a-toxin-based-counter-selection-system-for-markerless-gene-deletion

A Toxin-Based Counter-Selection System for Markerless Gene Deletion and High-Density Tn5 Transposon Mutagenesis in <em>Pectobacterium brasiliense</em>

Pectobacterium brasiliense is a highly pathogenic bacterium responsible for potato tuber soft rot, blackleg, and aerial stem rot. Elucidation of its ...

inner mongolia university

Currently, little is known about the host pathogen interaction between  spp. and citrus roots versus leaves. Therefore, we compared the molecular events occurring in citrus roots and leaves after zoospore inoculation with . We analyzed the physical characteristics and genetic responses to  infection of leaves and roots for susceptible and tolerant citrus rootstocks to examine the potential for leaves to model root responses to  infection. Leaves responded faster and stronger to  infection than roots, and leaves showed greater differential response than roots. In addition to differences in hormonal responses, sugar, phospholipase D (PLD), and phospholipase A (PLA) involvement in the interaction between citrus and  was identified. This work, for the first time, creates a solid  zoospore infection protocol, reports  infection on citrus leaves through stomata, and provides evidence that different host organs respond to the pathogen differentially in timing and magnitude. This work identifies the hormones, sugars, pathogenesis-related genes, PLDs, and PLAs that are involved in the molecular events occurring in citrus under infection of  zoospore, and advances our understanding of the mechanisms underlying the interaction.

 Infection of Citrus Leaves and Host Defense Activation Compared to Root Infection.

First Report of  Causing Blackleg on Potato in Inner Mongolia and Sichuan Province, China.

 spp. are the primary causative agents of aerial stem rot in potatoes in China. A nationwide survey revealed the widespread occurrence of aerial stem rot in the northern, southern, and southwestern cultivation regions, with occurrence rates ranging from 1 to 60%. In total, 36 strains were isolated and identified at the species level using multilocus sequence analysis of six housekeeping genes (, , , , , and ). Genome sequencing was conducted on one representative strain for each species, and further confirmation of their identities was achieved through average nucleotide identity and in silico digital DNA-DNA hybridization analysis. Five  species were identified, namely, , , , , and , with  and  being the most widely distributed. Pathogenicity tests demonstrated that, among the strains isolated in this study and those obtained from other studies,  and  are also the most virulent species. To the best of our knowledge, this is the first nationwide study describing the diversity and distribution of  spp. affecting potatoes in China. The information gathered will be utilized for disease diagnosis and the development of pathogen-specific integrated pest management strategies to protect potato production.

Species Diversity of  spp. Causing Potato Aerial Stem Rot in China.

Bacteriophages have emerged as promising alternatives to pesticides for controlling bacterial pathogens in crops. Among these pathogens, Streptomyces stelliscabiei (syn. S. stelliscabiei) is a primary causative agent of potato common scab (PCS), resulting in substantial global economic losses. The traditional management methods for PCS face numerous challenges, highlighting the need for effective and environmentally friendly control strategies. In this study, we successfully isolated three novel bacteriophages, namely Psst1, Psst2, and Psst4, which exhibited a broad host range encompassing seven S. stelliscabiei strains. Morphological analysis revealed their distinct features, including an icosahedral head and a non-contractile tail. These phages demonstrated stability across a broad range of temperatures (20-50°C), pH (pH 3-11), and UV exposure time (80 min). Genome sequencing revealed double-stranded DNA phage with open reading frames encoding genes for phage structure, DNA packaging and replication, host lysis and other essential functions. These phages lacked genes for antibiotic resistance, virulence, and toxicity. Average nucleotide identity, phylogenetic, and comparative genomic analyses classified the three phages as members of the Rimavirus genus, with Psst1 and Psst2 representing novel species. All three phages efficiently lysed S. stelliscabiei in the liquid medium and alleviated scab symptom development and reduced pathogen abundance on potato slices. Furthermore, phage treatments of radish seedlings alleviated the growth inhibition caused by S. stelliscabiei with no disease symptoms. In soil potted experiments, phages significantly reduced disease incidence by 40%. This decrease is attributed to a reduction in pathogen density and the selection of S. stelliscabiei strains with reduced virulence and slower growth rates in natural environments. Our study is the first to report the isolation of three novel phages that infect S. stelliscabiei as a host bacterium. These phages exhibit a broad host range, and demonstrate stability under a variety of environmental conditions. Additionally, they demonstrate biocontrol efficacy against bacterial infections in potato slices, radish seedlings, and potted experiments, underscoring their significant potential as biocontrol agents for the effective management of PCS.

Isolation, characterization and application of noble bacteriophages targeting potato common scab pathogen Streptomyces stelliscabiei.

Potato soft rot caused by  spp. are devastating diseases of potato which cause severe economic losses worldwide.  is considered as one of the most virulent species. However, the virulence mechanisms and pathogenicity factors of this strain have not been fully elucidated. Here, through pathogenicity screening, we identified two  isolates, SM and DQ, with distinct pathogenicity levels. SM exhibits higher virulence compared to DQ in inducing aerial stem rot, blackleg and tuber soft rot. Our genomic and transcriptomic analyses revealed that SM encodes strain specific genes with regard to plant cell wall degradation and express higher level of genes associated with bacterial motility and secretion systems. Our plate assays verified higher pectinase, cellulase, and protease activities, as well as fast swimming and swarming motility in SM. Importantly, a unique endoglucanase S specific to SM was identified. Expression of this cellulase in DQ greatly enhances its virulence compared to wild type strain. Our study sheds light on possible determinants causing different pathogenicity of  species with close evolutionary distance and provides new insight into the direction of genome evolution in response to host variation and environmental stimuli.

Comparative genomic and transcriptome analyses of two  strains revealed distinct virulence determinants and phenotypic features.

Citrus canker, caused by  subsp.  (Xcc), poses a substantial threat to global citrus production. Current disease control relies on the widespread application of copper-based bactericides. However, the excessive and recurrent use of copper has resulted in adverse outcomes, such as the emergence of copper-resistant strains and detrimental impacts on both the environment and human health. In this study, we demonstrate that propionate effectively inhibits the growth of  and is degradable by soil microbes, making it a promising and economic alternative for citrus canker management. Propionate exerts pleiotropic effects on Xcc by disrupting membrane potential, motility, and the type III secretion system. It acts as a bacteriostatic agent by acidifying the bacterial cytoplasm and disrupting proton homeostasis, which depletes intracellular ATP and induces cell dormancy. Through a Tn5 mutant library screen, we identified a propionate-sensitive mutant with a disrupted  gene, part of the  operon, which is involved in propionate metabolism and regulated by PrpR. PrpR represses the  operon by binding to a palindromic motif in the  promoter region. Our findings highlight propionate as a favorable control agent for citrus bacterial canker, offering effective disease management while mitigating the environmental and health concerns associated with copper-based treatments.IMPORTANCECitrus canker severely impacts citrus production, threatening a major fruit industry. Traditionally, managing this disease has depended on copper-based bactericides, which bring significant downsides, including copper resistance in pathogens and environmental toxicity. This study identifies propionate as a promising alternative to copper treatments for combating citrus canker caused by  subsp. . Propionate offers a dual benefit: it disrupts essential bacterial functions, effectively controlling the pathogen, and it is biodegradable in soil, which reduces environmental impact. Our findings show that propionate acidifies cytoplasm and disrupts critical bacterial processes such as membrane potential and motility, while depleting intracellular ATP and inducing cell dormancy. Propionate, therefore, emerges as an eco-friendly, cost-effective option for sustainable management of citrus bacterial canker, addressing both agricultural and environmental challenges in citrus production.

Propionate serves as a degradable control agent of citrus canker by acidifying cytoplasm and depleting intracellular ATP in .

Differentiating between potato common scab, powdery scab, and the physiological disorder of enlarged corky lenticels is challenging due to their similar visual symptoms. To address this, we propose YOLOv8-ST, an enhanced deep learning model that incorporates the Swin Transformer and Triplet Attention modules to effectively distinguish between these visually similar tuber blemishes.

Article	Year
Infection of Citrus Leaves and Host Defense Activation Compared to Root Infection. Phytopathology\| PubMed ID: 32228377	2020
First Report of Causing Blackleg on Potato in Inner Mongolia and Sichuan Province, China. Plant disease\| PubMed ID: 34739331	2022
Species Diversity of spp. Causing Potato Aerial Stem Rot in China. Plant disease\| PubMed ID: 38347734	2024
Isolation, characterization and application of noble bacteriophages targeting potato common scab pathogen Streptomyces stelliscabiei. Microbiological research\| PubMed ID: 38520838	2024
Comparative genomic and transcriptome analyses of two strains revealed distinct virulence determinants and phenotypic features. Frontiers in microbiology\| PubMed ID: 38800750	2024
Propionate serves as a degradable control agent of citrus canker by acidifying cytoplasm and depleting intracellular ATP in . mBio\| PubMed ID: 40298365	2025
Rapid detection of common scab, powdery scab, and enlarged lenticels in potato tubers using deep learning. Pest management science\| PubMed ID: 41351272	2026

Utpal Handique

Biography

JoVE Journal Publications

Other Publications