Campanumoea lancifolia (Roxb.) Merr. is a new plant species with both medicinal and edible values, demonstrating broad prospects for development and utilization. However, as its cultivation area expands, root rot has become increasingly severe. In the fields with severe root rot, the losses can reach up to 40%, accounting for 75% to 90% of the losses caused by all diseases affecting C. lancifolia. Root rot directly leads to declines in fruit yield and quality, affecting the commercial value of the fruits and reducing farmers' incomes. Thus, it is urgent to address the root rot in C. lancifolia. [Objective] To isolate and identify the pathogens causing root rot in C. lancifolia and investigate the inhibitory effects of essential oils extracted from four aromatic medicinal plants on the growth of these pathogens. [Methods] Pathogens were isolated from C. lancifolia plants displaying typical root rot symptoms by the tissue culture method. The pathogens were identified based on morphological and molecular evidence and verified according to Koch's postulates. Essential oils were extracted from four aromatic medicinal plants by steam distillation. The Oxford cup method was employed to examine the inhibitory effects of the essential oils on the pathogens, and the 96-well plate method was used to determine the minimum inhibitory concentrations (MICs) of the essential oils. [Results] Four pathogenic strains were isolated and identified from the roots of diseased C. lancifolia plants. Re-inoculation of these pathogens induced root rot symptoms consistent with those observed in the field. The pathogens were identified as Fusarium oxysporum, Fusarium solani, Colletotrichum liriopes, and Stagonosporopsis pogostemonis. The essential oils exhibited strong inhibitory effects on these pathogens, with inhibition rates ranging from 32.94% to 95.29%. Additionally, the MICs of the four essential oils against the pathogens ranged from 0.031 mg/mL to 4.000 mg/mL. [Conclusion] This study demonstrates that F. oxysporum, F. solani, C. liriopes, and S. pogostemonis are pathogenic to C. lancifolia. This is the first report of F. solani, C. liriopes, and S. pogostemonis causing root rot in C. lancifolia. Furthermore, the essential oils extracted from the selected four aromatic plants exhibited strong inhibitory effects on the pathogens causing root rot in C. lancifolia, which coincides with the theory of aromatic plants dispelling pathogens in traditional Chinese medicine. The findings lay a scientific foundation for the development of botanical pesticides against root rot in C. lancifolia and the eco-friendly cultivation of this plant.

[Objective] This study investigated the optimal proportion of green manure replacing chemical fertilizer and its effect on soil fungal community in the paddy field of Ultisol, aiming to achieve soil fertilization and sustainable utilization of Ultisol in southern China. [Methods] This study set seven treatments: no fertilizer (Control), application of Chinese milk vetch without chemical fertilizer in early season rice (G), conventional application of chemical fertilizer in early season rice (NPK100), application of Chinese milk vetch and conventional chemical fertilizer in early season rice (NPK100+G), application of Chinese milk vetch and 80% conventional chemical fertilizer in early season rice (NPK80+G), application of Chinese milk vetch and 60% conventional chemical fertilizer in early season rice (NPK60+G), and application of Chinese milk vetch and 40% conventional chemical fertilizer in early season rice (NPK40+G). The conventional chemical fertilizer was applied in late season rice for other treatments except the Control. The root surface soil samples of different treatments were collected at the maturity stage of late rice for the measurement of soil properties. At the same time, high-throughput sequencing (Illumina MiSeq) was employed to analyze the features of soil fungal community. [Results] Compared with NPK100, the treatments of green manure replacing chemical fertilizer increased the yields of rice and straw. Different treatments significantly altered the soil fungal community composition (P=0.001). Replacing medium and low amounts of chemical fertilizer with green manure increased the relative abundance of saprophytic fungi in soil, which increased the conversion rate of soil organic matter and nutrient turnover rate. Compared with NPK100, replacing 0, 20%, and 40% chemical fertilizer with green manure increased the relative abundance of saprophytic fungi in soil by 33.55%, 167.27%, and 55.28%, respectively. In addition, replacing medium and low amounts of chemical fertilizer with green manure decreased the relative abundance and diversity of potential plant pathogens in soil. [Conclusion] Replacing medium and low amounts (20%–40%) of chemical fertilizer with green manure not only increased rice yield but also reduced environmental pollution, improved soil nutrients, and optimized the fungal community in soil. This study systematically evaluated the effect of replacing different proportions of chemical fertilizer with green manure on the Ultisol paddy ecosystems. The results provided a theoretical basis for the sustainable development of agriculture in the Ultisol region of southern China.

Truffles must be symbiotic with plants to form ectomycorrhiza (ECM), which facilitates the formation of fruiting bodies by mutually beneficial exchanges of substances. [Objective] To elucidate the flow of substances between Tuber sinense and Pinus yunnanensis in a symbiosis relationship. [Methods] Liquid chromatography-mass spectrometry (LC-MS) was employed to analyze the metabolite profiles of freshly harvested T. sinense (SL) and P. yunnanensis roots (SG). [Results] In SL and SG, 1 304 and 1 516 substances were detected respectively, including 399 SL-specific substances (SLSs) and 611 SG-specific substances (SGSs). There were 294 common differential substances, including 93 up-regulated substances (DEMs-up) and 201 down-regulated substances in SL. The network correlation analysis revealed that 92 of the 100 substances with the relative content ≥2.0 (40 SLSs and SGSs and 60 DEMs-up) displayed significant correlations. Twenty-nine associated substances in SLSs and SGSs and 19 substances in DEMs-up were enriched in 15 important pathways, which mainly involved secondary metabolite biosynthesis, amino acid biosynthesis and metabolism, and vitamin biosynthesis and metabolism. [Conclusion] A network correlation existed between T. sinense and P. yunnanensis pine roots metabolites. Some substances in truffles and pine roots, including adenosine 3′, 5′-cyclic monophosphate (cAMP), phytosterol, and indole-3-acetic acid, had regulatory effects on the development of fruiting bodies. The findings provided a theoretical basis for further studying the potential signaling molecules associated with fruiting body development and elucidating the symbiotic mechanism between T. sinense and pines. Additionally, this study laid a practical basis for screening the substances promoting the growth of mycorrhizal seedlings and fruiting bodies and for large-scale artificial cultivation of truffles.

[Objective] Carbon and nitrogen sources play an important role in providing the energy required for fungal growth and increasing secondary metabolite production. This study aims to investigate the effects of carbon and nitrogen restriction on the growth of Inonotus obliquus and the production of triterpenoid secondary metabolites. [Methods] l-glutamine and d-anhydrous glucose were used as the nitrogen and carbon sources, respectively. A nutrient-sufficient medium (0.877 g/L l-glutamine and 20.000 g/L of d-anhydrous glucose) served as the control group (CK). The experimental groups included a nitrogen-limited medium (N-L) with 0.044 g/L l-glutamine and a carbon-limited medium (C-L) with 2.000 g/L d-anhydrous glucose. In the plate experiment, inoculated plates were incubated at 28 ℃ in a constant temperature incubator, and colony diameters were measured and recorded daily. In the shake flask fermentation experiment, samples were collected on the 5th and 10th days to measure biomass, triterpene content, and the expression levels of triterpene synthesis-related genes. [Results] The plate experiment showed that the C-L group had the fastest mycelium extension, but the mycelium was sparse, and the aging was greatly delayed. The N-L group exhibited faster mycelium extension than the CK group but slower than the C-L group, with the fastest mycelium aging. The shake flask fermentation results showed that the N-L group had the highest biomass. qPCR results demonstrated an upregulation trend in most triterpenoid synthase genes under C-L and N-L conditions. GC-MS analysis revealed that both lanosterol and inotodiol content increased in the C-L and N-L groups compared to the CK group. [Conclusion] Nutritional restriction conditions stimulate the accumulation of triterpenes in I. obliquus.

Inhibitors including sugar degradation products (e.g., 5-hydroxymethylfurfural and furfural) and phenols (e.g., 4-hydroxybenzoic acid and vanillin) from lignin degradation are inevitably formed in the pretreatment process of lignocellulose raw materials, exerting a negative impact on the fermentation efficiency. [Objective] To improve the tolerance of yeast to inhibitors in cellulose hydrolysates and ensure the efficient production of industrial biomass ethanol. [Methods] The model strain W303-1A was domesticated with the inhibitor furfural and p-hydroxybenzoic acid alone or in combination. The growth curves and ethanol fermentation performance of the domesticated strain and the original strain were compared under different inhibitor concentrations. We then conducted high-throughput genome resequencing of both the domesticated and original strains to identify the mutations in genes related to the glucose metabolism and drug resistance, thereby analyzing the variation points related to inhibitor tolerance. [Results] In the medium containing 2.0 g/L furfural, the ethanol yield of F-2 was 19.40 g/L, which was 2 times higher than that of the original strain. In the medium containing 1.6 g/L furfural and p-hydroxybenzoic acid, the highest ethanol yield of B-2 was 20.22 g/L, 7.6 times that of the original strain. Then, high-throughput genome resequencing of the original and domesticated strains revealed several mutations in the genes encoding ethanol dehydrogenase, fructose-1, 6-diphosphate aldolase, and pyruvate dehydrogenase in the glucose metabolism pathway. The mutations of YAP1 (transcriptional activator involved in oxidative stress response and REDOX homeostasis), PDR5 (pleiotropic ABC transporter tolerant to multiple chemicals), and RPN4 (zinc finger protein) genes played an important role in the inhibitor tolerance of Saccharomyces cerevisiae. [Conclusion] The findings provide more targets for further optimization and construction of model strains.

[Objective] Epoxide hydrolases (EHs) play a key role in the synthesis of chiral pharmaceuticals. We explored new EHs by engineering or gene retrieval, aiming to enrich and discover more high-performance EHs. [Methods] A novel epoxide hydrolase (Aspergillus carlsbadensis epoxide hydrolase, AcEH) from Aspergillus carlsbadensis was identified by gene retrieval technology. We then used AutoDock2 to predict the key hydrolysis sites of AcEH and employed computational design to clarify the influences of important sites on the structure and catalytic mechanism of AcEH. [Results] The primary structure of the novel EH had three characteristic α/β EH motifs: HGWP, GYTFS, and GGDIGS. AcEH exhibited high activity and could completely hydrolyze styrene oxide (SO) within 15 min, with a specific activity of 13 951 U/g. The K_m, V_max, and k_cat/K_m of AcEH were (107.07±57.98) mmol/L, (37.22±17.85) μmol/(min·mg), and 1.17 mmol/(L·s), respectively. The key hydrolysis sites of AcEH were Asp192-His372-Glu346, which catalyzed the triad, and two conserved tyrosine residues, Tyr251/314. The mutations R49L and R49Y caused enzyme inactivation, while the mutation Y45L resulted in the formation of inactive inclusion bodies. The interaction network revealed that changes in the 49th amino acid residue disrupted the interactions between key active site residues, leading to enzyme inactivation. On the other hand, the alteration of the 45th amino acid residue destabilized the enzyme structure, leading to the formation of inclusion bodies. [Conclusion] This study discovered a novel EH and analyzed its hydrolysis mechanism. The findings provide valuable insights for further research and engineering on this enzyme.

Metagenomics has enriched our understanding about the composition and functions of digestive tract microbiota in animals. Currently, metagenomic sequencing can generally achieve the classification rate of species between 15% and 45% at the read level. Therefore, improving the alignment rate of microbial reads in metagenomics can help to further mine microbial information from metagenome data. [Objective] To enhance the classification ability for digestive tract microbiota in ruminants by extending the Kraken2 standard database, thereby deeply mining the microbial information from metagenome data. [Methods] A total of 14 827 metagenome-assembled genomes (MAGs) of the rumen fluid, feces, and digestive tracts of cattle, sheep, and goats were collected. After quality control and filtering, 3 095 species-level genome bins (SGBs) were retained. These SGBs were integrated into the Kraken2 standard database following taxonomic classification and functional prediction, and the classification effect was evaluated. [Results] In the genome taxonomy database (GTDB), the 3 095 SGBs were identified as bacteria belonging to 782 genera of 28 phyla (3 053 SGBs) and archaea belonging to 8 genera of 2 phyla (42 SGBs). The functional prediction based on eggNOG annotated the SGBs into 26 clusters of orthologous groups of proteins (COGs). The Kyoto encyclopedia of genes and genomes (KEGG) enrichment categorized the top 25 ortholog groups (KO entries) into 14 pathways. The prediction of carbohydrate-active enzymes (CAZy) showed that 593 SGBs were annotated into six classes of CAZymes: auxiliary activities (AA), carbohydrate esterases (CE), glycosyltransferases (GT), carbohydrate-binding modules (CBM), glycoside hydrolases (GH), and polysaccharide lyases (PL). Among them, GH was the most common class. The addition of 3 095 SGBs to the Kraken2 standard database (May 2024) increased the number of species in the database by 5.00%, extending the size from 87.2 Gb to 98.2 Gb. Furthermore, a study about the effect of diet fiber-to-concentrate ratio on the rumen microbiota of Holstein cows by metagenomics was reassessed, which showed that the integration of SGBs into the database raised the species alignment rate of rumen metagenome reads from (19.35±1.81)% to (51.04±2.05)%. The principal component analysis results at the species level indicated that the extended database enhanced the ability to distinguish rumen microbiota structures under two different diet fiber-to-concentrate ratios. The linear discriminant analysis effect size results indicated that the microbial markers for low-fiber and high-fiber diets were Xylanibacter ruminicola and Aristaeella hokkaidonensis, respectively, in the standard database, whereas they were Prevotella sp. 902800365 and Prevotella sp. 900316445, respectively, in the extended database. [Conclusion] In summary, introducing SGBs to extend the Kraken2 standard database can increase species coverage and improve the alignment rate of species at the metagenome read level, thereby enhancing the understanding of microbial information in metagenome data.

Coastal wetlands are crucial blue carbon ecosystems and play an indispensable role in regulating global climate change and preserving biodiversity. Microorganisms are key drivers of biogeochemical cycles, maintaining ecosystem functions and stability by coupling carbon, nitrogen, and sulfur cycles. Synthetic microbial ecology aims to understand microbial interaction mechanisms and environmental applications. In view of the critical issues in coastal wetlands, this paper focuses on the theoretical studies of synthetic microbial ecology and explores the applications of microbiome engineering in reducing greenhouse gas emissions and enhancing carbon sinks, providing scientific and technological support for mitigating global climate change.

[Objective] Cold seeps and hydrothermal fields are typical chemosynthetic ecosystems in the ocean. With distinctive physicochemical properties, they harbor unique microbial communities. Dimethylsulfoniopropionate (DMSP), one of the most abundant organic sulfur-containing compounds on Earth, is synthesized and degraded by a variety of marine bacteria, which plays an important role in driving carbon and sulfur cycles in the ocean. In this study, we isolated and identified DMSP-synthesizing and degrading bacteria from the F-cold seep of the South China Sea and hydrothermal fields of the Okinawa Trough and analyzed their diversity and distribution, aiming to expand the understanding of these bacteria in the ocean. [Methods] Water, sediment, and animal samples were collected at different depths from both the F-cold seep of the South China Sea and the Yaeyama Knoll hydrothermal field of the Okinawa Trough. Three enrichment media (l-methionine addition and high salinity and low nitrogen for DMSP-synthesizing bacteria; DMSP addition for DMSP-degrading bacteria) and the 2216E medium were used for the enrichment and isolation of bacteria. The taxonomic status of strains was determined by 16S rRNA gene sequencing, and the abilities of representative strains to synthesize or degrade DMSP were assessed. [Results] A total of 874 culturable strains were obtained. Gammaproteobacteria emerged as the dominant class in the three media, and Marinobacter was the most abundant genus. The number and diversity of culturable strains obtained from cold seep samples after enrichment were higher than those from the hydrothermal field. The 14 strains of DMSP-synthesizing bacteria from the cold seep belonged to 7 genera, including 5 Thalassospira strains carrying the DMSP synthesis gene mmtN and 2 Pseudooceanicola strains carrying dsyB. A total of 130 DMSP-degrading bacterial strains were obtained from the cold seep, belonging to 39 genera, among which Glutamicibacter was the most abundant genus (24 strains) without known genes associated with DMSP degradation. There was only 1 strain of DMSP-synthetizing bacteria and 18 strains of DMSP-degrading bacteria from the hydrothermal field, both were much fewer than those from the cold seep. The strains with DMSP cleavage pathway accounted for 98.6% of the total DMSP-degrading strains (148), among which 55 strains had strong cleavage activity and were mainly Actinobacteria. Among the 40 strains with strong DMSP-degrading activity, 9 strains contained known cleavage genes and 3 strains contained known demethylation genes. [Conclusion] Abundant DMSP-synthesizing and -degrading bacteria exist in F-cold seep of the South China Sea and hydrothermal fields of the Okinawa Trough, including a variety of bacterial groups carrying potential novel DMSP synthesis/degradation genes. This study provides a basis for further understanding the microbial-driven organosulfur cycling in chemosynthetic ecosystems.