance to B deficiency is a pivotal goal in the breeding of rapeseed, yet the genetic basis for variations in B efficiency-related traits remains unclear. In this study, a natural population with 391 rapeseed accessions was used to investigate B efficiency-related traits through a nutrient solution system, including relative root length (RRL), shoot dry weight (SDW), root dry weight (RDW), and B efficiency coefficient (BEC), which exhibited extensive phenotypic variations under B deficiency.  Through a genome-wide association study (GWAS) of B efficiency-related traits using high-density SNP markers obtained from whole-genome resequencing, we identified 106 significantly associated SNPs by employing both the general linear model and the mixed linear model.  Among these SNP loci, two prominent SNP clusters were detected on chrA03:14087835–14764672 and chrC03:20110319–22135492 at low B level across three repeated experiments of multiple traits.  Integrated with the transcriptome analysis, four genes, BnaA03g29020D, BnaA03g29440D, BnaC03g33010D, and BnaC03g34490D, exhibiting higher differentially expressed fold-change along with favorable haplotypes within the promoter or coding region were identified as candidate genes that could potentially be involved in B efficient utilization, and their favorable haplotypes improved seedling growth and productivity under B deficiency.  In view of the lack of B mineral resources in China, rapid and accurate identification of more B-efficient alleles and the study of the genetic mechanism underlying crops in response to B deficiency have important theoretical and practical significance for cultivating B-efficient varieties and maintaining green, sustainable agriculture quickly and accurately.

Improving soil organic matter (SOM) maintenance is crucial for terrestrial carbon (C) sequestration and ecosystem functioning. Conservation tillage favors SOM pool buildup; however, it remains unclear how the decomposition of heterogeneous components is manipulated by microbial substrate utilization strategy from the view of SOM stability. Here, a one-year microcosm incubation was conducted using surface soils developed under 12 years of conservation tillage (high-C soil) and maize residue removal (low-C soil). Temporal changes in lignin phenols, neutral sugars, and amino sugars in the soil were monitored along with microbial phospholipid fatty acids (PLFAs) and enzyme activities. Throughout incubation, lignin phenols declined more (20.8-26.3%) than the SOM (12.3-14.5%) and amino sugars (10.6-12.3%), highlighting the key role of plant debris in SOM mineralization, and complementarily, the greater contribution of microbial necromass to SOM stabilization. Moreover, the decomposition dynamics of neutral sugars and lignin were strongly influenced by C availability. In the low-C soil, these two types of compounds decomposed with similar temporal patterns and extents, and such substrate co-metabolism was dominantly mediated by actinomycetes. In contrast, in the high-C soil, a lower oxidases-to-carbohydrolases ratio regulated the sequential decomposition of labile neutral sugars followed by recalcitrant lignin. Such microbial substrate selectivity was associated with a shift in microbial community from bacterial dominance toward increased fungal contribution. Overall, our findings underscore the significant interplay between soil C availability and flexible microbial substrate utilization strategy in regulating decomposition of heterogeneous SOM components, as well as their distinct contributions in SOM turnover and stabilization. 

Clostridium perfringens (Cp) is a major enteric pathogen in poultry, threatening both animal health and food safety. This study investigated the protective effects of Lactobacillus reuteri 21 (LR21) administered via in ovo injection against Cp infection in broilers. A total of 360 chicks, previously injected in ovo on embryonic day 18, were randomly allocated to four groups (n=6 replicates, 15 birds each): CON (PBS), Cp (PBS+Cp), IOF (LR21), and IOF-Cp (LR21+Cp). Birds were reared for 21 d. A two-way ANOVA was applied to determine the main and interaction effects for in vivo outcomes and one-way ANOVA for in vitro assays. Significant findings were followed by Tukey’s HSD for pairwise comparisons. Although in ovo injection of LR21 slightly mitigated Cp-induced growth suppression, it significantly increased the jejunal villus height and reduced epithelial apoptosis (P<0.05). LR21 also downregulated pro-inflammatory genes including NOD1, MyD88, NF-κB, and JNK, and inhibited the M1-type macrophage polarization in the jejunum compared to Cp challenge. Regarding gut microbiota, Cp challenge altered β-diversity and enriched Clostridium perfringens, whereas LR21 increased Roseburia, Lactobacillus, and specifically Lactobacillus reuteri. In addition, in ovo injection of LR21 enhanced the production of its signature metabolite, reuterin, in Cp-challenged broilers. In vitro, reuterin suppressed pro-inflammatory cytokines in macrophages and protected intestinal organoids from Cp-induced damage. Mechanistically, reuterin inhibited the TLR4/MAPK/NF-κB signaling pathway and activated the Nrf2/HO-1 pathway thereby alleviating inflammation response in Cp-infected macrophages. Reuterin aslo upregulated genes involved in glutathione metabolism (SLC7A11, GCLC, GCLM, GSR, PRDX6, IDH1) and increased antioxidant enzyme activities, thereby limiting ROS accumulation and cellular death of intestinal organoids. Taken together, these findings demonstrate that in ovo LR21 administration enhances intestinal resilience to Cp infection through reuterin-mediated coordination of effects on both macrophages and intestinal stem cells, leading to the attenuation of inflammatory responses and reinforcement of glutathione-dependent antioxidant defenses.