Virulence determinants are unevenly distributed within Streptomyces species and strains causing potato common scab in the province of Quebec, Canada.

Potato common scab is an important bacterial disease afflicting potatoes around the world. Better knowledge of the local Streptomyces spp. populations causing this disease is key to developing durable control strategies. In this study, we isolated 230 Streptomyces strains from scab-infected potato tubers harvested from commercial potato fields located across the province of Quebec, Canada. The genetic diversity of this collection was first studied using repetitive element-based PCR fingerprinting, and the genomes of 36 representative strains were sequenced using PacBio's sequencing technology. This enabled us to identify the strains to the species level, to study the distribution of previously characterized virulence-associated genes and clusters, and to explore the repertoires of putative plant cell wall-degrading enzymes. In parallel, the virulence of the 36 strains was evaluated using a potato tuber slice assay. The diversity was higher than previously reported, as eleven phytopathogenic species were found across the province. Among them, S. scabiei and S. acidiscabies were the most abundant as well as the most virulent. Strains belonging to these two species harbored numerous virulence determinants, including the thaxtomin biosynthetic gene cluster. By contrast, most weakly virulent strains lacked this cluster but harbored at least one known virulence determinant. The results obtained suggest that a higher number of virulence-associated genes and clusters in the genome of phytopathogenic Streptomyces spp. is associated with greater virulence. This study contributes to increasing the publicly available genomic resources of scab-causing Streptomyces spp., and expand our knowledge on the diversity and virulence of this important bacterial pathogen.

RNA disruption is a widespread phenomenon associated with stress-induced cell death in tumour cells.

We have previously shown that neoadjuvant chemotherapy can induce the degradation of tumour ribosomal RNA (rRNA) in patients with advanced breast cancer, a phenomenon we termed "RNA disruption". Extensive tumour RNA disruption during chemotherapy was associated with a post-treatment pathological complete response and improved disease-free survival. The RNA disruption assay (RDA), which quantifies this phenomenon, is now being evaluated for its clinical utility in a large multinational clinical trial. However, it remains unclear if RNA disruption (i) is manifested across many tumour and non-tumour cell types, (ii) can occur in response to cell stress, and (iii) is associated with tumour cell death. In this study, we show that RNA disruption is induced by several mechanistically distinct chemotherapy agents and report that this phenomenon is observed in response to oxidative stress, endoplasmic reticulum (ER) stress, protein translation inhibition and nutrient/growth factor limitation. We further show that RNA disruption is dose- and time-dependent, and occurs in both tumourigenic and non-tumourigenic cell types. Northern blotting experiments suggest that the rRNA fragments generated during RNA disruption stem (at least in part) from the 28S rRNA. Moreover, we demonstrate that RNA disruption is reproducibly associated with three robust biomarkers of cell death: strongly reduced cell numbers, lost cell replicative capacity, and the generation of cells with a subG1 DNA content. Thus, our findings indicate that RNA disruption is a widespread phenomenon exhibited in mammalian cells under stress, and that high RNA disruption is associated with the onset of cell death.

Pyoluteorin and 2,4-diacetylphloroglucinol are major contributors to Pseudomonas protegens Pf-5 biocontrol against Botrytis cinerea in cannabis.

Pseudomonas protegens Pf-5 is an effective biocontrol agent that protects many crops against pathogens, including the fungal pathogen Botrytis cinerea causing gray mold disease in Cannabis sativa crops. Previous studies have demonstrated the important role of antibiotics pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (DAPG) in Pf-5-mediated biocontrol. To assess the potential involvement of PLT and DAPG in the biocontrol exerted by Pf-5 against B. cinerea in the phyllosphere of C. sativa, two knockout Pf-5 mutants were generated by in-frame deletion of genes pltD or phlA, required for the synthesis of PLT or DAPG respectively, using a two-step allelic exchange method. Additionally, two complemented mutants were constructed by introducing a multicopy plasmid carrying the deleted gene into each deletion mutant. In vitro confrontation assays revealed that deletion mutant ∆pltD inhibited B. cinerea growth significantly less than wild-type Pf-5, supporting antifungal activity of PLT. However, deletion mutant ∆phlA inhibited mycelial growth significantly more than the wild-type, hypothetically due to a co-regulation of PLT and DAPG biosynthesis pathways. Both complemented mutants recovered in vitro inhibition levels similar to that of the wild-type. In subsequent growth chamber inoculation trials, characterization of gray mold disease symptoms on infected cannabis plants revealed that both ∆pltD and ∆phlA significantly lost a part of their biocontrol capabilities, achieving only 10 and 19% disease reduction respectively, compared to 40% achieved by inoculation with the wild-type. Finally, both complemented mutants recovered biocontrol capabilities in planta similar to that of the wild-type. These results indicate that intact biosynthesis pathways for production of PLT and DAPG are required for the optimal antagonistic activity of P. protegens Pf-5 against B. cinerea in the cannabis phyllosphere.

Comparative Genomics of Potato Common Scab-Causing Streptomyces spp. Displaying Varying Virulence.

Common scab of potato causes important economic losses worldwide following the development of necrotic lesions on tubers. In this study, the genomes of 14 prevalent scab-causing Streptomyces spp. isolated from Prince Edward Island, one of the most important Canadian potato production areas, were sequenced and annotated. Their phylogenomic affiliation was determined, their pan-genome was characterized, and pathogenic determinants involved in their virulence, ranging from weak to aggressive, were compared. 13 out of 14 strains clustered with Streptomyces scabiei, while the last strain clustered with Streptomyces acidiscabies. The toxicogenic and colonization genomic regions were compared, and while some atypical gene organizations were observed, no clear correlation with virulence was observed. The production of the phytotoxin thaxtomin A was also quantified and again, contrary to previous reports in the literature, no clear correlation was found between the amount of thaxtomin A secreted, and the virulence observed. Although no significant differences were observed when comparing the presence/absence of the main virulence factors among the strains of S. scabiei, a distinct profile was observed for S. acidiscabies. Several mutations predicted to affect the functionality of some virulence factors were identified, including one in the bldA gene that correlates with the absence of thaxtomin A production despite the presence of the corresponding biosynthetic gene cluster in S. scabiei LBUM 1485. These novel findings obtained using a large number of scab-causing Streptomyces strains are challenging some assumptions made so far on Streptomyces' virulence and suggest that other factors, yet to be characterized, are also key contributors.

Inhibition of Three Potato Pathogens by Phenazine-Producing Pseudomonas spp. Is Associated with Multiple Biocontrol-Related Traits.

Phenazine-producing Pseudomonas spp. are effective biocontrol agents that aggressively colonize the rhizosphere and suppress numerous plant diseases. In this study, we compared the ability of 63 plant-beneficial phenazine-producing Pseudomonas strains representative of the worldwide diversity to inhibit the growth of three major potato pathogens: the oomycete Phytophthora infestans, the Gram-positive bacterium Streptomyces scabies, and the ascomycete Verticillium dahliae. The 63 Pseudomonas strains are distributed among four different subgroups within the P. fluorescens species complex and produce different phenazine compounds, namely, phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide (PCN), 2-hydroxyphenazine-1-carboxylic acid, and 2-hydroxphenazine. Overall, the 63 strains exhibited contrasted levels of pathogen inhibition. Strains from the P. chlororaphis subgroup inhibited the growth of P. infestans more effectively than strains from the P. fluorescens subgroup. Higher inhibition was not associated with differential levels of phenazine production nor with specific phenazine compounds. The presence of additional biocontrol-related traits found in P. chlororaphis was instead associated with higher P. infestans inhibition. Inhibition of S. scabies by the 63 strains was more variable, with no clear taxonomic segregation pattern. Inhibition values did not correlate with phenazine production nor with specific phenazine compounds. No additional synergistic biocontrol-related traits were found. Against V. dahliae, PCN producers from the P. chlororaphis subgroup and PCA producers from the P. fluorescens subgroup exhibited greater inhibition. Additional biocontrol-related traits potentially involved in V. dahliae inhibition were identified. This study represents a first step toward harnessing the vast genomic diversity of phenazine-producing Pseudomonas spp. to achieve better biological control of potato pathogens. IMPORTANCE Plant-beneficial phenazine-producing Pseudomonas spp. are effective biocontrol agents, thanks to the broad-spectrum antibiotic activity of the phenazine antibiotics they produce. These bacteria have received considerable attention over the last 20 years, but most studies have focused only on the ability of a few genotypes to inhibit the growth of a limited number of plant pathogens. In this study, we investigated the ability of 63 phenazine-producing strains, isolated from a wide diversity of host plants on four continents, to inhibit the growth of three major potato pathogens: Phytophthora infestans, Streptomyces scabies, and Verticillium dahliae. We found that the 63 strains differentially inhibited the three potato pathogens. These differences are in part associated with the nature and the quantity of the phenazine compounds being produced but also with the presence of additional biocontrol-related traits. These results will facilitate the selection of versatile biocontrol agents against pathogens.

The RNA disruption assay is superior to conventional drug sensitivity assays in detecting cytotoxic drugs.

Conventional drug sensitivity assays used to screen prospective anti-cancer agents for cytotoxicity monitor biological processes associated with active growth and proliferation, used as proxies of cell viability. However, these assays are unable to distinguish between growth-arrested (but otherwise viable) cells and non-viable/dead cells. As a result, compounds selected based on the results of these assays may only be cytostatic, halting or slowing tumour progression temporarily, without tumour eradication. Because agents capable of killing tumour cells (cytotoxic drugs) are likely the most promising in the clinic, there is a need for drug sensitivity assays that reliably identify cytotoxic compounds that induce cell death. We recently developed a drug sensitivity assay, called the RNA disruption assay (RDA), which measures a phenomenon associated with tumour cell death. In this study, we sought to compare our assay's performance to that of current commonly used drug sensitivity assays (i.e, the clonogenic, the cell counting kit-8 and the Trypan blue exclusion assays). We found that RNA disruption occurred almost exclusively when total cell numbers decreased (cytotoxic concentrations), with little to no signal detected until cells had lost viability. In contrast, conventional assays detected a decrease in their respective drug sensitivity parameters despite cells retaining their viability, as assessed using a recovery assay. We also found that the RDA can differentiate between drug-sensitive and -resistant cells, and that it can identify agents capable of circumventing drug resistance. Taken together, our study suggests that the RDA is a superior drug discovery tool, providing a unique assessment of cell death.

Regulation of a muralytic enzyme-encoding gene by two non-coding RNAs.

Non-coding regulatory RNAs fine-tune gene expression post-transcriptionally. In the streptomycetes, rpfA - encoding a muralytic enzyme required for establishing and exiting dormancy - is flanked by non-coding regulatory RNA elements both upstream (riboswitch) and downstream [antisense small RNA (sRNA)]. In Streptomyces coelicolor, the upstream riboswitch decreases rpfA transcript abundance in response to the second messenger cyclic di-AMP, itself involved in cell wall metabolism and dormancy. There is, however, no obvious expression platform associated with this riboswitch and consequently, its mechanism of action is entirely unknown. Using in vitro transcription assays, we discovered that the rpfA riboswitch promoted premature transcription termination in response to cyclic di-AMP. Through an extensive mutational analysis, we determined that attenuation required ligand binding and involved an unusual extended stem-loop region unique to a subset of rpfA riboswitches in the actinobacteria. At the other end of the rpfA gene, an antisense sRNA, termed Scr3097, is expressed opposite the predicted rpfA terminator. Using northern blotting, we found that Scr3097 accumulation mirrored that of the rpfA mRNA. In liquid culture, we detected Scr3097 exclusively in exponential-phase cells, and in plate-grown culture, we observed the sRNA primarily in differentiating cultures. Using mutational analyses, we found that the sRNA increased rpfA mRNA abundance in cells. Taken together, our work revealed multiple regulatory RNAs controlling rpfA expression in the streptomycetes.

Nucleotide second messenger-mediated regulation of a muralytic enzyme in Streptomyces.

Peptidoglycan degradative enzymes have important roles at many stages during the bacterial life cycle, and it is critical that these enzymes be stringently regulated to avoid compromising the integrity of the cell wall. How this regulation is exerted is of considerable interest: promoter-based control and protein-protein interactions are known to be employed; however, other regulatory mechanisms are almost certainly involved. In the actinobacteria, a class of muralytic enzymes - the 'resuscitation-promoting factors' (Rpfs) - orchestrates the resuscitation of dormant cells. In this study, we have taken a holistic approach to exploring the mechanisms governing RpfA function using the model bacterium Streptomyces coelicolor and have uncovered unprecedented multilevel regulation that is coordinated by three second messengers. Our studies show that RpfA is subject to transcriptional control by the cyclic AMP receptor protein, riboswitch-mediated transcription attenuation in response to cyclic di-AMP, and growth stage-dependent proteolysis in response to ppGpp accumulation. Furthermore, our results suggest that these control mechanisms are likely applicable to cell wall lytic enzymes in other bacteria.

Resuscitation-promoting factors are cell wall-lytic enzymes with important roles in the germination and growth of Streptomyces coelicolor.

Dormancy is a common strategy adopted by bacterial cells as a means of surviving adverse environmental conditions. For Streptomyces bacteria, this involves developing chains of dormant exospores that extend away from the colony surface. Both spore formation and subsequent spore germination are tightly controlled processes, and while significant progress has been made in understanding the underlying regulatory and enzymatic bases for these, there are still significant gaps in our understanding. One class of proteins with a potential role in spore-associated processes are the so-called resuscitation-promoting factors, or Rpfs, which in other actinobacteria are needed to restore active growth to dormant cell populations. The model species Streptomyces coelicolor encodes five Rpf proteins (RpfA to RfpE), and here we show that these proteins have overlapping functions during growth. Collectively, the S. coelicolor Rpfs promote spore germination and are critical for growth under nutrient-limiting conditions. Previous studies have revealed structural similarities between the Rpf domain and lysozyme, and our in vitro biochemical assays revealed various levels of peptidoglycan cleavage capabilities for each of these five Streptomyces enzymes. Peptidoglycan remodeling by enzymes such as these must be stringently governed so as to retain the structural integrity of the cell wall. Our results suggest that one of the Rpfs, RpfB, is subject to a unique mode of enzymatic autoregulation, mediated by a domain of previously unknown function (DUF348) located within the N terminus of the protein; removal of this domain led to significantly enhanced peptidoglycan cleavage.

Children's perceptions of the Northern Fruit and Vegetable Program in Ontario, Canada.

OBJECTIVE: This study examined students' perceptions of and suggestions for the Northern Fruit and Vegetable Program, a free, school-based fruit and vegetable snack program implemented in elementary schools in 2 regions of northern Ontario, Canada. METHODS: This was a qualitative study involving 18 focus groups with students in 11 elementary schools in the Porcupine region and 7 schools in the Algoma region. One hundred thirty-nine students from grades 5-8 participated in this study. Inductive content analysis was used to identify key themes. RESULTS: Children perceived the Northern Fruit and Vegetable Program to be a valuable program that allowed them to try new fruits and vegetables (FVs). Participants stated they now eat more FVs at home and at school. Participants would like the program offered more frequently and with more variety. CONCLUSIONS AND IMPLICATIONS: Although children identified several areas for program improvement, they generally perceived the program to be positive and valuable in promoting FV consumption.

Northern Fruit and Vegetable Pilot Program: a process evaluation.

PURPOSE: A process evaluation was conducted of the Northern Fruit and Vegetable Pilot Program (NFVPP), a government-funded health promotion initiative. The objectives were to determine how the program was implemented and to identify program facilitators and challenges. METHODS: Facilitators and challenges in the implementation of the free fruit and vegetable snack program were assessed through qualitative interviews with school-level stakeholders (i.e., food preparers, teachers, and principals) and tracking wasted produce. The implementation of an enhanced nutrition education (ENE) component was assessed through a teacher survey. RESULTS: School-level stakeholders saw the NFVPP as a valuable program. Key facilitators included teacher role-modelling and sufficient funding for supplies and personnel. Key challenges included produce delivery, quality, wastage, and variety. The ENE component was minimally implemented. CONCLUSIONS: The study identified program strengths and areas that could be improved. As a result, changes were made to how the NFVPP was implemented in schools the following year. The use of qualitative methods enabled program planners to understand the program implementation process.

The ability of Pseudomonas sp. LBUM 223 to produce phenazine-1-carboxylic acid affects the growth of Streptomyces scabies, the expression of thaxtomin biosynthesis genes and the biological control potential against common scab of potato.

Streptomyces scabies causes common scab, an economical disease affecting potato crops world-wide, for which no effective control measure exists. This pathogen produces the plant toxin thaxtomin A, which is involved in symptom development on potato tubers. A biological control approach that can limit S. scabies growth and repress thaxtomin production represents an attractive alternative to classical control strategies. Pseudomonas sp. LBUM 223 produces phenazine-1-carboxylic acid (PCA), an antibiotic that inhibits the growth of plant pathogens and contributes to the biological control of plant diseases. In this study, the involvement of LBUM 223's PCA-producing ability in the growth inhibition of S. scabies, repression of thaxtomin biosynthesis genes (txtA and txtC) and the biological control of common scab of potato was investigated using a mutant defective in PCA production (LBUM 223phzC(-) ). Streptomyces scabies growth was inhibited to a significantly lesser degree by LBUM 223phzC(-) than by the wild type. LBUM 223 also significantly repressed txtA and txtC expression in S. scabies and protected potato against disease, whereas LBUM 223phzC(-) did not. These results suggest that PCA production is central to the ability of LBUM 223 to limit pathogen growth, repress the expression of key pathogenicity genes and control common scab of potato.

Impact evaluation of the Northern Fruit and Vegetable Pilot Programme - a cluster-randomised controlled trial.

OBJECTIVE: The purpose of this impact evaluation was to measure the influence of a government of Ontario, Canada health promotion initiative, the Northern Fruit and Vegetable Pilot Programme (NFVPP), on elementary school-aged children's psychosocial variables regarding fruit and vegetables, and fruit and vegetable consumption patterns. DESIGN: A cluster-randomised controlled trial design was used. The NFVPP consisted of three intervention arms: (i) Intervention I: Free Fruit and Vegetable Snack (FFVS) + Enhanced Nutrition Education; (ii) Intervention II: FFVS-alone; and (iii) Control group. Using the Pro-Children Questionnaire, the primary outcome measure was children's fruit and vegetable consumption, and the secondary outcome measures included differences in children's awareness, knowledge, self-efficacy, preference, intention and willingness to increase fruit and vegetable consumption. SETTING/SUBJECTS: Twenty-six elementary schools in a defined area of Northern Ontario were eligible to participate in the impact evaluation. A final sample size of 1,277 students in grades five to eight was achieved. RESULTS: Intervention I students consumed more fruit and vegetables at school than their Control counterparts by 0.49 serving/d (P < 0.05). Similarly, Intervention II students consumed more fruit and vegetables at school than Control students by 0.42 serving/d, although this difference was not statistically significant. Among students in both intervention groups, preferences for certain fruit and vegetables shifted from 'never tried it' towards 'like it'. CONCLUSIONS: The NFVPP resulted in positive changes in elementary school-aged children's fruit and vegetable consumption at school, and favourable preference changes for certain fruit and vegetables.

Genetic diversity of Streptomyces spp. causing common scab of potato in eastern Canada.

Common scab is an important disease of potato caused by Streptomyces scabies and other closely related species. In this study, the genetic diversity of Streptomyces spp. causing common scab of potato in eastern Canada was for the first time investigated. Forty-one Streptomyces spp. isolates were retrieved from necrotic lesions of potato tubers harvested from different regions of the Canadian provinces New-Brunswick, Nova Scotia and Prince-Edward-Island. Most isolates were closely related to known pathogenic S. scabies strains on the basis of partial 16S ribosomal (r) RNA and rpoB gene sequence analyses. Two isolates were identified as pathogenic species of Streptomyces acidiscabies. To our knowledge, this species has never been previously isolated in these areas. Genome fingerprinting studies using repetitive elements (rep) polymerase chain reactions (PCR) revealed 10 distinct genetic groups in eastern Canada. The geographical distribution of the genetic groups was region-dependant. Pathogenicity- and virulence-related genes (txtA, txtC, and tomA) were PCR-amplified from each isolate, and nucleotide sequence analysis of partial gene fragments revealed slight polymorphisms in both txtA and txtC genes. No genetic variation was noted in the partial tomA gene sequences.

Habitus, stress, and the body: the everyday production of health and cardiovascular risk.

The incidence of coronary heart disease (CHD) and the distribution of contributory risk factors are closely linked with social patterns of advantage and disadvantage. The authors conducted eight focus groups in urban, northern, and rural sites in Ontario, Canada. Participants were all at high absolute risk for or had been diagnosed with CHD. Analysis centered on habitus, which forms the pivotal link between the person and "place." The authors focused on participants' dialogue about stress because it dealt with the impingements of the social world and resultant constraints on health-related activities in everyday places. Participants described four types of places or social positions in their "stress talk": work-places, transitional spaces, gendered situations, and exclusions. Places can support or constrain health related activities in many ways. Habits and practices linked with stress by participants were enduringly associated with these contexts, suggesting that place, body, and health are inseparable and coconstituted.