Vascular Colonization Research Articles

Contrasting interactions of the aggressive Verticillium longisporum lineage A1/D1 and the non‐aggressive A1/D2 lineage with roots of oilseed rape

AbstractVerticillium longisporum is a soilborne fungal vascular pathogen of Brassica crops. Three hybrid lineages originating from three independent hybridization events of four haploid Verticillium parents are known. V. longisporum lineage A1/D2 has only been found in horseradish and previous pathogenicity tests have shown that it is non‐aggressive on oilseed rape (Brassica napus). The aim of this study was to investigate the interaction of the non‐aggressive lineage A1/D2 with oilseed rape in comparison with the aggressive A1/D1 lineage. Although A1/D2 did not cause severe disease symptoms, the study revealed restricted root infection and systemic colonization by this lineage, as assessed by quantitative PCR of fungal DNA. Assessment of root surface colonization by confocal laser microscopy indicated early sporulation, disorganized loosely attached hyphal growth and hyphal coiling of A1/D2. By contrast, A1/D1 showed organized hyphal growth tight to the root surface, as well as hyphal swellings at points of penetration. Vascular colonization was studied with transmission electron microscopy, which revealed less vessel colonization, higher intensity of vascular responses and lower plant cell wall degradation in A1/D2‐treated plants in comparison with A1/D1. Vascular responses consisted of strong coating of vessel walls and fungal hyphae with an electron‐dense material, indicating higher intensity of plant defence in comparison with A1/D1. The results, for the first time, demonstrate stem colonization and thus a basic level of pathogenic interaction of non‐aggressive strains of A1/D2 with oilseed rape associated with a lack of severe disease symptoms, which indicate tolerance in oilseed rape to lineage A1/D2.

Live-Cell Visualization of Early Stages of Root Colonization by the Vascular Wilt Pathogen Fusarium oxysporum.

Fungal phytopathogens induce a variety of pathogenicity symptoms on their hosts. The soilborne vascular wilt pathogen Fusarium oxysporum infects roots of more than 150 different crop species. Initial colonization stages are asymptomatic, likely representing a biotrophic phase of infection, followed by a necrotrophic switch after vascular colonization which results in blockage of the plant xylem and killing of the host. Live-cell microscopy techniques have been successfully employed to study interaction events during fungal colonization of root tissues. This technique is widely used to track fungal development during disease progression. Here, we describe a well-established protocol for generation and screening of fluorescently tagged F. oxysporum transformants, as well as for live-cell imaging of the early colonization stages of F. oxysporum on tomato (Solanum lycopersicum) seedlings. The presented experimental design and techniques involved are also applicable to other root infecting fungi.

Disease caused by Neofusicoccum parvum in pruning wounds of grapevine shoots and its control by Trichoderma spp. and Xenorhabdus szentirmaii

Neofusicoccum parvum, is a fungal pathogen and one of the etiological agents of dieback disease in grapevines. The fungus causes deterioration of vines due to vascular colonization and/or production of toxins. We report herein the inhibitory effects of Trichoderma spp. isolates and the antifungal effects of cell-free supernatants (CFS) from Xenorhabdus and Photorhabdus bacteria against N. parvum in agar plates. We also evaluated the effects of the most effective fungi and bacteria against the pathogen in pruning wounds of vine shoots. All isolates of Trichoderma exhibited antifungal activity ranging between 82 and 97.5% at 14 days of post-treatment. All Xenorhabdus and Photorhabdus CFS at 10 and 33% concentrations inhibited mycelial growth with X. szentirmaii PAM 11 and PAM 25 causing the highest inhibition (>74%). In the shoot experiments, T. asperellum IB 01/13 and T. asperellum Quality®, X. szentirmaii PAM 11 (undiluted growth culture and CFS) suppressed the fungus by ≥ 93%. Our study highlights the potential of Trichoderma and X. szentirmaii PAM 11 for use as biofungicides in the management of N. parvum in grapevines. Further studies should be conducted to develop formulations of Trichoderma and Xenorhabdus that enhance stability in shelf-life and increase the efficacy of N. parvum control in grapevines under field conditions.

Deciphering Fluid Transport Within Leaf-Inspired Capillary Networks Based on a 3D Computational Model.

Leaf venation provides a promising template for engineering capillary-like vasculature in vitro due to its highly efficient fluid transport capability and remarkable similarities to native capillary networks. A key challenge in exploring the potential biological applications of leaf-inspired capillary networks (LICNs) is to accurately and quantitively understand its internal fluid transport characteristics. Here, a centerline-induced partition-assembly modeling strategy is proposed to establish a 3D computational model, which can accurately simulate the flow conditions in LICNs. Based on the 3D flow simulation, the authors demonstrate the excellent defect-resistant fluid transport capability of LICNs. Interestingly, structural defects in the primary channel can effectively accelerate the overall perfusion efficiency. Flow patterns in LICNs with multiple defects can be estimated by simple superposition of the simulation results derived from the corresponding single-defect models. The 3D computational model is further used to determine the optimal perfusion parameter for the in-vitro formation of endothelialized capillary networks by mimicking native microvascular flow conditions. The endothelialized networks can recapitulate the vascular colonization process and reveal a strong correlation between cancer cell adhesion and flow-induced shear stress. This study offers a quantitative tool to scrutinize the fluid and biological transport mechanisms within LICNs for various biomedical applications.

Reduction of verticillium wilt in tomato by an arbuscular mycorrhizal fungus - Rhizophagus intraradices and an endophytic fungus - Penicillium pinophilum is cultivar dependent

Verticillium wilt is a major concern in vegetable production due to its broad host range, world-wide distribution, vascular colonization, and resilient microsclerotia. This study was conducted to evaluate the growth and tolerance to Verticillium dahliae in two tomato cultivars inoculated with arbuscular mycorrhizal fungi (AMF)- Rhizophagus intraradices 15S-1 and an endophytic fungus- Penicillium pinophilum EU0013. In a randomized factorial layout, seedlings of tomato cultivars (Momotaro, Rodeo) were inoculated with beneficial microbes and V. dahliae pathogen and raised until maturity under greenhouse condition. In vitro dual culture indicated the growth inhibition of V. dahliae by P. pinophilum after one month on PDA media. Root colonization by R. intraradices and P. pinophilum were modulated in response to V. dahliae in a cultivar-dependent manner. While Momotaro showed greater foliar and internal disease symptoms than Rodeo, P. pinophilum and R. intraradices significantly reduced disease incidence, compared to control. Verticillium-induced reductions in root and fruit dry weights in Rodeo and Momotaro were reduced by P. pinophilum, while AMF generally boosted fruit weights. Co-inoculation of R. intraradices and P. pinophilum did not consistently improve host growth indices compared to single inoculation, and modulations in mineral element nutrition by both beneficial microbes in response to V. dahliae was cultivar dependent. In Momotaro, R. intraradices and P. pinophilum increased SlSOD2, SlSOD5 and SlSOD9 gene expression, but this was not generally the case in Rodeo, where lower expression levels were observed. Cultivar-dependent modulation of root colonization, element nutrition, and SlSODs gene expression, by R. intraradices and P. pinophilum underpin their promotion of host growth and tolerance to verticillium wilt.

Colonization of dermal arterioles by Neisseria meningitidis provides a safe haven from neutrophils

The human pathogen Neisseria meningitidis can cause meningitis and fatal systemic disease. The bacteria colonize blood vessels and rapidly cause vascular damage, despite a neutrophil-rich inflammatory infiltrate. Here, we use a humanized mouse model to show that vascular colonization leads to the recruitment of neutrophils, which partially reduce bacterial burden and vascular damage. This partial effect is due to the ability of bacteria to colonize capillaries, venules and arterioles, as observed in human samples. In venules, potent neutrophil recruitment allows efficient bacterial phagocytosis. In contrast, in infected capillaries and arterioles, adhesion molecules such as E-Selectin are not expressed on the endothelium, and intravascular neutrophil recruitment is minimal. Our results indicate that the colonization of capillaries and arterioles by N. meningitidis creates an intravascular niche that precludes the action of neutrophils, resulting in immune escape and progression of the infection.

The endophytic microbiota of Citrus limon is transmitted from seed to shoot highlighting differences of bacterial and fungal community structures

Citrus limon (L.) Burm. F. is an important evergreen fruit crop whose rhizosphere and phyllosphere microbiota have been characterized, while seed microbiota is still unknown. Bacterial and fungal endophytes were isolated from C. limon surface-sterilized seeds. The isolated fungi—belonging to Aspergillus, Quambalaria and Bjerkandera genera—and bacteria—belonging to Staphylococcus genus—were characterized for indoleacetic acid production and phosphate solubilization. Next Generation Sequencing based approaches were then used to characterize the endophytic bacterial and fungal microbiota structures of surface-sterilized C. limon seeds and of shoots obtained under aseptic conditions from in vitro growing seedlings regenerated from surface-sterilized seeds. This analysis highlighted that Cutibacterium and Acinetobacter were the most abundant bacterial genera in both seeds and shoots, while Cladosporium and Debaryomyces were the most abundant fungal genera in seeds and shoots, respectively. The localization of bacterial endophytes in seed and shoot tissues was revealed by Fluorescence In Situ Hybridization coupled with Confocal Laser Scanning Microscopy revealing vascular bundle colonization. Thus, these results highlighted for the first time the structures of endophytic microbiota of C. limon seeds and the transmission to shoots, corroborating the idea of a vertical transmission of plant microbiota and suggesting its crucial role in seed germination and plant development.

Acibenzolar-S-methyl induces protection against the vascular wilt pathogen Fusarium oxysporum in cape gooseberry (Physalis peruviana L.)

Vascular wilt caused by Fusarium oxysporum is one of the most devastating diseases that affect cape gooseberry plants. In order to contribute to its management, the objective of this work was to evaluate the plant activator acibenzolar-S-methyl (ASM) as a reducer of vascular wilt symptoms in cape gooseberry. Sixty-day-old plants were sprayed twice with eleven concentrations of ASM ranging from 10 to 200 mg L−1, respectively. The phytotoxic effect was assessed for a period of eight weeks. The appearance of symptoms such as stunting, a decrease in the shoot and roots dry weight, leaf malformation and defoliation was associated with the ASM concentration. The two lowest concentrations of ASM assessed (10 and 20 mg L−1) were selected to evaluate their effectiveness against F. oxysporum in greenhouse conditions. Both concentrations reduced vascular colonization and necrosis generated by seven isolates of F. oxysporum, and in consequence, the area under the disease progress curve (AUDPC) decreased significantly during the eight weeks of evaluation. These results suggest that ASM induces resistance to F. oxysporum in cape gooseberry under greenhouse conditions.

LTBP3 promotes early metastatic events during cancer cell dissemination.

Latent Transforming Growth Factor β (TGFβ) Binding Proteins (LTBPs) are important for the secretion, activation and function of mature TGFβ, especially so in cancer cell physiology. However, specific roles of the LTBPs remain understudied in the context of the primary tumor microenvironment. Herein, we investigated the role of LTBP-3 in the distinct processes involved in cancer metastasis. By using three human tumor cell lines of different tissue origin (epidermoid HEp-3 and prostate PC-3 carcinomas and HT-1080 fibrosarcoma) and several metastasis models conducted in both mammalian and avian settings, we show that LTBP-3 is involved in the early dissemination of primary cancer cells, namely in the intravasation step of the metastatic cascade. Knockdown of LTBP-3 in all tested cell lines led to significant inhibition of tumor cell intravasation, but did not affect primary tumor growth. LTBP-3 was dispensable in the late steps of carcinoma cell metastasis that follow tumor cell intravasation, including vascular arrest, extravasation and tissue colonization. However, LTBP-3 depletion diminished the angiogenesis-inducing potential of HEp-3 cells in vivo, which was restorable by exogenous delivery of LTBP-3 protein. A similar compensatory approach rescued the dampened intravasation of LTBP-3-deficient HEp-3 cells, suggesting that LTBP-3 regulates the induction of the intravasation-supporting angiogenic vasculature within developing primary tumors. Using our recently developed microtumor model, we confirmed that LTBP-3 loss resulted in the development of intratumoral vessels with an abnormal microarchitecture incompatible with efficient intravasation of HEp-3 carcinoma cells. Collectively, these findings demonstrate that LTBP-3 represents a novel oncotarget that has distinctive functions in the regulation of angiogenesis-dependent tumor cell intravasation, a critical process during early cancer dissemination. Our experimental data are also consistent with the survival prognostic value of LTBP3 expression in early stage head and neck squamous cell carcinomas, further indicating a specific role for LTBP-3 in cancer progression towards metastatic disease.

Vascular Cell Co-Culture on Silk Fibroin Matrix.

Silk fibroin (SF), a natural polymer material possessing excellent biocompatibility and biodegradability, and has been widely used in biomedical applications. In order to explore the behavior of vascular cells by co-culturing on regenerated SF matrix for use as artificial blood vessels, human aorta vascular smooth muscle cells (HAVSMCs) were co-cultured with human arterial fibroblasts (HAFs) or human umbilical vein endothelial cells (HUVECs) on SF films and SF tubular scaffolds (SFTSs). Analysis of cell morphology and deoxyribonucleic acid (DNA) content showed that HUVECs, HAVSMCs and HAFs adhered and spread well, and exhibited high proliferative activity whether cultured alone or in co-culture. Immunofluorescence and scanning electron microscopy (SEM) analysis showed that HUVECs and HAFs co-existed well with HAVSMCs on SF films or SFTSs. Cytokine expression determined by reverse transcription-polymerase chain reaction (RT-PCR) indicated that the expression levels of α-smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (SM-MHC) in HAVSMCs were inhibited on SF films or SFTSs, but expression could be obviously promoted by co-culture with HUVECs or HAFs, especially that of SM-MHC. On SF films, the expression of vascular endothelial growth factor (VEGF) and platelet endothelial cell adhesion molecule-1 (CD31) in HUVECs was promoted, and the expression levels of both increased obviously when co-cultured with HAVSMCs, with the expression levels of VEGF increasing with increasing incubation time. The expression levels of VEGF and CD31 in cells co-cultured on SFTSs improved significantly from day 3 compared with the mono-culture group. These results were beneficial to the mechanism analysis on vascular cell colonization and vascular tissue repair after in vivo transplantation of SFTSs.

Direct Root Penetration and Rhizome Vascular Colonization by Fusarium oxysporum f. sp. cubense are the Key Steps in the Successful Infection of Brazil Cavendish.

Fusarium wilt of banana, which is caused by Fusarium oxysporum f. sp. cubense, is one of the most serious diseases of banana. F. oxysporum f. sp. cubense race 1 (Foc1) and race 4 (Foc4) are the most prevalent pathogens of banana cultivars in the world. To understand the differences in the infection processes between Foc1 and Foc4, green fluorescent protein-tagged strains of F. oxysporum f. sp. cubense tropical race 4 (FocTR4) and Foc1 were used to inoculate 'Brazil Cavendish' banana. At 2 days postinoculation (dpi), it was observed that the spores and hyphae of both Foc1 and Foc4 attached to the root hairs and root epidermis. At 3 dpi, the hyphae of both Foc1 and Foc4 were found in the vascular tissues of roots. However, Foc4 was observed in the parenchymal cells of banana root, whereas Foc1 was not found in parenchymal cells at 7 dpi. Furthermore, few Foc1 hyphae were observed in a few xylems whereas many more Foc4 hyphae were present in many xylems and phloems. Foc4 was observed in the vascular tissues of banana rhizomes, whereas no Foc1 was found in rhizomes 2 months after inoculation. The attachment process in F. oxysporum f. sp. cubense infection was further studied with scanning electron microscopy. Foc4 was observed to penetrate into banana roots from the intercellular space of the epidermis and wounds, whereas Foc1 mainly penetrated from the wounds but not from the intercellular space of the epidermis. Therefore, direct root penetration and rhizome vascular colonization by F. oxysporum f. sp. cubense are the key steps in the successful infection of Brazil Cavendish.

Colonization of Durum Wheat (Triticum turgidum L. var. durum) Culms Exhibiting Premature Senescence (Dead Heads) Associated with Fusarium pseudograminearum Crown Rot.

Fusarium crown rot is a significant disease of durum wheat (Triticum turgidum L. var. durum), which exhibits high levels of disease susceptibility. The most extreme symptom of crown rot is a prematurely senescing culm that typically fails to set grain. Individual crown rot-affected durum wheat plants displaying both nonsenescent and prematurely senescent culms were harvested to compare visual discoloration, Fusarium pseudograminearum biomass, and vascular colonization in culm sections sampled at three different heights above the crown. Field samples of EGA Bellaroi were collected at Wellcamp, QLD, in 2011, 2012, 2013, and 2014, and of Hyperno at Narrabri, NSW, in 2014. Prematurely senescent culms exhibited greater visual discoloration, F. pseudograminearum biomass, and vascular colonization than nonsenescent culms in each year they were examined. The extent of these differences varied between environments and timing of collection in each year. Vascular colonization initially occurred in xylem vessels and spread into phloem tissues as disease severity increased. The increased presence of hyphae in vascular bundles of prematurely senescing culms provides strong evidence for the hypothesis that restriction of water and nutrient movement in a diseased culm is a key factor in crown rot severity.

Vascular plant diversity structures bryophyte colonization in experimental grassland

AbstractQuestionsNumerous grassland biodiversity experiments have explored how plant diversity influences colonization by vascular plants, but no such studies have examined how sown vascular plant diversity structures colonization by bryophytes, which can contribute greatly to grassland diversity.LocationGrassland biodiversity experiment (Jena Experiment), Germany.MethodsWe studied bryophyte composition in experimental grasslands encompassing a gradient of vascular plant species richness (1, 2, 4, 8, 16 and 60 species), functional group richness and composition (one to four; grasses, legumes, tall herbs, small herbs) as well as in additional bare ground and spontaneously colonized plots and semi‐natural control meadows.ResultsIncreasing vascular plant species richness and functional group richness as well as the presence of legume species decreased bryophyte species richness and increased the spatial variability of this richness. Bryophyte species richness and cover responded positively to grass presence, which also decreased the spatial variability of both. The proportion of acrocarpous species was largest in bare ground plots and decreased with increasing vascular plant species richness and with grass and legume presence. Non‐metric multidimensional scaling revealed that bryophyte species composition followed the gradient in sown vascular plant species richness and was dependent on grass and legume presence. The effect of plant diversity on bryophytes was only partly attributable to increased vascular plant species cover. Bryophyte habitat indicator values suggested that conditions in communities of higher vascular plant diversity that included grasses were more shady, moist and nutrient‐rich than in communities with lower vascular plant diversity and without grasses, whereas bryophyte assemblages in communities with legumes indicated well‐lit nutrient‐rich conditions. Even after 6 yr, bryophyte species richness and cover was considerably lower in experimental grasslands than in semi‐natural meadows, suggesting a role for dispersal limitation or habitat filtering.ConclusionsOur study shows that bryophyte colonization is not random, rather it is structured by vascular plant species diversity and composition. Not all components of a plant community respond positively to increased vascular plant diversity and advocating increased richness of one taxonomic group over another in grassland may reduce net species diversity.

Verticillium dahliae transcription factor VdFTF1 regulates the expression of multiple secreted virulence factors and is required for full virulence in cotton.

Fungal transcription factors (TFs) implicated in the regulation of virulence gene expression have been identified in a number of plant pathogens. In Verticillium dahliae, despite its agricultural importance, few regulators of transcription have been characterized. In this study, a T-DNA insertion mutant with significantly reduced virulence towards cotton was identified. The T-DNA was traced to VdFTF1, a gene encoding a TF containing a Fungal_trans domain. Transient expression in onion epidermal cells indicated that VdFTF1 is localized to the nucleus. The VdFTF1-deletion strains displayed normal vegetative growth, mycelial pigmentation and conidial morphology, but exhibited significantly reduced virulence on cotton, suggesting that VdFTF1 is required exclusively for pathogenesis. Comparisons of global transcription patterns of wild-type and VdFTF1-deletion strains indicated that VdFTF1 affected the expression of 802 genes, 233 of which were associated with catalytic processes. These genes encoded 69 potentially secreted proteins, 43 of which contained a carbohydrate enzyme domain known to participate in pathogenesis during infection of cotton. Targeted gene deletion of one VdFTF1-regulated gene resulted in significantly impaired vascular colonization, as measured by quantitative polymerase chain reaction, as well as aggressiveness and symptom severity in cotton. In conclusion, VdFTF1, which encodes a TF containing a Fungal_trans domain, regulates the gene expression of plant cell wall degradation enzymes in V. dahliae, which are required for full virulence on cotton.

Strength of Neisseria meningitidis binding to endothelial cells requires highly-ordered CD147/\u03b22-adrenoceptor clusters assembled by alpha-actinin-4

Neisseria meningitidis (meningococcus) is an invasive bacterial pathogen that colonizes human vessels, causing thrombotic lesions and meningitis. Establishment of tight interactions with endothelial cells is crucial for meningococci to resist haemodynamic forces. Two endothelial receptors, CD147 and the β2-adrenergic receptor (β2AR), are sequentially engaged by meningococci to adhere and promote signalling events leading to vascular colonization, but their spatiotemporal coordination is unknown. Here we report that CD147 and β2AR form constitutive hetero-oligomeric complexes. The scaffolding protein α-actinin-4 directly binds to the cytosolic tail of CD147 and governs the assembly of CD147–β2AR complexes in highly ordered clusters at bacterial adhesion sites. This multimolecular assembly process increases the binding strength of meningococci to endothelial cells under shear stress, and creates molecular platforms for the elongation of membrane protrusions surrounding adherent bacteria. Thus, the specific organization of cellular receptors has major impacts on host–pathogen interaction.

Borrelia burgdorferi Keeps Moving and Carries on: A Review of Borrelial Dissemination and Invasion.

Borrelia burgdorferi is the etiological agent of Lyme disease, a multisystemic, multistage, inflammatory infection resulting in patients experiencing cardiac, neurological, and arthritic complications when not treated with antibiotics shortly after exposure. The spirochetal bacterium transmits through the Ixodes vector colonizing the dermis of a mammalian host prior to hematogenous dissemination and invasion of distal tissues all the while combating the immune response as it traverses through its pathogenic lifecycle. The innate immune response controls the borrelial burden in the dermis, but is unable to clear the infection and thereby prevent progression of disease. Dissemination in the mammalian host requires temporal regulation of virulence determinants to allow for vascular interactions, invasion, and colonization of distal tissues. Virulence determinants and/or adhesins are highly heterogenetic among environmental B. burgdorferi strains with particular genotypes being associated with the ability to disseminate to specific tissues and the severity of disease, but fail to generate cross-protective immunity between borrelial strains. The unique motility of B. burgdorferi rendered by the endoflagella serves a vital function for dissemination and protection from immune recognition. Progress has been made toward understanding the chemotactic regulation coordinating the activity of the two polar localized flagellar motors and their role in borrelial virulence, but this regulation is not yet fully understood. Distinct states of motility allow for dynamic interactions between several B. burgdorferi adhesins and host targets that play roles in transendothelial migration. Transmigration across endothelial and blood–brain barriers allows for the invasion of tissues and elicits localized immune responses. The invasive nature of B. burgdorferi is lacking in proactive mechanisms to modulate disease, such as secretion systems and toxins, but recent work has shown degradation of host extracellular matrices by B. burgdorferi contributes to the invasive capabilities of the pathogen. Additionally, B. burgdorferi may use invasion of eukaryotic cells for immune evasion and protection against environmental stresses. This review provides an overview of B. burgdorferi mechanisms for dissemination and invasion in the mammalian host, which are essential for pathogenesis and the development of persistent infection.

The vascular plant colonization on decaying Picea abies logs in Karkonosze mountain forest belts: the effects of forest community type, cryptogam cover, log decomposition and forest management

Among the vascular plants there is a lack of the typical epixylous species but they are a constant component on decaying wood. Their distribution patterns on this kind of substrate seem to be the least known among phototrophs. A total of 454 dead logs of Picea abies were analyzed with regard to cover of vascular plants and the independent morphometric features of logs and altitude. Four types of forest were compared, and the frequency and cover of the most frequent species were analyzed across the forest communities along the decomposition stage. Among the logs that were studied, 292 were colonized by vascular plants. The highest number of colonized logs was recorded in Calamagrostio villosae-Piceetum and the lowest in a deciduous beech forest of the Fagetalia order. Detrended correspondence analysis revealed that the dead logs occurring in the four forest communities differed significantly in species composition. Constrained correspondence analysis showed that six variables significantly explained the species variation, i.e., altitude, shade, moisture, decomposition stage, cover of bryophytes and status of forest (protected vs. managed). The results of the indicator value method indicated that of the 34 found species, ten could be treated as indicator species for the forest communities that were analyzed. The statistical analysis did not confirm significant role of coarse woody debris as a secondary habitat for rare and protected vascular plants.

A quorum sensing-defective mutant of Pectobacterium carotovorum ssp. brasiliense 1692 is attenuated in virulence and unable to occlude xylem tissue of susceptible potato plant stems.

Pectobacterium carotovorum ssp. brasiliense 1692 (Pcb1692) is an important emerging pathogen of potatoes causing blackleg in the field and soft rot during post-harvest storage. Blackleg diseases involve the bacterial colonization of vascular tissue and the formation of aggregates, also known as biofilms. To understand the role of quorum sensing in vascular colonization by Pcb1692, we generated a Pcb1692ΔexpI mutant strain. Inactivation of expI led to the reduced production of plant cell wall-degrading enzymes (PCWDEs), the inability to produce acyl homoserine lactone (AHL) and reduced virulence in potato tubers and stems. Complementation of the mutant strain with the wild-type expI gene intrans successfully restored AHL and PCWDE production as well as virulence. Transmission electron microscopy and invitro motility assays demonstrated hyperpiliation and loss of flagella and swimming motility in the mutant strain compared with the wild-type Pcb1692. Furthermore, we noted that, in the early stages of infection, Pcb1692 wild-type cells had intact flagella which were shed at the later stages of infection. Confocal laser microscopy of PcbΔexpI-inoculated plants showed that the mutant strain tended to aggregate in intercellular spaces, but was unable to transit to xylem tissue. On the contrary, the wild-type strain was often observed forming aggregates within xylem tissue of potato stems. Gene expression analyses confirmed that flagella are part of the quorum sensing regulon, whereas fimbriae and pili appear to be negatively regulated by quorum sensing. The relative expression levels of other important putative virulence genes, such as those encoding different groups of PCWDEs, were down-regulated in the mutant compared with the wild-type strain.

Vascular catheter colonization: surveillance based on culture of needleless connectors.

BackgroundSuperficial culture has a high negative predictive value in the assessment of catheter tip colonization (CC) and catheter-related bloodstream infection (C-RBSI). However, the process of hub culture requires the hubs to be swabbed, and this carries a risk of dislodging the biofilm. At present, most catheter hubs are closed by needleless connectors (NCs) that are periodically replaced. Our objective was to compare the yield of SC (skin + hub culture) with that of skin + NC culture in the assessment of CC and C-RBSI.MethodsDuring 5 months, we included the patients on the Major Heart Surgery ICU when a central venous catheter (CVC) remained in place ≥7 days after insertion. SCs were taken simultaneously when the NC was withdrawn and processed by the semi-quantitative method, even when the catheter was not removed. All catheter tips were cultured. All NCs belonging to a single catheter lumen were individually flushed with 100 μl of brain-heart infusion (BHI) broth. We considered the lumen to be colonized when ≥1 NC culture from the lumen flush was positive. We collected a total of 60 catheters.ResultsThe overall CC rate was 15.0 %, and we confirmed two episodes of C-RBSI. The validity values after the comparison of SCs with skin + NC culture for prediction of CC were the following: sensitivity 66.7 % vs. 77.8 %, and negative predictive value 93.6 % vs. 93.1 %. The sensitivity and negative predictive value for prediction of C-RBSI was 100 % for both SC and skin + NC culture.ConclusionThe combination of skin and flushed NC culture can be an alternative to conventional SC for ruling out CC and C-RBSI.

Influence of seed- borne and soil- carried inocula of Dickeya spp. on potato plant transpiration and symptom expression

Pectinolytic bacteria such as Dickeya spp. and Pectobacterium spp. represent a significant threat for seed potato production in Europe. Dickeya spp. induces various symptoms such as plant wilting, stem rot (usually referred to as blackleg) and tuber soft rot. Expression of wilting and blackleg symptoms depends on a range of factors: the susceptibility of the cultivar, the aggressiveness of the Dickeya strain and environmental factors such as soil moisture, temperature and plant evapotranspiration. Using the flow of sap driven by plant transpiration, these bacteria can colonize the entire plant vascular system. However, it is not known whether this vascular colonization differs according to the nature of the inoculum (soil-carried or tuber-borne) and whether this has an impact on the expression of symptoms. To better characterize the consequences of colonization of the xylem vessels by Dickeya, greenhouse and field trials were carried out with plants infected via mother tuber inoculation or root inoculation. The development of symptoms was tracked, as were changes in plants’ transpiration rate, and plant transpiration was used as an index of the early stages of symptom expression. Our trials revealed that seed- and soil- carried inocula can both induce wilting and blackleg symptoms in potato plants. It was also found that blackleg symptoms appearing during the first three weeks after plant emergence are caused mainly by a tuber-borne inoculum. Finally, it was established that the presence of a latent inoculum in the xylem vessels does not alter the plant transpiration process, as long as there are no wilting symptoms on rotting lesions.