Seasonal Dynamics of Various Scab-Causing Streptomyces Genotypes Among Potato Fields on Prince Edward Island.

Potato common scab is an important bacterial plant disease caused by numerous Streptomyces species and strains. A better understanding of the genetic diversity and population dynamics of these microorganisms in the field is crucial to develop effective control methods. Our research group previously studied the genetic diversity of scab-causing Streptomyces spp. in Prince Edward Island, one of Canada's most important potato-growing provinces. Fourteen distinct Streptomyces genotypes were identified and displayed contrasting aggressiveness toward potato tubers. To better understand the distribution and occurrence of these genotypes over time under field conditions, the population dynamics were studied in nine commercial potato fields throughout a growing season. A comparative genomic-driven approach was used to design genotype-specific primers and probes, allowing us to quantify, using quantitative polymerase chain reaction, the abundance of each of the 14 genotypes in field soil. Thirteen of the previously identified genotypes were detected in at least one soil sample, with various frequencies and population sizes across the different fields under study. Interestingly, weakly virulent genotypes dominated, independent of time or location. Among them, three genotypes accounted for more than 80% of the genotypes' combined population. Although the highly virulent genotypes were detected in lower relative abundance than the weakly virulent ones, an increase in the highly virulent genotypes' population size was observed over the growing season in most fields. The results will ultimately be useful for the development of targeted common scab control strategies.

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.

Biocontrol Activity of Bacillus spp. and Pseudomonas spp. Against Botrytis cinerea and Other Cannabis Fungal Pathogens.

Gray mold caused by Botrytis cinerea is one of the most widespread and damaging diseases in cannabis crops worldwide. With challenging restrictions on pesticide use and few effective control measures, biocontrol agents are needed to manage this disease. The aim of this study was to identify bacterial biocontrol agents with wide-spectrum activity against B. cinerea and other cannabis fungal pathogens. Twelve Bacillus and Pseudomonas strains were first screened with in vitro confrontational assays against 10 culturable cannabis pathogens, namely B. cinerea, Sclerotinia sclerotiorum, Fusarium culmorum, F. sporotrichoides, F. oxysporum, Nigrospora sphaerica, N. oryzae, Alternaria alternata, Phoma sp., and Cercospora sp. Six strains displaying the highest inhibitory activity, namely Bacillus velezensis LBUM279, FZB42, LBUM1082, Bacillus subtilis LBUM979, P. synxantha LBUM223, and P. protegens Pf-5, were further assessed in planta where all, except LBUM223, significantly controlled gray mold development on cannabis leaves. Notably, LBUM279 and FZB42 reduced disease severity by at least half compared with water-treated plants and prevented lesion development and/or sporulation up to 9 days after pathogen inoculation. Genomes of LBUM279, LBUM1082, and LBUM979 were sequenced de novo and taxonomic affiliations were determined to ensure nonrelatedness with pathogenic strains. Moreover, the genomes were exempt of detrimental genes encoding major toxins and virulence factors that could otherwise pose a biosafety risk when used on crops. Eighteen gene clusters of potential biocontrol interest were also identified. To our knowledge, this is the first reported attempt to control cannabis fungal diseases in planta by direct antagonism with beneficial bacteria.

Seasonal changes in the abundance and activity of bacterial and fungal denitrifying communities associated with different compost amendments.

Composts can be efficient organic amendments in potato culture as they can supply carbon and nutrients to the soil. However, more information is required on the effects of composts on denitrification and nitrous oxide emissions (N2O) and emission-producing denitrifying communities. The effects of three compost amendments (municipal source separated organic waste compost (SSOC), forestry waste mixed with poultry manure compost (FPMC), and forestry residues compost (FRC)) on fungal and bacterial denitrifying communities and activity was examined in an agricultural field cropped to potatoes during the fall, spring, and summer seasons. The denitrification enzyme activity (DEA), N2O emissions, and respiration were measured in parallel. N2O emission rates were greater in FRC-amended soils in the fall and summer, whereas soil respiration was highest in the SSOC-amended soil in the fall. A large number of nirK denitrifying fungal transcripts were detected in the fall, coinciding with compost application, while the greatest nirK bacterial transcripts were measured in the summer when plants were actively growing. Denitrifying community and transcript levels were poor predictors of DEA, N2O emissions, or respiration rates in compost-amended soil. Overall, the sampling date was driving the population and activity levels of the three denitrifying communities under study.

Diversity and Virulence of Streptomyces spp. Causing Potato Common Scab in Prince Edward Island, Canada.

Common scab (CS) is a potato disease that significantly decreases the market value of potato tubers after the development of necrotic lesions on their surface. Streptomyces scabiei is the main causal agent of CS; however, other closely related species, including S. acidiscabies and S. turgidiscabies, have also been shown to cause the disease. In this study, we characterized the genetic and phenotypic diversity of Streptomyces spp. causing CS in Prince Edward Island, the main potato-producing province in Canada. Two hundred and ninety-six pathogenic Streptomyces spp. isolates were retrieved from diseased tubers harvested from six fields located across a longitudinal geographical gradient. Genome fingerprinting analyses using repetitive elements PCR (ERIC- and BOX-PCR) revealed 14 distinct genetic groups. Thirteen groups were taxonomically affiliated with S. scabiei, whereas the fourteenth group was affiliated with S. acidiscabies. Their geographical distribution was characterized and revealed that on average between six and eight different genetic groups were detected per field, with variable abundance. Virulence assays showed strong differences in virulence between the genetic groups, ranging from low to highly virulent. Interestingly, pathogenic Streptomyces spp. populations in each field seem to be dominated by the most virulent genetic groups. The results obtained will contribute to better understanding of the population dynamic of pathogenic Streptomyces spp. causing CS of potato and promoting the development of more efficient detection and intervention tools to manage this important potato disease.

Metabolic and Genomic Traits of Phytobeneficial Phenazine-Producing Pseudomonas spp. Are Linked to Rhizosphere Colonization in Arabidopsis thaliana and Solanum tuberosum.

Bacterial rhizosphere colonization is critical for phytobeneficial rhizobacteria such as phenazine-producing Pseudomonas spp. To better understand this colonization process, potential metabolic and genomic determinants required for rhizosphere colonization were identified using a collection of 60 phenazine-producing Pseudomonas strains isolated from multiple plant species and representative of the worldwide diversity. Arabidopsis thaliana and Solanum tuberosum (potato) were used as host plants. Bacterial rhizosphere colonization was measured by quantitative PCR using a newly designed primer pair and TaqMan probe targeting a conserved region of the phenazine biosynthetic operon. The metabolic abilities of the strains were assessed on 758 substrates using Biolog phenotype microarray technology. These data, along with available genomic sequences for all strains, were analyzed in light of rhizosphere colonization. Strains belonging to the P. chlororaphis subgroup colonized the rhizospheres of both plants more efficiently than strains belonging to the P. fluorescens subgroup. Metabolic results indicated that the ability to use amines and amino acids was associated with an increase in rhizosphere colonization capability in A. thaliana and/or in S. tuberosum The presence of multiple genetic determinants in the genomes of the different strains involved in catabolic pathways and plant-microbe and microbe-microbe interactions correlated with increased or decreased rhizosphere colonization capabilities in both plants. These results suggest that the metabolic and genomic traits found in different phenazine-producing Pseudomonas strains reflect their rhizosphere competence in A. thaliana and S. tuberosum Interestingly, most of these traits are associated with similar rhizosphere colonizing capabilities in both plant species.IMPORTANCE Rhizosphere colonization is crucial for plant growth promotion and biocontrol by antibiotic-producing Pseudomonas spp. This colonization process relies on different bacterial determinants which partly remain to be uncovered. In this study, we combined a metabolic and a genomic approach to decipher new rhizosphere colonization determinants which could improve our understanding of this process in Pseudomonas spp. Using 60 distinct strains of phenazine-producing Pseudomonas spp., we show that rhizosphere colonization abilities correlated with both metabolic and genomic traits when these bacteria were inoculated on two distant plants, Arabidopsis thaliana and Solanum tuberosum Key metabolic and genomic determinants presumably required for efficient colonization of both plant species were identified. Upon further validation, these targets could lead to the development of simple screening tests to rapidly identify efficient rhizosphere colonizers.

Diversity of phytobeneficial traits revealed by whole-genome analysis of worldwide-isolated phenazine-producing Pseudomonas spp.

Plant-beneficial Pseudomonas spp. competitively colonize the rhizosphere and display plant-growth promotion and/or disease-suppression activities. Some strains within the P. fluorescens species complex produce phenazine derivatives, such as phenazine-1-carboxylic acid. These antimicrobial compounds are broadly inhibitory to numerous soil-dwelling plant pathogens and play a role in the ecological competence of phenazine-producing Pseudomonas spp. We assembled a collection encompassing 63 strains representative of the worldwide diversity of plant-beneficial phenazine-producing Pseudomonas spp. In this study, we report the sequencing of 58 complete genomes using PacBio RS II sequencing technology. Distributed among four subgroups within the P. fluorescens species complex, the diversity of our collection is reflected by the large pangenome which accounts for 25 413 protein-coding genes. We identified genes and clusters encoding for numerous phytobeneficial traits, including antibiotics, siderophores and cyclic lipopeptides biosynthesis, some of which were previously unknown in these microorganisms. Finally, we gained insight into the evolutionary history of the phenazine biosynthetic operon. Given its diverse genomic context, it is likely that this operon was relocated several times during Pseudomonas evolution. Our findings acknowledge the tremendous diversity of plant-beneficial phenazine-producing Pseudomonas spp., paving the way for comparative analyses to identify new genetic determinants involved in biocontrol, plant-growth promotion and rhizosphere competence.

Transcriptome alteration in Phytophthora infestans in response to phenazine-1-carboxylic acid production by Pseudomonas fluorescens strain LBUM223.

BACKGROUND: Phytophthora infestans is responsible for late blight, one of the most important potato diseases. Phenazine-1-carboxylic acid (PCA)-producing Pseudomonas fluorescens strain LBUM223 isolated in our laboratory shows biocontrol potential against various plant pathogens. To characterize the effect of LBUM223 on the transcriptome of P. infestans, we conducted an in vitro time-course study. Confrontational assay was performed using P. infestans inoculated alone (control) or with LBUM223, its phzC- isogenic mutant (not producing PCA), or exogenically applied PCA. Destructive sampling was performed at 6, 9 and 12 days and the transcriptome of P. infestans was analysed using RNA-Seq. The expression of a subset of differentially expressed genes was validated by RT-qPCR. RESULTS: Both LBUM223 and exogenically applied PCA significantly repressed P. infestans' growth at all times. Compared to the control treatment, transcriptomic analyses showed that the percentages of all P. infestans' genes significantly altered by LBUM223 and exogenically applied PCA increased as time progressed, from 50 to 61% and from to 32 to 46%, respectively. When applying an absolute cut-off value of 3 fold change or more for all three harvesting times, 207 genes were found significantly differentially expressed by PCA, either produced by LBUM223 or exogenically applied. Gene ontology analysis revealed that both treatments altered the expression of key functional genes involved in major functions like phosphorylation mechanisms, transmembrane transport and oxidoreduction activities. Interestingly, even though no host plant tissue was present in the in vitro system, PCA also led to the overexpression of several genes encoding effectors. The mutant only slightly repressed P. infestans' growth and barely altered its transcriptome. CONCLUSIONS: Our study suggests that PCA is involved in P. infestans' growth repression and led to important transcriptomic changes by both up- and down-regulating gene expression in P. infestans over time. Different metabolic functions were altered and many effectors were found to be upregulated, suggesting their implication in biocontrol.

Phenazines in plant-beneficial Pseudomonas spp.: biosynthesis, regulation, function and genomics.

Plant-beneficial phenazine-producing Pseudomonas spp. are proficient biocontrol agents of soil-dwelling plant pathogens. Phenazines are redox-active molecules that display broad-spectrum antibiotic activity toward many fungal, bacterial and oomycete plant pathogens. Phenazine compounds also play a role in the persistence and survival of Pseudomonas spp. in the rhizosphere. This mini-review focuses on plant-beneficial phenazine-producing Pseudomonas spp. from the P. fluorescens species complex, which includes numerous well-known phenazine-producing strains of biocontrol interest. In this review the current knowledge on phenazine biosynthesis and regulation, the role played by phenazines in biocontrol and rhizosphere colonization, as well as exciting new advances in the genomics of plant-beneficial phenazine-producing Pseudomonas spp. will be discussed.

Interaction between 2,4-Diacetylphloroglucinol- and Hydrogen Cyanide-Producing Pseudomonas brassicacearum LBUM300 and Clavibacter michiganensis subsp. michiganensis in the Tomato Rhizosphere.

We have previously demonstrated that inoculation of tomato plants with 2,4-diacetylphloroglucinol (DAPG)- and hydrogen cyanide (HCN)-producing Pseudomonas brassicacearum LBUM300 could significantly reduce bacterial canker symptoms caused by Clavibacter michiganensis subsp. michiganensis In this study, in order to better characterize the population dynamics of LBUM300 in the rhizosphere of tomato plants, we characterized the role played by DAPG and HCN production by LBUM300 on rhizosphere colonization of healthy and C. michiganensis subsp. michiganensis-infected tomato plants. The impact of C. michiganensis subsp. michiganensis presence on the expression of DAPG and HCN biosynthetic genes in the rhizosphere was also examined. In planta assays were performed using combinations of C. michiganensis subsp. michiganensis and wild-type LBUM300 or DAPG (LBUM300ΔphlD) or HCN (LBUM300ΔhcnC) isogenic mutant strains. Populations of LBUM300 and phlD and hcnC gene expression levels were quantified in rhizosphere soil at several time points up to 264 h postinoculation using culture-independent quantitative PCR (qPCR) and reverse transcriptase quantitative PCR (RT-qPCR) TaqMan assays, respectively. The presence of C. michiganensis subsp. michiganensis significantly increased rhizospheric populations of LBUM300. In C. michiganensis subsp. michiganensis-infected tomato rhizospheres, the populations of wild-type LBUM300 and strain LBUM300ΔhcnC, both producing DAPG, were significantly higher than the population of strain LBUM300ΔphlD A significant upregulation of phlD expression was observed in the presence of C. michiganensis subsp. michiganensis, while hcnC expression was only slightly increased in the mutant strain LBUM300ΔphlD when C. michiganensis subsp. michiganensis was present. Additionally, biofilm production was found to be significantly reduced in strain LBUM300ΔphlD compared to the wild-type and LBUM300ΔhcnC strains.IMPORTANCE The results of this study suggest that C. michiganensis subsp. michiganensis infection of tomato plants contributes to increasing rhizospheric populations of LBUM300, a biocontrol agent, as well as the overexpression of the DAPG biosynthetic operon in this bacterium. The increasing rhizospheric populations of LBUM300 represent one of the key factors in controlling C. michiganensis subsp. michiganensis in tomato plants, as DAPG-producing bacteria have shown the ability to decrease bacterial canker symptoms in tomato plants.

Phenazine-1-Carboxylic Acid Production by Pseudomonas fluorescens LBUM636 Alters Phytophthora infestans Growth and Late Blight Development.

Phytophthora infestans causes late blight of potato, one of the most devastating diseases affecting potato production. Alternative approaches for controlling late blight are being increasingly sought due to increasing environmental concerns over the use of chemical pesticides and the increasing resistance of P. infestans to fungicides. Our research group has isolated a new strain of Pseudomonas fluorescens (LBUM636) of biocontrol interest producing the antibiotic phenazine-1-carboxylic acid (PCA). Wild-type LBUM636 was shown to significantly inhibit the growth of Phytophthora infestans in in vitro confrontational assays whereas its isogenic mutant (phzC-; not producing PCA) only slightly altered the pathogen's growth. Wild-type LBUM636 but not the phzC- mutant also completely repressed disease symptom development on tubers. A pot experiment revealed that wild-type LBUM636 can significantly reduce P. infestans populations in the rhizosphere and in the roots of potato plants, as well as reduce in planta disease symptoms due to PCA production. The expression of eight common plant defense-related genes (ChtA, PR-1b, PR-2, PR-5, LOX, PIN2, PAL-2, and ERF3) was quantified in tubers, roots, and leaves by reverse-transcription quantitative polymerase chain reaction and revealed that the biocontrol observed was not associated with the induction of a plant defense response by LBUM636. Instead, a direct interaction between P. infestans and LBUM636 is required and PCA production appears to be a key factor for LBUM636's biocontrol ability.

Novel P450nor Gene Detection Assay Used To Characterize the Prevalence and Diversity of Soil Fungal Denitrifiers.

UNLABELLED: Denitrifying fungi produce nitrous oxide (N2O), a potent greenhouse gas, as they generally lack the ability to convert N2O to dinitrogen. Contrary to the case for bacterial denitrifiers, the prevalence and diversity of denitrifying fungi found in the environment are not well characterized. In this study, denitrifying fungi were isolated from various soil ecosystems, and novel PCR primers targeting the P450nor gene, encoding the enzyme responsible for the conversion of nitric oxide to N2O, were developed, validated, and used to study the diversity of cultivable fungal denitrifiers. This PCR assay was also used to detect P450nor genes directly from environmental soil samples. Fungal denitrification capabilities were further validated using an N2O gas detection assay and a PCR assay targeting the nirK gene. A collection of 492 facultative anaerobic fungi was isolated from 15 soil ecosystems and taxonomically identified by sequencing the internal transcribed spacer sequence. Twenty-seven fungal denitrifiers belonging to 10 genera had the P450nor and the nirK genes and produced N2O from nitrite. N2O production is reported in strains not commonly known as denitrifiers, such as Byssochlamys nivea, Volutella ciliata, Chloridium spp., and Trichocladium spp. The prevalence of fungal denitrifiers did not follow a soil ecosystem distribution; however, a higher diversity was observed in compost and agricultural soils. The phylogenetic trees constructed using partial P450nor and nirK gene sequences revealed that both genes clustered taxonomically closely related strains together. IMPORTANCE: A PCR assay targeting the P450nor gene involved in fungal denitrification was developed and validated. The newly developed P450nor primers were used on fungal DNA extracted from a collection of fungi isolated from various soil environments and on DNA directly extracted from soil. The results indicated that approximatively 25% of all isolated fungi possessed this gene and were able to convert nitrite to N2O. All soil samples from which denitrifying fungi were isolated also tested positive for the presence of P450nor The P450nor gene detection assay was reliable in detecting a large diversity of fungal denitrifiers. Due to the lack of homology existing between P450nor and bacterial denitrification genes, it is expected that this assay will become a tool of choice for studying fungal denitrifiers.

Biocontrol of Potato Common Scab is Associated with High Pseudomonas fluorescens LBUM223 Populations and Phenazine-1-Carboxylic Acid Biosynthetic Transcript Accumulation in the Potato Geocaulosphere.

Pseudomonads are often used as biocontrol agents because they display a broad range of mechanisms to control diseases. Common scab of potato, caused by Streptomyces scabies, was previously reported to be controlled by Pseudomonas fluorescens LBUM223 through phenazine-1-carboxylic acid (PCA) production. In this study, we aimed at characterizing the population dynamics of LBUM223 and the expression of phzC, a key gene involved in the biosynthesis of PCA, in the rhizosphere and geocaulosphere of potato plants grown under controlled and field conditions. Results obtained from controlled experiments showed that soil populations of LBUM223 significantly declined over a 15-week period. However, at week 15, the presence of S. scabies in the geocaulosphere was associated with significantly higher populations of LBUM223 than when the pathogen was absent. It also led to the detection of significantly higher phzC gene transcript numbers. Under field conditions, soil populations of LBUM223 followed a similar decline in time when a single inoculation was applied in spring but remained stable when reinoculated biweekly, which also led to greater phzC gene transcripts accumulation. Taken together, our findings suggest that LBUM223 must colonize the potato geocaulosphere at high levels (10(7) bacteria/g of soil) in order to achieve biocontrol of common scab through increased PCA production.

Tillage Management and Seasonal Effects on Denitrifier Community Abundance, Gene Expression and Structure over Winter.

Tillage effects on denitrifier communities and nitrous oxide (N2O) emissions were mainly studied during the growing season. There is limited information for the non-growing season, especially in northern countries where winter has prolonged periods with sub-zero temperatures. The abundance and structure of the denitrifier community, denitrification gene expression and N2O emissions in fields under long-term tillage regimes [no-tillage (NT) vs conventional tillage (CT)] were assessed during two consecutive winters. NT exerted a positive effect on nirK and nosZ denitrifier abundance in both winters compared to CT. Moreover, the two contrasting managements had an opposite influence on nirK and nirS RNA/DNA ratios. Tillage management resulted in different denitrifier community structures during both winters. Seasonal changes were observed in the abundance and the structure of denitrifiers. Interestingly, the RNA/DNA ratios were greater in the coldest months for nirK, nirS and nosZ. N2O emissions were not influenced by management but changed over time with two orders of magnitude increase in the coldest month of both winters. In winter of 2009-2010, emissions were mainly as N2O, whereas in 2010-2011, when soil temperatures were milder due to persistent snow cover, most emissions were as dinitrogen. Results indicated that tillage management during the growing season induced differences in denitrifier community structure that persisted during winter. However, management did not affect the active cold-adapted community structure.

Pseudomonas fluorescens LBUM223 Increases Potato Yield and Reduces Common Scab Symptoms in the Field.

Common scab of potato, caused by pathogenic Streptomyces spp., is an important disease not efficiently controlled by current methods. We previously demonstrated that Pseudomonas fluorescens LBUM223 reduces common scab development under controlled conditions through phenazine-1-carboxylic (PCA) production, leading to reduced thaxtomin A production by the pathogen, a key pathogenicity and virulence factor. Here, we aimed at determining if LBUM223 is able to increase potato yield and control common scab under field conditions, while characterizing the biocontrol mechanisms involved. We investigated if a reduction in pathogen soil populations, activation of induced systemic resistance in potato, and/or changes in txtA gene expression, involved in thaxtomin A biosynthesis in pathogenic Streptomyces spp. were involved in common scab control by LBUM223. Common scab symptoms were significantly reduced and total tuber weight increased by 46% using biweekly applications of LBUM223. LBUM223 did not reduce pathogen soil populations, nor was potato systemic defense-related gene expression significantly altered between treatments. However, a significant down-regulation of txtA expression occurred in the geocaulosphere. This is the first demonstration that a Pseudomonas strain can directly alter the transcriptional activity of a key pathogenesis gene in a plant pathogen under field conditions, contributing to disease control.

New Developments in Quantitative Real-time Polymerase Chain Reaction Technology.

Real time-quantitative PCR (RT-qPCR) technology has revolutionized the detection landscape in every area of molecular biology. The fundamental basis of this technology has remained unchanged since its inception, however various modifications have enhanced the overall performance of this highly versatile technology. These improvements have ranged from changes in the individual components of the enzymatic reaction cocktail (polymerizing enzymes, reaction buffers, probes, etc.) to the detection system itself (instrumentation, software, etc.). The RT-qPCR technology currently available to researchers is more sensitive, faster and affordable than when this technology was first introduced. In this article, we summarize the developments of the last few years in RT-qPCR technology and nucleic acid amplification.

Long-term induction of defense gene expression in potato by pseudomonas sp. LBUM223 and streptomyces scabies.

Streptomyces scabies is a causal agent of common scab of potato, which generates necrotic tuber lesions. We have previously demonstrated that inoculation of potato plants with phenazine-1-carboxylic acid (PCA)- producing Pseudomonas sp. LBUM223 could significantly reduce common scab symptoms. In the present study, we investigated whether LBUM223 or an isogenic phzC- mutant not producing PCA could elicit an induced systemic resistance response in potato. The expression of eight defense-related genes (salicylic acid [SA]-related ChtA, PR-1b, PR-2, and PR-5; and jasmonic acid and ethylene-related LOX, PIN2, PAL-2, and ERF3) was quantified using newly developed TaqMan reverse-transcription quantitative polymerase chain reaction assays in 5- and 10-week-old potted potato plants. Although only wild-type LBUM223 was capable of significantly reducing common scab symptoms, the presence of both LBUM223 and its PCA-deficient mutant were equally able to upregulate the expression of LOX and PR-5. The presence of S. scabies overexpressed all SA-related genes. This indicates that (i) upregulation of potato defense-related genes by LBUM223 is unlikely to contribute to common scab's control and (ii) LBUM223's capacity to produce PCA is not involved in this upregulation. These results suggest that a direct interaction occurring between S. scabies and PCA-producing LBUM223 is more likely involved in controlling common scab development.

Identification and genomic characterization of Pseudomonas spp. displaying biocontrol activity against Sclerotinia sclerotiorum in lettuce.

Lettuce is an economically major leafy vegetable that is affected by numerous diseases. One of the most devastating diseases of lettuce is white mold caused by Sclerotinia sclerotiorum. Control methods for this fungus are limited due to the development of genetic resistance to commonly used fungicides, the large number of hosts and the long-term survival of sclerotia in soil. To elaborate a new and more sustainable approach to contain this pathogen, 1,210 Pseudomonas strains previously isolated from agricultural soils in Canada were screened for their antagonistic activity against S. sclerotiorum. Nine Pseudomonas strains showed strong in vitro inhibition in dual-culture confrontational assays. Whole genome sequencing of these strains revealed their affiliation with four phylogenomic subgroups within the Pseudomonas fluorescens group, namely Pseudomonas corrugata, Pseudomonas asplenii, Pseudomonas mandelii, and Pseudomonas protegens. The antagonistic strains harbor several genes and gene clusters involved in the production of secondary metabolites, including mycin-type and peptin-type lipopeptides, and antibiotics such as brabantamide, which may be involved in the inhibitory activity observed against S. sclerotiorum. Three strains also demonstrated significant in planta biocontrol abilities against the pathogen when either inoculated on lettuce leaves or in the growing substrate of lettuce plants grown in pots. They however did not impact S. sclerotiorum populations in the rhizosphere, suggesting that they protect lettuce plants by altering the fitness and the virulence of the pathogen rather than by directly impeding its growth. These results mark a step forward in the development of biocontrol products against S. sclerotiorum.

Quantitative Real-time Polymerase Chain Reaction for Tracking Microbial Gene Expression in Complex Environmental Matrices.

Environmental matrices are highly diverse in their composition and range from simple (e.g. water) to highly complex (e.g. organic soils/biosolids). Analysis of microbial gene expression from such substrates is done for variety of purposes which could range from bio-surveillance to elucidation of biological function of a target microbe. Quantitative real-time PCR (RT-qPCR) has become a technique of choice for studying such bio-processes, due to its unique ability to both detect and quantify a target transcript in real-time. Challenges in extracting inhibitor-free, structurally intact RNA, amenable for a sensitive technique like RT-qPCR, has however proved to be a major impediment in our ability to rigorously implement this highly versatile technology. Despite these 'substrate defined' limitations, many attempts have been made to implement the RT-qPCR technology. Efforts like these have given us invaluable insight into the expression status of a particular transcript and hence, the biological functioning of the microbe, specifically under natural in situ conditions. As a result, it has enhanced our understanding of the role and diversity of many microbial populations which, previously was not possible using conventional molecular approaches. In this article, we have sought to summarize such technical problems faced by molecular environmental microbiologist and solutions developed to mitigate those challenges.

Development of a versatile TaqMan™ real-time quantitative PCR (RT-qPCR) compliant anchor sequence to quantify bacterial gene transcripts from RNA samples containing carryover genomic DNA.

BACKGROUND: In bacterial systems, the sequence congruence of genomic DNA (gDNA) and cDNA obtained following reverse transcription of RNA, makes gDNA an automatic target for qPCR primers. This could lead to aberrant gene expression quantification. This is why a rigorous treatment of bacterial RNA with DNase I is usually required to remove any traces of carryover gDNA. As bacterial RNA is known to be extremely labile, any procedure that affects RNA yield, such as DNase I treatment, can be logically assumed to also influence detection and quantification of gene transcripts, leading to either an underestimation or no detection at all. To address such problems, we have developed a novel and versatile TaqMan RT-qPCR compliant anchor sequence (MYT4) for quantifying bacterial gene transcripts without the need for DNase I treatment. RESULTS: A non-genomic anchor sequence, henceforth referred to as MYT4 was designed using a synthetic DNA sequence called myIC, previously shown to share no significant homology to any known accession in the GenBank database. The sequence characteristic of MYT4 was kept within the design parameters required for the TaqMan RT-qPCR platform. The specificity and robustness of the novel MYT4 sequence was validated on RNA extracted from the bacterium Pseudomonas sp. LBUM300, grown under liquid culture and spiked soil conditions. Two transcripts, namely hcnC and phlD, were quantified from these two experimental systems. Using the MYT4 anchor, no RT-qPCR signal was detected from non-DNase I treated RNA, while strong signals were obtained using conventional reverse primers and RT-qPCR, indicating the presence of carryover gDNA in the RNA, extracted from either liquid culture or soil. Serial treatment of the RNA samples with DNase I (required to achieve absolute gDNA elimination) resulted in 50-70% loss of RNA which, when submitted to conventional RT-qPCR, significantly altered the transcript numbers detected when compared to the MYT4-based approach. CONCLUSIONS: Implementation of the versatile approach described in this study, which can be "retrofitted" to any existing TaqMan RT-qPCR system, should contribute to reducing the time and lowering the costs required to perform adequate bacterial RNA purification for downstream quantification of gene transcripts.