Previous studies revealed that the type VI secretion system (T6SS) has an essential role in bacterial competition and virulence in many gram-negative bacteria. However, the role of T6SS in virulence in Pectobacterium atrosepticum remains controversial. We examined a closely related strain, PccS1, and discovered that its T6SS comprises a single-copy cluster of 17 core genes with a higher identity to homologs from P. atrosepticum. Through extensive phenotypic and functional analyses of over 220 derivatives of PccS1, we found that three of the five VgrGs could be classified into group I VgrGs. These VgrGs interacted with corresponding DUF4123 domain proteins, which were secreted outside of the membrane and were dependent on either the T6SS or type IV secretion system (T4SS). This interaction directly governed virulence and competition. Meanwhile, supernatant proteomic analyses with strains defective in the T6SS and/or T4SS confirmed that effectors, such as FhaB, were secreted redundantly to control the virulence and suppress host callose deposition in the course of infection. Notably, this redundant secretion mechanism between the T6SS and T4SS is believed to be the first of its kind in bacteria.

Biological control is an effective and environmentally friendly strategy for managing plant diseases. In this study, a broad-spectrum antagonistic bacterium, designated strain AN6, was isolated from rice plants and exhibited potent inhibitory activity against a variety of phytopathogens. In Oxford cup assays, AN6 suppressed the growth of Xanthomonas oryzae pv. oryzae (Xoo) by 73.60%, and its cell-free culture filtrate caused pronounced morphological deformation in the bacterial cells. Further in vitro assays, including dual-culture assays, volatile organic compound (VOC) assays, and cell-free supernatant (CFS) assays, demonstrated that AN6 also exerted strong antifungal effects against several pathogenic fungi. In addition, the strain was found to produce proteases and siderophores, which may contribute to its antagonistic capabilities. Taxonomic identification based on morphological traits, 16S rRNA and gyrA gene sequencing, average nucleotide identity (ANI), in silico DNA–DNA hybridization (isDDH), and phylogenetic analysis classified strain AN6 as Bacillus velezensis. Whole-genome sequencing revealed that AN6 harbors a 3,929,788 bp genome comprising 4025 protein-coding genes with a GC content of 46.50%. Thirteen biosynthetic gene clusters (BGCs) associated with the production of secondary metabolites—such as nonribosomal peptides, polyketides, and dipeptide antibiotics—were identified. The pot experiment further validated the biocontrol potential of AN6, achieving an 80.49% reduction in rice bacterial blight caused by Xanthomonas oryzae pv. oryzae. Collectively, these results indicate that B. velezensis AN6 is a promising candidate for development as a highly effective biocontrol agent for the integrated management of diverse plant diseases.

Strains from the genus of Pectobacterium can cause soft rot in numerous important plants, leading to significant losses. 16S rRNA gene sequences reliably identify genera, but species identification is sometimes challenging due to indistinguishable species or strains and unrecognized new taxa. Therefore, a dependable and straightforward classification method is needed. In this study, we analyzed the complete genome sequence and predicted genome components of Pectobacterium strains. We also assessed their genetic relationships using average nucleotide identity and in silico DNA–DNA hybridization, alongside phylogenetic analysis. This was done by examining the whole-genome sequences of 14 new strains isolated in our laboratory, responsible for plant soft rot, and comparing them with 64 strains’ genome sequences available in GenBank. The results reveal that three subspecies of P. carotovorum with genome sequences deposited in GenBank (PccS1, PCC21, and strain 67) need to be reclassified as separate species and also confirm that our new isolated strains are accurately categorized at the species level. Additionally, the virulence and adaptability of certain strains of Pectobacterium (PccS1, SCRI1043, and SCC3193) are influenced by horizontal acquisition genes. Furthermore, our findings suggest that the diversity in the car gene cluster among Pectobacterium strains likely stems from gene losing, as well as the auto-induced regulatory mechanisms underlying virulence determinant gene activation, and Car biosynthesis might act in different ways in PccS1, enhancing our knowledge of their genomic traits through comparative studies.

Strains from the genus of Pectobacterium are significant bacterial phytopathogens causing substantial economic losses globally. To understanding molecular pathogenic mechanism of Pectobacterium, genetic manipulation is often the initial work as in many other pathogens. We previously obtained many single gene deletion mutants carrying some different special antibiotic markers using homologous recombination for Pectobacterium. However, many biological processes involve multiple functionally genes, so multiple-gene deletion mutants are needed, but the limitation in types of antibiotic markers have controlled us to do so, even though the antibiotic markers might result in some unexpected effects on our understanding about the pathogenicity. In this study, we firstly introduced Cre-loxP system to construct marker-less multiple-gene deletion mutants in Pectobacterium strains.

Paenibacillus polymyxa is a promising biocontrol agent based on many applications in agriculture. In this study, characterization of a novel strain JX-1 was performed by various comparative phenotypic assays including cell wall-degrading enzyme activity assay, antagonism in vitro and in planta and multi-omics analysis consisting of whole-genome sequencing and metabolomic analysis. The results showed that JX-1 produced protease, cellulase, and pectinase, with protease activity exceeding other P. polymyxa strains. The supernatant and VOCs produced by JX-1 both contain components that effectively antagonize both bacteria and fungi. JX-1 inhibited Fusarium verticillioides, Sclerotinia sclerotiorum, Botrytis cinerea and Sclerotium rolfsii. Furthermore, it significantly reduced the severity of clubroot in Arabidopsis thaliana and Brassica rapa in planta, resulting in an improvement in the total shoot fresh weight. The closely related P. polymyxa strain JX-2 (ANI = 99.96% compared to strain JX-1) was used for comparative phenotyping and multi-omics analysis that implicated extracellular proteases and some small peptides could be essential for clubroot biocontrol. Genomic analysis of strain JX-1 confirmed that it is indeed P. polymyxa (ANI > 95% compared to reference strains). Moreover, JX-1 harbors predicted carbohydrate-active enzymes (CAZymes), 16 extracellular protease genes, and 17 gene clusters with biosynthetic potential (NRPS/PKS hybrids). Metabolomic profiling of the culture supernatant further identified differential accumulation of hydrophobic-amino-acid-containing small peptides, providing a metabolic basis for the observed antagonism and offering leads for future mechanistic studies. These findings establish JX-1 as a broad-spectrum biocontrol agent against clubroot disease.

Bacterial pathogens secrete effector proteins that suppress plant immune responses and facilitate infection. This study focuses on Pectobacterium brasiliense NJAU180, a bacterial pathogen causing severe blackleg disease in potato plants in Inner Mongolia, China. Using exoproteomic analysis, plant-induced extracellular proteins were identified by comparing culture supernatants from P. brasiliense NJAU180 grown in minimal medium (MM) alone and in the presence of aseptically grown potato plantlets at an early growth stage (OD₆₀₀ ≈ 0.5). The results reveal PurK as a novel plant-induced extracellular protein, and deletion of purK markedly reduces virulence. PurK, N⁵-carboxyaminoimidazole ribonucleotide synthetase, is a key enzyme in de novo purine biosynthesis. Its impact on virulence is distinct from the conventional production of plant cell wall–degrading enzymes: PurK promotes motility by modulating transcription of flagellar genes, acting through its three domains as an integrated unit to infect successfully. Extracellularly detected PurK suppresses callose deposition, a PAMP-triggered immunity (PTI)-like defense, while it also triggers a strong hypersensitive response and upregulates expression of PTI marker genes such as PR2 and WRKY7 when secreted into the host plant. Although PurK interacts specifically with PurE, our data indicate that PurK’s pathogenic effects operate independently of purine biosynthesis. This study reveals a reliable experimental model for more accurate assessment of microbe–plant interactions and highlights new functional roles for PurK in P. brasiliense NJAU180 pathogenesis and identifies potential targets for disease control strategies.