Pine wilt disease is a devastating forest disease caused by Bursaphelenchus xylophilus infection. The associated bacterium Pseudomonas fluorescens and its flagellin protein have been implicated in this process, but the role of the flagellin-encoding gene fliC in pathogenesis remains unclear. Current gene knockout strategies in P. fluorescens are limited by low transformation efficiency and cumbersome procedures, which constrain functional studies of this gene. To address this methodological gap, we established a fliC knockout method based on the pT18sacB-sacB counterselection system. Short homologous arms were constructed by splice overlap extension PCR, and the knockout mutant ΔfliC was obtained through S17-1 λpir-mediated conjugation and sucrose counterselection. The mutant was verified by junction PCR, internal PCR, and sequencing. Using Pinus massoniana callus as the experimental material, we preliminarily compared the wild-type strain and the ΔfliC mutant in individual pathogenicity and in promoting complex infection by B. xylophilus. The ΔfliC mutant showed delayed symptom onset and lower disease severity than the wild type, and its ability to promote complex infection also appeared reduced. These results provide preliminary evidence that fliC contributes to symptom development in this callus assay. The established fliC knockout technique provides a methodological basis for further investigating the role of associated bacteria in pine wilt disease.

Scirpus × mariqueter (Tang & F.T.Wang) Tatanov, which is endemic to eastern estuaries in China, is a tidal zone-engineering species with great promise for managing greenhouse gases and enhancing ecosystem resilience against invasive species. Although S. mariqueter is widely recognized as a hybrid species derived from Bolboschoenus planiculmis (F. Schmidt) T.V. Egorova and Schoenoplectus triqueter (L.) Palla, its speciation remains highly controversial. The lack of a reference genome is the major cause of this ambiguity. We generated the first chromosome-level genome assembly for S. mariqueter combining PacBio long-reads, Illumina short-reads, and the Hi-C method. The genome assembly consisted of 227.75 Mb (contig N50: 3.89 Mb). We also constructed a haploid karyotype comprising 54 pseudochromosomes. The average size of these pseudochromosomes was small (4.05 Mb). Thirty-two pseudochromosomes were assembled to a telomere to telomere level. Repetitive elements represented approximately 54.12% of the genome. We predicted and annotated 25,239 protein-coding genes. The overall BUSCO score was 95.10%, with notably few duplicated genes (1.70%). This high-quality genome provides critical data for future studies.

Schoenoplectus tabernaemontani (C. C. Gmelin) Palla is a typical macrophyte in diverse wetland ecosystems. This species holds great potential in decontamination applications and carbon sequestration. Previous studies have shown that this species may have experienced recent polyploidization. This would make S. tabernaemontani a unique model to study the processes and consequences of whole-genome duplications in the context of the well-documented holocentric chromosomes and dysploidy events in Cyperaceae. However, the inference was not completely solid because it lacked homology information that is essential to ascertain polyploidy. We present here the first chromosome-level genome assembly for S. tabernaemontani. By combining Oxford Nanopore Technologies (ONT) long reads and Illumina short reads, plus chromatin conformation via the Hi-C method, we assembled a genome spanning 507.96 Mb, with 99.43% of Hi-C data accurately mapped to the assembly. The assembly contig N50 value was 3.62 Mb. The overall BUSCO score was 94.40%. About 68.94% of the genome was comprised of repetitive elements. A total of 36,994 protein-coding genes were predicted and annotated. Long terminal repeat retrotransposons accounted for ∼26.99% of the genome, surpassing the content observed in most sequenced Cyperid genomes. Our well-supported haploid assembly comprised 21 pseudochromosomes, each harboring putative holocentric centromeres. Our findings corroborated a karyotype of 2n = 2X = 42. We also confirmed a recent whole-genome duplication occurring after the divergence between Schoenoplecteae and Bolboschoeneae. Our genome assembly expands the scope of sequenced genomes within the Cyperaceae family, encompassing the fifth genus. It also provides research resources on Cyperid evolution and wetland conservation.

Bolboschoenus planiculmis (F.Schmidt) T.V.Egorova is a typical wetland plant in the species-rich Cyperaceae family. This species contributes prominently to carbon dynamics and trophic integration in wetland ecosystems. Previous studies have reported that the chromosomes of B. planiculmis are holocentric; i.e. they have kinetic activity along their entire length and carry multiple centromeres. This feature was suggested to lead to a rapid genome evolution through chromosomal fissions and fusions and participate to the diversification and ecological success of the Bolboschoenus genus. However, the specific mechanism remains uncertain, partly due to the scarcity of genetic information on Bolboschoenus. We present here the first chromosome-level genome assembly for B. planiculmis. Through the integration of high-quality long-read and short-read data, together with chromatin conformation using Hi-C technology, the ultimate genome assembly was 238.01 Mb with a contig N50 value of 3.61 Mb. Repetitive elements constituted 37.04% of the genome, and 18,760 protein-coding genes were predicted. The low proportion of long terminal repeat retrotransposons (similar to 9.62%) was similar to that reported for other Cyperaceae species. The Ks (synonymous substitutions per synonymous site) distribution suggested no recent large-scale genome duplication in this genome. The haploid assembly contained a large number of 54 pseudochromosomes with a small mean size of 4.10 Mb, covering most of the karyotype. The results of centromere detection support that not all the chromosomes in B. planiculmis have multiple centromeres, indicating more efforts are needed to fully reveal the specific style of holocentricity in cyperids and its evolutionary significance.

Objective To use genomic analysis to identify Acinetobacter spp. and to explore the distribution characteristics of ß-lactamase oxallicinases (blaOXA) among Acinetobacter species globally. Methods Genomes of global Acinetobacter spp. were downloaded from GenBank using Aspera batch. After quality check using CheckM and QUAST software, the genomes were annotated using Prokka software to investigate the distribution of blaOXAs across Acinetobacter spp.; a phylogenetic tree was constructed to explore the evolutionary relationship among the blaOXA genes in Acinetobacter spp. Average-nucleotide identification (ANI) was performed to re-type the Acinetobacter spp. BLASTN comparison analysis was implemented to determine the sequence type (ST) of Acinetobacter baumannii strain. Results A total of 7,853 genomes were downloaded, of which only 6,639 were left for further analysis after quality check. Among them, 282 blaOXA variants were identified from the genomes of 5,893 Acinetobacter spp.; blaOXA-23 (n = 3,168, 53.8%) and blaOXA-66 (2,630, 44.6%) were the most frequent blaOXAs, accounting for 52.6% (3,489/6639), and the co-carriage of blaOXA-23 and blaOXA-66 was seen in 2223 (37.7%) strains. The 282 blaOXA variants were divided into 27 clusters according to the phylogenetic tree. The biggest clade was blaOXA-51-family carbapenem-hydrolyzing enzymes composed of 108 blaOXA variants. Overall, 4,923 A. baumannii were identified out of the 6,639 Acinetobacter spp. strains and 291 distinct STs were identified among the 4,904 blaOXA-carrying A. baumannii. The most prevalent ST was ST2 (n = 3,023, 61.6%) followed by ST1 (n = 228, 4.6%). Conclusion OXA-like carbapenemases were the main blaOXA-type β-lactamase spread widely across Acinetobacter spp. Both blaOXA-23 and blaOXA-66 were the predominant blaOXAs, among all A. baumannii strains, with ST2 (belonging to CC2) being the main clone disseminated globally.

Abstract Kobresia species are common in meadows on the Qinghai–Tibet Plateau. They are important food resources for local livestock, and serve a critical foundation for ecosystem integration. Genetic resources of Kobresia species are scarce. Here, we generated a chromosome-level genome assembly for K. myosuroides (Cyperaceae), using PacBio long-reads, Illumina short-reads, and Hi–C technology. The final assembly had a total size of 399.9 Mb with a contig N50 value of 11.9 Mb. The Hi–C result supported a 29 pseudomolecules model which was in consistent with cytological results. A total of 185.5 Mb (44.89% of the genome) transposable elements were detected, and 26,748 protein-coding genes were predicted. Comparative analysis revealed that Kobresia plants have experienced recent diversification events during the late Miocene to Pliocene. Karyotypes analysis indicated that the fission and fusion of chromosomes have been a major driver of speciation, which complied with the lack of whole-genome duplication (WGD) in K. myosuroides genome. Generally, this high-quality reference genome provides insights into the evolution of alpine sedges, and may be helpful to endemic forage improvement and alpine ecosystem preservation.

The marine pathogen Vibrio parahaemolyticus has caused huge economic losses to aquaculture. Flagellin is a key bacterial virulence factor that induces an inflammatory response via activation of Toll-like receptor 5 (TLR5) signaling. Herein, to explore the inflammatory activity of V. parahaemolyticus flagellins (flaA, flaB, flaC, flaD, flaE, and flaF), we investigated their ability to induce apoptosis in a fish cell line. All six flagellins induced severe apoptosis. Moreover, treatment with V. parahaemolyticus flagellins increased TLR5 and myeloid differentiation factor 88 (MyD88) expression and the production of TNF-α and IL-8 significantly. This indicated that flagellins might induce a TLR5-meditated immune response via an MyD88-dependent pathway. FlaF exhibited the strongest immunostimulatory effect; therefore, the interaction between TLR5 and flaF was screened using the yeast two-hybrid system. A significant interaction between the two proteins was observed, indicating that flaF binds directly to TLR5. Finally, the amino acids that participate in the TLR5-flaF interaction were identified using molecular simulation, which indicated three binding sites. These results deepen our understanding of the immunogenic properties of flagellins from V. parahaemolyticus, which could be used for vaccine development in the future.

Eria lasiopetala (Willd.) Ormerod is an important ornamental plant and traditional Chinese medicine resource, but it is currently listed as a class II protected species due to its few resources in China. To further clarify the taxonomic status as well as provide genetic information for its conservation, the chloroplast genome of E. lasiopetala was assembled and characterized in this study. Results show that the genome is 158,740 bp in length, including a large single copy, two inverted repeats, and a small single copy, their lengths are 88,062, 26,213, and 18,252 bp, respectively. There are 132 genes in the chloroplast genome, among which the number of encoding protein genes, rRNA genes, and tRNA genes are 86, 8, and 38, respectively. The phylogenetic tree clearly shows that E. lasiopetala and Eria corneri cluster together with 100% support, rather than with Dendrobium group.

Agrimonia pilosa var. nepalensis (D. Don) Nakai is an herbaceous species of Rosaceae distributed in China. It has ornamental and ecological values. Lack of genetic background seriously hinders its further research and utilization. To provide genetic information for further study of it, complete chloroplast (cp) genome was characterized in this study. The genome is a circular molecule of 155,147 bp in length with overall GC content of 36.9%, which contains 85 protein-coding genes, eight ribosomal RNA genes, and 37 transfer RNA genes. It contains a typical tetrad structure, including a large single copy, a small single copy, and two inverted repeat regions. Phylogenetic analysis revealed that A. pilosa var. nepalensis and A. pilosa are closely related. Result of this study could provide genetic information for further research of A. pilosa var. nepalensis.

With the environmental problems brought about by the excessive use of fertilizers and pesticides, numerous biological amendments have been developed and used in recent years. This study, through in-depth analysis of the effects of two different microbial amendments on the microbial diversity in the garlic rhizosphere, provides a theoretical basis and data support for farmers to select microbial amendments. In the experiment, two different microbial amendments were applied to garlic, and its rhizosphere soil was collected after 10, 20, 30, and 40 days. The polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) was used to analyze bacterial diversity in the garlic rhizosphere soil. The total abundance and diversity of microbial flora in the rhizosphere soil of garlic increased after application of microbial amendments to soil or plants. Plant growth was significantly better in the soil treatment than the plant treatment and the water control. The two dominant bacteria of uncultured gamma proteobacterium and Uncultured Gemmatimonadete existed only in the soil treatment. After 40 day of treatment, the abundance in the rhizosphere soil of these four bacterial strains (Uncultured Bacillus sp. clone D.an-22, Mesorhizobium sp., uncultured gamma proteobacterium, and Pseudomonas boreopolis) was greater in the soil than the plant treatment and the control. The irrigation of microbial amendments not only influenced the structure and abundance of rhizosphere microorganisms, and also promoted the colonization by microorganisms. The five bacterial strains of uncultured gamma proteobacterium, Uncultured Gemmatimonadetes, Uncultured Bacillus sp. clone D.an-22, Mesorhizobium sp., Pseudomonas boreopolis could facilitate the growth and enhance resistance of garlic, establishing a foundation for the use of microbial amendments and providing new ideas and methods for environmental management and protection.

As one of the major forest diseases in the world today, Bursaphelenchus xylophilus is difficult to diagnose and treat, causing huge economic losses. At present, preliminary studies have been made on the early diagnosis of Bursaphelenchus xylophilus through changes in the structure of the microbial flora. However, according to the complex wild growing environment, there are still no related scholars to study and report the differences in the microbial flora structure with different degrees of disease and different sampling locations. In the experiment of this study, by analyzing bacteria in different ecological tissue parts of different disease levels of Pinus massoniana in the field with high-throughput sequencing technology, it was found that the structures of the in vivo flora of healthy Pinus massoniana and different parts of the diseased pine affected by pine wood nematode were significantly different. The results showed that the bacteria in healthy Pinus massoniana was mainly composed of unidentified Oxyphotobacteria, Sphingomonas leidyi, Beijerinckia sp M016010, Methylobacterium komagatae, and unknown bacteria. After inoculation with pine wood nematodes, the bacterial flora diversity in the Pinus massoniana samples increased with the increase of the infection time and disease level. Along with the sharp decrease in unidentified Oxyphotobacteria, the bacteria abundance of Sphingomonas leidyi, Paradoxia multiseta, Luteibacter rhizovicinus, Burkholderia sp symbiont of Dicranocephalus medius, Elliptochloris bilobata, Beijerinckia sp M016010, Clostridium disporicum, Methylobacterium komagatae and Amantichitinumursilacus all increased. With horizontal comparison of three different tissue niches of Pinus massoniana, it was found that during the pinewood nematode infestation, the flora structures of pine needles and pine branches were similar, showing a sharp decrease in the abundance of unidentified Oxyphotobacteria, but a significant increase in the abundance of Paradoxia multiseta, Burkholderia sp symbiont of Dicranocephalus medius, Beijerinckia sp M016010, and unknown bacteria. However, the structural diversity of pine stems was slightly different, which was mainly manifested by a significant increase in the abundance of Clostridium disporicum, Luteibacter rhizovicinus, and unknown bacteria. At the same time, after different tissue parts were infected by pine wood nematode, the flora structure of Pinus massoniana changed significantly when the first-level disease characterization occurred. From the diversity and richness of the detected flora structure, it can be seen that the bacterial flora structures of pine branches and pine needlesare similar, and their bacteria abundancesare higher than that of pine stems, and the abundance of the pine branches is the highest. By analyzing the differences in the bacterial flora structure in the three major ecological nichesofdiseased Pinus massoniana with different levels of Bursaphelenchus xylophilus, this study provides data support for the further use of the differences in microbial flora structure to diagnose Bursaphelenchus xylophilus in Pinus massoniana. Meanwhile, it provides technical guidance at the selection of sampling and detecting sites.

With the environmental and ecological security problems caused by the continuous use of chemical fertilizers and pesticides, more and more biological agents have been developed and used in recent years. Through in-depth analysis of the effects of two kinds of biological agents on microbial diversity in the rhizosphere of pepper seedlings, this study exploresprobiotic strains that promote growth and disease resistance, and provides some theoretical basis and data support for farmers to purchase and select biological agents. In the experiment of this study, two different kinds of biological agent were applied to the roots of pepper seedlings, whose rhizosphere soil was collected after 10 days, 20 days, 30 days and 40 days respectively. The total DNA, of bacteria in rhizosphere soil was extracted by soil genomic DNA extraction kit with magnetic bead method. Besides, the experiment used polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique to analyze the effects of two biological agents on bacterial diversity in the rhizosphere soil of pepper seedlings. The results showed that the total abundance and species of microflora in the rhizosphere soil of pepper seedlings irrigated with Bacillus subtilis and Acinetobacter sp. increased, while the plant growth in the Bacillus subtilis treatment group and Acinetobacter sp. treatment group was significantly better than that of the blank control group, and the Bacillus subtilis treatment group was the best. It was further found that Uncultured bacterium clone (JF2360 35), Bacillus firmus OrfA gene (U61539), Uncultured Gemmatimonadete (AY922177), and Uncultured methylibium sp. (KF469187) these four kinds of bacteria exist alone in Acinetobacter sp. treatment group, while Uncultured pseudomonas sp. (JQ279040) existed separately in the Bacillus subtilis treatment group. At the same time, it was found that the abundance of Bacillus firmus strain sctcc471(HQ622343), Uncultured bacterium clone (FJ719098), and Uncultured alpha proteobacterium (KM978288) in rhizosphere soil of Bacillus subtilis treatment group was much higher than that of Acinetobacter sp.treatment group and clear water control group on the 40th day.To sum up, root irrigation with biological agents of Bacillus subtilis and Acinetobacter sp. can affect the structure and abundance of microorganisms in crop rhizosphere and promote the colonization of microflora and the growth of pepper seedlings to a certain extent. The high abundance of five bacterial strains, Acinetobacter sp. (AM295822), Uncultured pseudomonas sp. (JQ279040),Bacillus firmus strain sctcc471(HQ622343),Uncultured bacterium clone(FJ719098),and Uncultured alpha proteobacterium (KM978288), may play a positive role in promoting the growth of pepper seedlings, which lays a foundation for further research and development of biological agents, and provide new ideas and methods for environmental control and ecological security protection.