A novel virulence gene, cviA1 of Clavibacter michiganensis for necrosis development in the Nicotiana benthamiana plant.

Clavibacter michiganensis is a Gram-positive bacterium that causes diverse disease symptoms in tomatoes and Nicotiana benthamiana, a surrogate host plant, including canker, blister lesions, and wilting. Previously, we reported that C. michiganensis also causes necrosis in N. benthamiana leaves. Here, to identify novel virulence genes of C. michiganensis required for necrosis development in N. benthamiana leaves, we screened 1,862 transposon-inserted mutants and identified a mutant strain that exhibited weak and delayed necrosis, whereas there was no discernible difference in blister lesions, canker, or wilting symptoms. Notably, this mutant caused canker similar to that of the wild-type strain, but caused mild wilting in tomato. This mutant carried a transposon in a chromosomal gene, called Clavibactervirulence gene A1 (cviA1). CviA1 encodes a 180-amino acid protein with a signal peptide (SP) at the N-terminus and two putative transmembrane domains (TMs) at the C-terminus. Interestingly, deletion of the SP or the C-terminus, including the two putative TMs, in CviA1 failed to restore full necrosis in the mutant, highlighting the importance of protein secretion and the putative TMs for necrosis. A paralog of cviA1, cviA2 is located on the large plasmid pCM2 of C. michiganensis. Despite its high similarity to cviA1, the introduction of cviA2 into the cviA1 mutant strain did not restore virulence. Similarly, the introduction of cviA1 into the Clavibacter capsici type strain PF008, which initially lacks cviA1, did not enhance necrosis symptoms. These results reveals that the chromosomal cviA1 gene in C. michiganensis plays an important role in necrosis development in N. benthamiana leaves.

Label-Free Surface-Enhanced Raman Scattering Detection of Fire Blight Pathogen Using a Pathogen-Specific Bacteriophage.

Fire blight is one of the most devastating plant diseases, causing severe social and economic problems. Herein, we report a novel method based on label-free surface-enhanced Raman scattering (SERS) combined with an Erwinia amylovora-specific bacteriophage that allows detecting efficiently fire blight bacteria E. amylovora for the first time. To achieve the highest SERS signals for E. amylovora, we synthesized and compared plasmonic nanoparticles (PNPs) with different sizes, i.e., bimetallic gold core-silver shell nanoparticles (Au@AgNPs) and monometallic gold nanoparticles (AuNPs) and utilized the coffee-ring effect for the self-assembly of PNPs and enrichment of fire blight bacteria. Furthermore, we investigated the changes in the SERS spectra of E. amylovora after incubation with an E. amylovora-specific bacteriophage, and we found considerable differences in the SERS signals as a function of the bacteriophage incubation time. The results indicate that our bacteriophage-based label-free SERS analysis can specifically detect E. amylovora without the need for peak assignment on the SERS spectra but simply by monitoring the changes in the SERS signals over time. Therefore, our facile method holds great potential for the label-free detection of pathogenic bacteria and the investigation of viral-bacterial interactions.

A Putative Apoplastic Effector of Clavibacter capsici, ChpGCc as Hypersensitive Response and Virulence (Hrv) Protein in Plants.

Clavibacter bacteria use secreted apoplastic effectors, such as putative serine proteases, for virulence in host plants and for hypersensitive response (HR) induction in nonhost plants. Previously, we have shown that Clavibacter capsici ChpGCc is important for the necrosis development in pepper (Capsicum annuum) leaves. Here, we determine the function of ChpGCc, along with three paralogous proteins, for HR induction in the apoplastic space of a nonhost plant, Nicotiana tabacum. The full-length and signal peptide-deleted (ΔSP) mature forms of all proteins fused with the tobacco PR1b signal sequence were generated. The full-length and ΔSP forms of ChpGCc and only the ΔSP forms of ChpECc and Pat-1Cc, but none of the ChpCCc, triggered HR. Based on the predicted protein structures, ChpGCc carries amino acids for a catalytic triad and a disulfide bridge in positions like Pat-1Cm. Substituting these amino acids of ChpGCc with alanine abolished or reduced HR-inducing activity. To determine whether these residues are important for necrosis development in pepper, alanine-substituted chpGCc genes were transformed into the C. capsici PF008ΔpCM1 strain, which lacks the intact chpGCc gene. The strain with any variants failed to restore the necrosis-causing ability. These results suggest that ChpGCc has a dual function as a virulence factor in host plants and an HR elicitor in nonhost plants. Based on our findings and previous results, we propose Clavibacter apoplastic effectors, such as ChpGCc, Pat-1Cm, Chp-7Cs, and ChpGCm, as hypersensitive response and virulence (Hrv) proteins that display phenotypic similarities to the hypersensitive response and pathogenicity (Hrp) proteins found in gram-negative bacteria. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Bacteriophage cocktail for biocontrol of soft rot disease caused by Pectobacterium species in Chinese cabbage.

Pectobacterium spp. are necrotrophic plant pathogens that cause the soft rot disease in Chinese cabbage, resulting in severe yield loss. The use of conventional antimicrobial agents, copper-based bactericides, and antibiotics has encountered several limitations, such as bioaccumulation on plants and microbial resistance. Bacteriophages (phages) are considered promising alternative antimicrobial agents against diverse phytopathogens. In this study, we isolated and characterized two virulent phages (phiPccP-2 and phiPccP-3) to develop a phage cocktail. Morphological and genomic analyses revealed that two phages belonged to the Tevenvirinae and Mccorquodalevirinae subfamilies, respectively. The phiPccP-2 and phiPccP-3 phages, which have a broad host range, were stable at various environmental conditions, such as various pHs and temperatures and exposure to ultraviolet light. The phage cocktail developed using these two lytic phages inhibited the emergence of phage-resistant bacteria compared to single-phage treatments in in vitro challenge assays. The phage cocktail treatment effectively prevented the development of soft rot symptom in matured Chinese cabbage leaves. Additionally, the phage cocktail comprising three phages (phiPccP-1, phiPccP-2, and phiPccP-3) showed superior biocontrol efficacy against the mixture of Pectobacterium strains in Chinese cabbage seedlings. These results suggest that developing phage cocktails is an effective approach for biocontrol of soft rot disease caused by Pectobacterium strains in crops compared to single-phage treatments. KEY POINTS: •Two newly isolated Pectobacterium phages, phiPccP-2 and phiPccP-3, infected diverse Pectobacterium species and effectively inhibited the emergence of phage-resistant bacteria. •Genomic and physiological analyses suggested that both phiPccP-2 and phiPccP-3 are lytic phages and that their lytic activities are stable in the environmental conditions under which Chinese cabbage grows. •Treatment using a phage cocktail comprising phiPccP-2 and phiPccP-3 efficiently suppressed soft rot disease in detached mature leaves and seedlings of Chinese cabbage, indicating the applicability of the phage cocktail as an alternative antimicrobial agent.

On-site applicable diagnostic fluorescent probe for fire blight bacteria.

Fire blight is a representative plant infection that contaminates edible plants and causes socio-economic problems in agricultural and livestock industries globally. It is caused by the pathogen Erwinia amylovora (E. amylovora) creates lethal plant necrosis and spreads rapidly across plant organs. We newly disclose the fluorogenic probe B-1 for real-time on-site detection of fire blight bacteria for the first time. B-1 exhibited no emission signals but manifested bright emission properties in the presence of fire blight bacteria. Based on these features, fluorescence imaging of the fire blight bacteria and its real-time detection from the infected host plant tissues were conducted. The detection limit against E. amylovora was 102 CFU/mL, which had excellent sensitivity. The fluorogenic probe-based on-site diagnostic technology was supplemented by introducing a new portable UV device. This work holds enormous potential to be a new advanced tool for detecting fire blight in agricultural and livestock industries.

Developing Core Elements and Checklist Items for Implementing Antimicrobial Stewardship Programs in Korean General Hospitals: A Modified Delphi Survey.

BACKGROUND: Antimicrobial stewardship programs (ASPs) aim to optimize antimicrobial use by minimizing the spread of antimicrobial resistance. The core elements for implementing ASPs in healthcare facilities have been developed by the World Health Organization, international research group and government agencies of various countries. However, to date, there is no documented core elements for implementation of ASP in Korea. This survey aimed to establish a national consensus on a set of core elements and their related checklist items for the implementation of ASPs in Korean general hospitals. MATERIALS AND METHODS: The survey was conducted from July 2022 to August 2022 by the Korean Society for Antimicrobial Therapy with support from the Korea Disease Control and Prevention Agency. A literature review was conducted by searching Medline and relevant websites to retrieve a list of core elements and checklist items. These core elements and checklist items were evaluated by a multidisciplinary panel of experts using a structured modified Delphi consensus procedure, using two-step survey included online in-depth questionnaires and in-person meeting. RESULTS: The literature review identified 6 core elements (Leadership commitment, Operating system, Action, Tracking, Reporting, and Education) and 37 related checklist items. Fifteen experts participated in the consensus procedures. Ultimately, all 6 core elements were retained, and 28 checklist items were proposed, all with ≥80% agreement; in addition 9 items were merged into 2 items, 2 items were deleted, and 15 items were rephrased. CONCLUSION: This Delphi survey provides useful indicators for the implementation of ASP in Korea and suggests national policy improvement about the barriers (e.g., shortage of staffing and financial support) existing in Korea for optimal implementation of ASPs.

Visceral obesity as a risk factor of incisional hernia after single-port laparoscopic gynecologic surgery.

OBJECTIVE: We aimed to evaluate associations between abdominal fat distribution (AFD) parameters and incisional hernia (IH) in patients who underwent transumbilical single-port laparoscopic surgery (SPLS) for gynecological disease. METHODS: Medical records of 2116 patients who underwent SPLS for gynecological disease at Daejeon St. Mary's Hospital between March 2014 and February 2021 were reviewed. Among 21 (1.0%) patients who developed IH requiring surgical treatment after SPLS, 18 had preoperative abdominopelvic computed tomography (CT) images. As a control group, we randomly selected 72 patients who did not develop IH and who had undergone preoperative abdominopelvic CT scan, matched to test patients by type of surgery. Total fat area (TFA), visceral fat area (VFA), subcutaneous fat area (SFA), visceral-to-subcutaneous fat ratio (VSR), and waist circumference (WC) were measured at the level of the third lumbar vertebral body on the preoperative abdominopelvic CT images, using National Institutes of Health (NIH) ImageJ version 1.53 k. RESULTS: Receiver operating curve analysis showed that VFA has the highest predictive value for IH among AFD parameters (AUC = 0.749, 95% CI 0.630-0.869, p < 0.001). Univariate analysis showed that age, BMI, hypertension, dyslipidemia, TFA, VFA, VSR and WC were significant factors for IH. In multivariate analysis, only high VFA was identified as an independent risk factor for IH (HR 6.18, 95% CI 1.13-33.87, p = 0.04), whereas BMI, TFA, SFA, VSR, and WC failed to show statistical significance. CONCLUSION: We could find high VFA as an independent risk factor of IH in patients who underwent SPLS for gynecologic disease.

Advanced parental age is an independent risk factor for term low birth weight and macrosomia.

We aimed to investigate association between parental age and the risks of term low birth weight and macrosomia. This was a retrospective cohort study using a national database including 2,245,785 term singleton live births with complete parental age data. Old parental age was defined as 35 years or older. Odd ratios (OR) for term low birth weight and macrosomia were analyzed using univariate and multivariate logistic regression analysis. Neonatal sex, maternal occupation, parity, nationality, age, and paternal age were significant factors of term low birth weight and macrosomia, in univariate analysis. In multivariate analysis, old maternal age (≥35 years old) showed increased odds of term low birth weight and macrosomia (aOR = 1.122, 95% CI: 1.083 -1.162; and aOR = 1.166, 95% CI: 1.143 - 1.189, respectively). Similarly, old paternal age (≥35 years old) showed increased odds of term low birth weight and macrosomia (aOR = 1.090, 95% CI: 1.058 -1.122; and aOR = 1.101, 95% CI: 1.083 - 1.119, respectively). Maternal education that lasted more than 12 years had reduced odds of term low birth weight and macrosomia (OR = 0.817, 95% CI: 0.792 -0.842; and OR = 0.894, 95% CI: 0.879 - 0.91, respectively). Paternal education that lasted more than 12 years also had reduced odds of term low birth weight and macrosomia (OR = 0.865, 95% CI: 0.84 -0.892; and OR = 0.897, 95% CI: 0.881 - 0.913, respectively). This study suggests that not only maternal age but also paternal age are significantly associated with term low birth weight and macrosomia. In addition, parental education levels are also associated with term low birth weight and macrosomia.

Nicotiana benthamiana, a Surrogate Host to Study Novel Virulence Mechanisms of Gram-Positive Bacteria, Clavibacter michiganensis, and C. capsici in Plants.

Clavibacter michiganensis is a Gram-positive bacterium that causes bacterial canker and wilting in host plants like tomato. Two major virulence genes encoding a cellulase (celA) and a putative serine protease (pat-1) have been reported. Here we show that Nicotiana benthamiana, a commonly used model plant for studying molecular plant-pathogen interactions, is a surrogate host of C. michiganensis and C. capsici. When a low concentration of two Clavibacter species, C. michiganensis and C. capsici, were infiltrated into N. benthamiana leaves, they caused blister-like lesions closely associated with cell death and the generation of reactive oxygen species and proliferated significantly like a pathogenic bacterium. By contrast, they did not cause any disease symptoms in N. tabacum leaves. The celA and pat-1 mutants of C. michiganensis still caused blister-like lesions and cankers like the wild-type strain. When a high concentration of two Clavibacter species and two mutant strains were infiltrated into N. benthamiana leaves, all of them caused strong and rapid necrosis. However, only C. michiganensis strains, including the celA and pat-1 mutants, caused wilting symptoms when it was injected into stems. When two Clavibacter species and two mutants were infiltrated into N. tabacum leaves at the high concentration, they (except for the pat-1 mutant) caused a strong hypersensitive response. These results indicate that C. michiganensis causes blister-like lesions, canker, and wilting in N. benthamiana, and celA and pat-1 genes are not necessary for the development of these symptoms. Overall, N. benthamiana is a surrogate host of Clavibacter species, and their novel virulence factors are responsible for disease development in this plant.

Relationship between metabolic syndrome and follicle-stimulating hormone in postmenopausal women.

ABSTRACT: Depletion of ovarian reserve during menopausal transition raises follicle-stimulating hormone (FSH) markedly and menopause is related to an increased risk for metabolic syndrome (MetS). This study examined the relationship between FSH and MetS in postmenopausal women.We evaluated the anthropometric values, lipid profiles, high-sensitivity C-reactive protein (hs-CRP) level, Homeostasis model assessment for insulin resistance (HOMA-IR), and serum adipokines levels in 219 postmenopausal women. Serum FSH and estradiol levels were significantly lower in the MetS group than in the non-MetS group. An inverse correlation was observed between FSH with body fat mass (BFM), and HOMA-IR, and a positive correlation was found between FSH and adiponectin level after adjustment for age, years since menopause, BMI, and serum estradiol.The odds ratio for MetS was higher significantly in the lowest quartile of FSH level than the highest quartile of FSH level (odd ratio = 1.32, 95% CI = 1.09-1.75). Our study showed an increased FSH level favored insulin sensitivity with a higher adiponectin and lower HOMA-IR as well as a lower incidence of MetS in postmenopausal women.These findings suggest a new approach to the role of FSH for regulating energy metabolism and for use as a biomarker of MetS risk in postmenopausal women.This systematic review is based on published researches, so there is no ethical approval required.

An Apoplastic Effector Pat-1Cm of the Gram-Positive Bacterium Clavibacter michiganensis Acts as Both a Pathogenicity Factor and an Immunity Elicitor in Plants.

Clavibacter michiganensis, a Gram-positive plant-pathogenic bacterium, utilizes apoplastic effectors for disease development in host plants. Here, we determine the roles of Pat-1Cm (a putative serine protease) in pathogenicity and plant immunity. Pat-1Cm was found to be a genuine secreted protein, and the secreted mature form did not carry the first 33 amino acids predicted to be a signal peptide (SP). The pat-1Cm mutant impaired to cause wilting, but still caused canker symptom in tomato. Moreover, this mutant failed to trigger the hypersensitive response (HR) in a nonhost Nicotiana tabacum. Among orthologs and paralogs of pat-1Cm , only chp-7Cs from Clavibacter sepedonicus, a potato pathogen, successfully complemented pat-1Cm function in pathogenicity in tomato, whereas all failed to complement pat-1Cm function in HR induction in N. tabacum. Based on the structural prediction, Pat-1Cm carried a catalytic triad for putative serine protease, and alanine substitution of any amino acids in the triad abolished both pathogenicity and HR-inducing activities of Pat-1Cm in C. michiganensis. Ectopic expression of pat-1Cm with an SP from tobacco secreted protein triggered HR in N. tabacum, but not in tomato, whereas a catalytic triad mutant failed to induce HR. Inoculation of the pat-1Cm mutant mixed with the mutant of another apoplastic effector CelA (cellulase) caused severe wilting in tomato, indicating that these two apoplastic effectors can functionally cooperate in pathogenicity. Overall, these results indicate that Pat-1Cm is a distinct secreted protein carrying a functional catalytic triad for serine protease and this enzymatic activity might be critical for both pathogenicity and HR-eliciting activities of Pat-1Cm in plants.

Core Elements for Implementing Antimicrobial Stewardship Programs in Korean General Hospitals.

Currently, antimicrobial resistance (AMR) is a major threat to global public health. The antimicrobial stewardship program (ASP) has been proposed as an important approach to overcome this crisis. ASP supports the optimal use of antimicrobials, including appropriate dosing decisions, administration duration, and administration routes. In Korea, efforts are being made to overcome AMR using ASPs as a national policy. The current study aimed to develop core elements of ASP that could be introduced in domestic medical facilities. A Delphi survey was conducted twice to select the core elements through expert consensus. The core elements for implementing the ASP included (1) leadership commitment, (2) operating system, (3) action, (4) tracking, (5) reporting, and (6) education. To ensure these core elements are present at medical facilities, multiple departments must collaborate as teams for ASP operations. Establishing a reimbursement system and a workforce for ASPs are prerequisites for implementing ASPs. To ensure that ASP core elements are actively implemented in medical facilities, it is necessary to provide financial support for ASPs in medical facilities, nurture the healthcare workforce in performing ASPs, apply the core elements to healthcare accreditation, and provide incentives to medical facilities by quality evaluation criteria.

Primary signet ring cell carcinoma of the uterine cervix: A case report.

RATIONALE: Primary signet ring cell carcinoma of the uterine cervix is extremely rare and the clinical characteristics and prognosis are not well known and there are no specific guidelines for treatment. PATIENT CONCERNS: A 43-year-old woman was referred to our hospital for abnormal uterine bleeding lasting 1 month. DIAGNOSES: Histological examination revealed a signet ring cell carcinoma of the uterine cervix. After evaluation of extragenital origin, the patient was diagnosed International Federation of Gynecology and Obstetrics stage IIIC1 primary signet ring cell carcinoma or the uterine cervix. INTERVENTION: The patient was prescribed concomitant chemo-radiation followed by intracavitary brachytherapy. OUTCOMES: She showed no evidence of disease after treatment but, it recurred after 7 months of last treatment. LESSONS: Different approaches to diagnosis and treatment of this rare disease are needed and molecular pathological studies related to the onset of the disease are required.

Impact of Progesterone on Molecular Mechanisms of Preterm Premature Rupture of Membranes.

The role and mechanisms of progesterone in preterm premature rupture of membranes (PPROM) remains unclear. This study aims to investigate the molecular mechanisms of action of progesterone in pre-labor full-term fetal amniotic membrane cells with and without stimulation by microbial, pro-inflammatory, or thrombogenic agents. Fetal amniotic membranes were collected from 30 women with a normal singleton pregnancy undergoing elective cesarean section at term prior to the onset of labor. The human amniotic epithelial cells isolated were pretreated with and without medroxyprogesterone acetate for 24 h. Then, cells were treated with and without TLR/NLR agonists, pro-inflammatory cytokines, or thrombin for 48 h. Semi-quantitative RT-PCR, Western blot, and caspase-3 activity measurement were performed. Progesterone stimulation decreased the expression of TLR2, TLR5, and Nod2 genes (alone and/or in combination with TLR/NLR agonists) and decreased the expression of IL-1ß and IL-8 genes increased by stimulation with specific agonists for TLR2, TLR4, TLR5, Nod1, and Nod2. Moreover, progesterone decreased thrombin-induced IL-8 gene expression. Progesterone also decreased expression of Bax and Bid proteins (pro-apoptotic factors) increased by stimulation with pro-inflammatory cytokines (TNF-α, NGAL, IL-18, and IL-1ß) and thrombin. Progesterone stimulation alone as well as co-stimulation with TNF-α, NGAL, IL-18, IL-1ß, or thrombin with progesterone either increased, decreased, or did not change the expression of Bcl-2, Bcl-XL, or XIAP genes (anti-apoptotic factors). These data suggest progesterone plays protective roles against PPROM through anti-microbial, anti-inflammatory, and anti-thrombogenic actions on human-term fetal amniotic membrane cells. Progesterone alters pro-inflammatory cytokine- and thrombin-induced apoptosis by controlling the expression of pro-apoptotic and anti-apoptotic factors.

Phage PPPL-1, A New Biological Agent to Control Bacterial Canker Caused by Pseudomonas syringae pv. actinidiae in Kiwifruit.

Pseudomonas syringae pv. actinidiae (Psa) is a Gram-negative bacterium that causes bacterial canker disease in kiwifruit. Copper or antibiotics have been used in orchards to control this disease, but the recent emergence of antibiotic-resistant Psa has called for the development of a new control agent. We previously reported that the bacteriophage (or phage) PPPL-1 showed antibacterial activity for both biovar 2 and 3 of Psa. To investigate the possibility of PPPL-1 to control bacterial canker in kiwifruit, we further tested the efficacy of PPPL-1 and its phage cocktail with two other phages on suppressing disease development under greenhouse conditions using 6 weeks old kiwifruit plants. Our results showed that the disease control efficacy of PPPL-1 treatment was statistically similar to those of phage cocktail treatment or AgrimycinTM, which contains streptomycin and oxytetracycline antibiotics as active ingredients. Moreover, PPPL-1 could successfully kill streptomycin-resistant Psa isolates, of which the treatment of BuramycinTM carrying only streptomycin as an active ingredient had no effect in vitro. The phage PPPL-1 was further characterized, and stability assays showed that the phage was stable in the field soil and at low temperature of 0 ± 2 °C. In addition, the phage could be scaled up quickly up to 1010 pfu/mL at 12 h later from initial multiplicity of infection of 0.000005. Our results indicate that PPPL-1 phage is a useful candidate as a biocontrol agent and could be a tool to control the bacterial canker in kiwifruit by Psa infection in the field conditions.

Biocontrol of Soft Rot Caused by Pectobacterium odoriferum with Bacteriophage phiPccP-1 in Kimchi Cabbage.

Pectobacterium odoriferum has recently emerged as a widely infective and destructive pathogen causing soft-rot disease in various vegetables. Bacteriophage phiPccP-1 isolated from Pyeongchang, South Korea, showed lytic activity against P. odoriferum Pco14 and two other Pectobacterium species. The transmission electron microscopy and genome phylograms revealed that phiPccP-1 belongs to the Unyawovirus genus, Studiervirinae subfamily of the Autographivirinae family. Genome comparison showed that its 40,487 bp double-stranded DNA genome shares significant similarity with Pectobacterium phage DU_PP_II with the identity reaching 98% of the genome. The phiPccP-1 application significantly inhibited the development of soft-rot disease in the mature leaves of the harvested Kimchi cabbage up to 48 h after Pco14 inoculation compared to the untreated leaves, suggesting that phiPccP-1 can protect Kimchi cabbage from soft-rot disease after harvest. Remarkably, bioassays with phiPccP-1 in Kimchi cabbage seedlings grown in the growth chamber successfully demonstrated its prophylactic and therapeutic potential in the control of bacterial soft-rot disease in Kimchi cabbage. These results indicate that bacteriophage phiPccP-1 can be used as a potential biological agent for controlling soft rot disease in Kimchi cabbage.

Comparative Genome Analyses of Clavibacter michiganensis Type Strain LMG7333T Reveal Distinct Gene Contents in Plasmids From Other Clavibacter Species.

Clavibacter michiganensis, a Gram-positive, plant-pathogenic bacterium belonging to Actinobacteria, is a causal agent of bacterial canker in tomatoes. Although LMG7333T is the type strain of C. michiganensis, it has not been used in many studies, probably because of a lack of the complete genome sequence being available. Therefore, in this study, the complete genome sequence of this type strain was obtained, and comparative genome analysis was conducted with the genome sequences of two other C. michiganensis strains and type strains of Clavibacter species, of which their complete genome sequences are available. C. michiganensis LMG7333T carries one chromosome and two plasmids, pCM1 and pCM2, like two other C. michiganensis strains. All three chromosomal DNA sequences were almost identical. However, the DNA sequences of two plasmids of LMG7333T are similar to those of UF1, but different from those of NCPPB382, indicating that both plasmids carry distinct gene content among C. michiganensis strains. Moreover, 216 protein-coding sequences (CDSs) were only present in the LMG7333T genome compared with type strains of other Clavibacter species. Among these 216 CDSs, approximately 83% were in the chromosome, whereas others were in both plasmids (more than 6% in pCM1 and 11% in pCM2). However, the ratio of unique CDSs of the total CDSs in both plasmids were approximately 38% in pCM1 and 30% in pCM2, indicating that the high gene content percentage in both plasmids of C. michiganensis are different from those of other Clavibacter species, and plasmid DNAs might be derived from different origins. A virulence assay with C. michiganensis LMG7333T using three different inoculation methods, root-dipping, leaf-clipping, and stem injection, resulted in typical disease symptoms, including wilting and canker in tomato. Altogether, our results indicate that two plasmids of C. michiganensis carry distinct gene content, and the genome information of the type strain LMG7333T will help to understand the genetic diversity of the two plasmids of Clavibacter species, including C. michiganensis.

Transcriptional Changes of Plant Defense-Related Genes in Response to Clavibacter Infection in Pepper and Tomato.

Pepper and tomato plants infected with two Clavibacter species, C. capsici and C. michiganensis have shown different patterns of disease development depending on their virulence. Here, we investigated how pepper and tomato plants respond to infection by the high-virulent or low-virulent Clavibacter strains. For this, we chose two strains of each Clavibacter species to show different virulence level in the host plants. Although low-virulent strains showed less disease symptoms, they grew almost the same level as the high-virulent strains in both plants. To further examine the response of host plants to Clavibacter infection, we analyzed the expression patterns of plant defense-related genes in the leaves inoculated with different strains of C. capsici and C. michiganensis. Pepper plants infected with high-virulent C. capsici strain highly induced the expression of CaPR1, CaDEF, CaPR4b, CaPR10, and CaLOX1 at 5 days after inoculation (dai), but their expression was much less in low-virulent Clavibacter infection. Expression of CaSAR8.2 was induced at 2 dai, regardless of virulence level. Expression of GluA, Pin2, and PR2 in tomato plants infected with high-virulent C. michiganensis were much higher at 5 dai, compared with mock or low-virulent strain. Expression of PR1a, Osmotin-like, Chitinase, and Chitinase class 2 was increased, regardless of virulence level. Expression of LoxA gene was not affected by Clavibacter inoculation. These results suggested that Clavibacter infection promotes induction of certain defense-related genes in host plants and that differential expression of those genes by low-virulent Clavibacter infection might be affected by their endophytic lifestyle in plants.

Plasmid composition and the chpG gene determine the virulence level of Clavibacter capsici natural isolates in pepper.

The gram-positive bacterial species Clavibacter capsici causes necrosis and canker in pepper plants. Genomic and functional analyses of C. capsici type strain PF008 have shown that multiple virulence genes exist in its two plasmids. We aimed to identify the key determinants that control the virulence of C. capsici. Pepper leaves inoculated with 54 natural isolates exhibited significant variation in the necrosis. Six isolates showed very low virulence, but their population titres in plants were not significantly different from those of the highly virulent isolates. All six isolates lacked the pCM1Cc plasmid that carries chpG, which has been shown to be required for virulence and encodes a putative serine protease, but two of them, isolates 1,106 and 1,207, had the intact chpG elsewhere in the genome. Genomic analysis of these two isolates revealed that chpG was located in the pCM2Cc plasmid, and two highly homologous regions were present next to the chpG locus. The chpG expression in isolate 1,106 was not induced in plants. Introduction of chpG of the PF008 strain into the six low-virulence isolates restored their virulence to that of PF008. Our findings indicate that there are at least three different variant groups of C. capsici and that the plasmid composition and the chpG gene are critical for determining the virulence level. Moreover, our findings also indicate that the virulence level of C. capsici does not directly correlate with bacterial titres in plants.

Natural Variation in Virulence of Acidovorax citrulli Isolates That Cause Bacterial Fruit Blotch in Watermelon, Depending on Infection Routes.

Acidovorax citrulli causes bacterial fruit blotch in Cucurbitaceae, including watermelon. Although A. citrulli is a seed-borne pathogen, it can cause diverse symptoms in other plant organs like leaves, stems and fruits. To determine the infection routes of A. citrulli, we examined the virulence of six isolates (Ac0, Ac1, Ac2, Ac4, Ac8, and Ac11) on watermelon using several inoculation methods. Among six isolates, DNA polymorphism reveals that three isolates Ac0, Ac1, and Ac4 belong to Clonal Complex (CC) group II and the others do CC group I. Ac0, Ac4, and Ac8 isolates efficiently infected seeds during germination in soil, and Ac0 and Ac4 also infected the roots of watermelon seedlings wounded prior to inoculation. Infection through leaves was successful only by three isolates belonging to CC group II, and two of these also infected the mature watermelon fruits. Ac2 did not cause the disease in all assays. Interestingly, three putative type III effectors (Aave_2166, Aave_2708, and Aave_3062) with intact forms were only found in CC group II. Overall, our results indicate that A. citrulli can infect watermelons through diverse routes, and the CC grouping of A. citrulli was only correlated with virulence in leaf infection assays.