Extraction-free clinical detection of SARS-CoV-2 virus from saline gargle samples using Hamilton STARlet liquid handler.

As part of the COVID-19 pandemic, clinical laboratories have been faced with massive increases in testing, resulting in sample collection systems, reagent, and staff shortages. We utilized self-collected saline gargle samples to optimize high throughput SARS-CoV-2 multiplex polymerase chain reaction (PCR) testing in order to minimize cost and technologist time. This was achieved through elimination of nucleic acid extraction and automation of sample handling on a widely available robotic liquid handler, Hamilton STARlet. A customized barcode scanning script for reading the sample ID by the Hamilton STARlet's software system was developed to allow primary tube sampling. Use of pre-frozen SARS-CoV-2 assay reaction mixtures reduced assay setup time. In both validation and live testing, the assay produced no false positive or false negative results. Of the 1060 samples tested during validation, 3.6% (39/1060) of samples required retesting as they were either single gene positive, had internal control failure or liquid aspiration error. Although the overall turnaround time was only slightly faster in the automated workflow (185 min vs 200 min), there was a 76% reduction in hands-on time, potentially reducing staff fatigue and burnout. This described process from sample self-collection to automated direct PCR testing significantly reduces the total burden on healthcare systems in terms of human resources and reagent requirements.

Evaluation of observed and unobserved self-collection of saline gargle samples for the detection of SARS-CoV-2 in outpatients.

The diagnostic sensitivity of observed and unobserved self-collected saline gargle samples for the molecular detection of SARS-CoV-2 in adults and school-aged children was evaluated against a reference standard of health care worker collected nasopharyngeal flocked swab. A total of 46 participants had a positive nasopharyngeal swab sample; of these, 10 were in the observed phase and 36 were in the unobserved phase. Only one matching saline gargle sample tested negative and this was in the unobserved phase, giving an overall sensitivity of 98%. Average viral target Ct values were higher in the saline gargle samples. RNaseP Ct values were lower in unobserved collected samples compared to observed collected samples. Unobserved self-collection of saline gargle samples is a promising outpatient testing method for COVID-19 diagnosis. The self-collection method has potential to simplify the diagnostic cycle and facilitate implementation of COVID-19 testing, particularly in settings with limited access to health care workers.

Development and validation of a new triplex real-time quantitative reverse Transcriptase-PCR assay for the clinical detection of SARS-CoV-2.

To increase the repertoire of PCR based laboratory developed tests (LDTs) for the detection of SARS-CoV-2, we describe a new multiplex assay (SORP), targeting the SARS-CoV-2's, Spike and ORF8 genes. The widely used human RNaseP internal control was modified to specifically co-amplify the RNaseP mRNA. The SORP triplex assay was tested on a cohort (n = 372; POS = 144/NEG = 228) of nasopharyngeal flocked swab (NPFS) specimens, previously tested for the presence of SARS-CoV-2 using a PCR assay targeting E and RdRp genes. The overall sensitivity and specificity of the SORP assay was: 99.31% (95% CI: 96.22-99.98%), 100.0% (95% CI: 98.4-100%) respectively. The SORP assay could also detect a panel of variants of concern (VOC) from the B1.1.7 (UK) and B1.351 (SA) lineage. In summary, access to a repertoire of new SARS-CoV-2 LDT's would assist diagnostic laboratories in developing strategies to overcome some of the testing issues encountered during high-throughput SARS-CoV-2 testing.

Self-Collected Saline Gargle Samples as an Alternative to Health Care Worker-Collected Nasopharyngeal Swabs for COVID-19 Diagnosis in Outpatients.

We assessed the performance, stability, and user acceptability of swab-independent self-collected saliva and saline mouth rinse/gargle sample types for the molecular detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in adults and school-aged children. Outpatients who had recently been diagnosed with COVID-19 or were presenting with suspected COVID-19 were asked to have a nasopharyngeal (NP) swab collected and provide at least one self-collected sample type. Participants were also asked about sample acceptability using a five-point Likert scale. For those previously diagnosed with COVID-19, all samples underwent real-time PCR testing using a lab-developed assay, and the majority were also tested using an FDA-authorized assay. For those presenting with suspected COVID-19, only those with a positive nasopharyngeal swab sample went on to have other samples tested. Saline mouth rinse/gargle and saliva samples were tested daily at time zero, day 1, and day 2 to assess nucleic acid stability at room temperature. Fifty participants (aged 4 to 71 years) were included; of these, 40 had at least one positive sample and were included in the primary sample yield analysis. Saline mouth rinse/gargle samples had a sensitivity of 98% (39/40), while saliva samples had a sensitivity of 79% (26/33). Both saline mouth rinse/gargle and saliva samples showed stable viral RNA detection after 2 days of room temperature storage. Mouth rinse/gargle samples had the highest (mean, 4.9) and health care worker (HCW)-collected NP swabs had the lowest acceptability scores (mean, 3.1). In conclusion, saline mouth rinse/gargle samples demonstrated higher combined user acceptability ratings and analytical performance than saliva and HCW-collected NP swabs. This sample type is a promising swab-independent option, particularly for outpatient self-collection in adults and school-aged children.

Asymptomatic Pharyngeal Carriage of Kingella kingae Among Young Children in Vancouver, British Columbia, Canada.

BACKGROUND: Kingella kingae has emerged as a significant cause of osteoarticular infections in young children. Pharyngeal colonization is considered a prerequisite for invasive K. kingae infection. We conducted a prospective study to estimate the prevalence of pharyngeal carriage of K. kingae among healthy young children in Vancouver. METHODS: From March 2016 to May 2017, children between 6 and 48 months of age visiting British Columbia Children's Hospital outpatient clinics for noninfectious causes were included in the study. Another set of participants was enrolled from a day-care center located at British Columbia Children's Hospital. A single-throat swab was collected after obtaining consent from parent/guardian. The samples were stored at -70°C and tested using an in-house developed real-time polymerase chain reaction assay. Epidemiologic characteristics and risk factors for K. kingae colonization were collected via a study questionnaire. RESULTS: A total of 179 children were enrolled in the study, but only 174 samples were eligible for testing. Of the 174 samples, 5 had indeterminate results and the remaining 169 samples were negative by K. kingae polymerase chain reaction. The median age of participants was 23 months. About 36% of children were attending day care and had another sibling <5 years of age. Previous history of cold symptoms and antibiotic use was reported in 42% and 12%, respectively. CONCLUSIONS: The results of our study showed no prevalence of asymptomatic pharyngeal carriage of K. kingae in young children in Vancouver. Additional multicenter studies may help to understand the differences in pharyngeal carriage rate among healthy children.

Real-time Detection and Monitoring of Loop Mediated Amplification (LAMP) Reaction Using Self-quenching and De-quenching Fluorogenic Probes.

Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification (iNAAT) technique known for its simplicity, sensitivity and speed. Its low-cost feature has resulted in its wide scale application, especially in low resource settings. The major disadvantage of LAMP is its heavy reliance on indirect detection methods like turbidity and non-specific dyes, which often leads to the detection of false positive results. In the present work, we have developed a direct detection approach, whereby a labelled loop probe quenched in its unbound state, fluoresces only when bound to its target (amplicon). Henceforth, referred to as Fluorescence of Loop Primer Upon Self Dequenching-LAMP (FLOS-LAMP), it allows for the sequence-specific detection of LAMP amplicons. The FLOS-LAMP concept was validated for rapid detection of the human pathogen, Varicella-zoster virus, from clinical samples. The FLOS-LAMP had a limit of detection of 500 copies of the target with a clinical sensitivity and specificity of 96.8% and 100%, respectively. The high level of specificity is a major advance and solves one of the main shortcomings of the LAMP technology, i.e. false positives. Self-quenching/de-quenching probes were further used with other LAMP primer sets and different fluorophores, thereby demonstrating its versatility and adaptability.

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.

Complete Genome Sequence of Pseudomonas fluorescens LBUM636, a Strain with Biocontrol Capabilities against Late Blight of Potato.

Herein provided is the full-genome sequence of Pseudomonas fluorescens LBUM636. This strain is a plant growth-promoting rhizobacterium (PGPR) which produces phenazine-1-carboxylic acid, an antibiotic involved in the biocontrol of numerous plant pathogens, including late blight of potato caused by the plant pathogen Phytophthora infestans.

Complete Genome Sequence of Pseudomonas brassicacearum LBUM300, a Disease-Suppressive Bacterium with Antagonistic Activity toward Fungal, Oomycete, and Bacterial Plant Pathogens.

Pseudomonas brassicacearum LBUM300, a plant rhizosphere-inhabiting bacterium, produces 2,4-diacetylphloroglucinol and hydrogen cyanide and has shown antagonistic activity against the plant pathogens Verticillium dahliae, Phytophthora cactorum, and Clavibacter michiganensis subsp. michiganensis. Here, we report the complete genome sequence of P. brassicacearum LBUM300.

Complete Genome Sequence of Biocontrol Strain Pseudomonas fluorescens LBUM223.

Pseudomonas fluorescens LBUM223 is a plant growth-promoting rhizobacterium (PGPR) with biocontrol activity against various plant pathogens. It produces the antimicrobial metabolite phenazine-1-carboxylic acid, which is involved in the biocontrol of Streptomyces scabies, the causal agent of common scab of potato. Here, we report the complete genome sequence of P. fluorescens LBUM223.

Validation of endogenous reference genes in Buglossoides arvensis for normalizing RT-qPCR-based gene expression data.

Selection of a stably expressed reference gene (RG) is an important step for generating reliable and reproducible quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) gene expression data. We, in this study, have sought to validate RGs for Buglossoides arvensis, a high nutraceutical value plant whose refined seed oil is entering the market under the commercial trade name Ahiflower™. This weed plant has received attention for its natural ability to significantly accumulate the poly-unsaturated fatty acid (PUFA) stearidonic acid (SDA, C18:4n-3) in its seeds, which is uncommon for most plant species. Ten candidate RGs (ß-Act, 18S rRNA, EF-1a, α-Tub, UBQ, α-actin, CAC, PP2a, RUBISCO, GAPDH) were isolated from B. arvensis and TaqMan™ compliant primers/probes were designed for RT-qPCR analysis. Abundance of these gene transcripts was analyzed across different tissues and growth regimes. Two of the most widely used algorithms, geNorm and NormFinder, showed variation in expression levels of these RGs. However, combinatorial analysis of the results clearly identified CAC and α-actin as the most stable and unstable RG candidates, respectively. This study has for the first time identified and validated RGs in the non-model system B. arvensis, a weed plant projected to become an important yet sustainable source of dietary omega-3 PUFA.

Fidelity and representativeness of two isothermal multiple displacement amplification systems to preamplify limiting amounts of total RNA.

In this study we investigated the fidelity and representativeness of two novel multiple displacement amplification (MDA) protocols leading to whole transcriptome amplification (WTA). WTA is used to amplify a limiting amount of experimental RNA, allowing its use in downstream applications. Using Phi29 and Bst DNA polymerase-based MDA, henceforth referred to as WTA-Phi and WTA-Bst, respectively, we successfully amplified very low amounts of linearly concatenated cDNA originating from 10 to 100 ng of starting RNA. The average yield obtained from 10 ng was 3.5 and 4.7 µg for WTA-Phi and WTA-Bst, respectively, while 100 ng of starting RNA yielded 7.0 and 12.4 µg for WTA-Phi and WTA-Bst, respectively. Representational distortion of the templates, analyzed via conventional PCR, showed robust amplification of 11 different transcripts when either WTA-Phi or WTA-Bst synthesized templates were used, while some transcripts were not detected from unamplified templates. Loci representation, a measure of amplification consistency, was evaluated using TaqMan RT-qPCR amplification of five different transcripts, yielding values ranging from 96.4 to 189.3 %, comparable to those obtained using genomic target-based MDA systems. The two MDA protocols described in this study efficiently lead to representative WTA, using as little as 10 ng of starting RNA.

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.

A linear concatenation strategy to construct 5'-enriched amplified cDNA libraries using multiple displacement amplification.

In various experimental systems, limiting available amounts of RNA may prevent a researcher from performing large-scale analyses of gene transcripts. One way to circumvent this is to 'pre-amplify' the starting RNA/cDNA, so that sufficient amounts are available for any downstream analysis. In the present study, we report the development of a novel protocol for constructing amplified cDNA libraries using the Phi29 DNA polymerase based multiple displacement amplification (MDA) system. Using as little as 200 ng of total RNA, we developed a linear concatenation strategy to make the single-stranded cDNA template amenable for MDA. The concatenation, made possible by the template switching property of the reverse transcriptase enzyme, resulted in the amplified cDNA library with intact 5' ends. MDA generated micrograms of template, allowing large-scale polymerase chain reaction analyses or other large-scale downstream applications. As the amplified cDNA library contains intact 5' ends, it is also compatible with 5' RACE analyses of specific gene transcripts. Empirical validation of this protocol is demonstrated on a highly characterized (tomato) and an uncharacterized (corn gromwell) experimental system.

Production of DAPG and HCN by Pseudomonas sp. LBUM300 contributes to the biological control of bacterial canker of tomato.

Bacterial canker caused by Clavibacter michiganensis subsp. michiganensis is known to cause significant economic losses to tomato production worldwide. Biological control has been proposed as an alternative to current chemical containment methods, which are often inefficient and may leave adverse effects on the environment. However, only little headway has so far been made in developing biocontrol strategies against C. michiganensis subsp. michiganensis. To address this knowledge gap, we investigated the antagonistic capacity of PCA, produced by Pseudomonas sp. LBUM223, and DAPG and HCN, both produced by Pseudomonas sp. LBUM300, on C. michiganensis subsp. michiganensis under in vitro and in planta conditions. Nonsynthesizing isogenic mutants of the producer strains were also developed to further dissect the role of each individual metabolite on C. michiganensis subsp. michiganensis biological control. Novel specific quantitative polymerase chain reaction TaqMan assays allowed quantification of C. michiganensis subsp. michiganensis in tomato plants and rhizospheric soil. Pseudomonas spp. LBUM223 and LBUM300 significantly repressed C. michiganensis subsp. michiganensis growth in vitro, while their respective nonproducing mutants showed less or no significant antagonistic activity. In planta, only Pseudomonas sp. LBUM300 was capable of significantly reducing disease development and C. michiganensis subsp. michiganensis rhizospheric population, suggesting that the production of both DAPG and HCN was involved. In summary, simultaneous DAPG/HCN production by Pseudomonas sp. LBUM300 shows great potential for controlling bacterial canker of tomato.

A novel method to perform genomic walks using a combination of single strand DNA circularization and rolling circle amplification.

Characterization of regions flanking a known sequence within a genome, known as genome walking, is a cornerstone technique in modern genetic analysis. In the present work we have developed a new PCR-dependent, directional genome walking protocol based on the unique circularization property of a novel DNA ligase, CircLigase. In the first step, PCR based primer extension is performed using a phosphorylated primer, designed to extend from the boundary of the known sequence, into the flanking region. This linear amplification results in the generation of single-stranded (ss) DNA, which is then circularized using CircLigase. Using the hyperbranching activity of Phi29 DNA polymerase, the circular ssDNA is then linearized by rolling circle amplification, resulting in copious amounts of double stranded concatameric DNA. Nested primers are used to amplify the flanking sequence using inverse PCR. The products are resolved on an agarose gel and the bands whose mobility change due to the nested location of the primer combination used are identified, extracted, and cloned into a plasmid vector for sequencing. Empirical proof for this concept was generated on two antimicrobial biosynthetic genes in Pseudomonas sp. LBUM300. Using the hcnB and phlD genes as starting points, ca 1 kb of flanking sequences were successfully isolated. The use of locus specific primers ensured both directionality and specificity of the walks, alleviating the generation of spurious amplicons, typically observed in randomly primed walking protocols. The presented genome walking protocol could be applied to any microbial genome and requires only 100-150 bp of prior sequence information. The proposed methodology does not entail laborious testing of restriction enzymes or adaptor ligation. This is the first report of a successful application of the novel ligase enzyme, CircLigase for genomic walking purposes.

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.

Relative and absolute quantitative real-time PCR-based quantifications of hcnC and phlD gene transcripts in natural soil spiked with Pseudomonas sp. strain LBUM300.

Transcriptional analysis of microbial gene expression using relative quantitative real-time PCR (qRT-PCR) has been hampered by various technical problems. One such problem is the unavailability of an exogenous standard robust enough for use in a complex matrix like soil. To circumvent this technical issue, we made use of a recently developed artificial RNA (myIC) as an exogenous "spike-in" control. Nonsterile field soil was inoculated with various concentrations of the test bacterium Pseudomonas sp. strain LBUM300, ranging from 4.3- to 8.3-log bacterial cells per gram of soil. Total soil RNA was extracted at days 0, 7, and 14 postinoculation, and using two-step TaqMan assays, phlD (encoding the production of 2,4-diacetylphloroglucinol) and hcnC (encoding the production of hydrogen cyanide) gene expression was monitored. For relative quantification, a defined quantity of in vitro-synthesized myIC RNA was spiked during the RNA extraction procedure. Absolute qRT-PCR was also performed in parallel. Both the absolute and relative quantifications showed similar transcriptional trends. Overall, the transcriptional activity of phlD and hcnC changed over time and with respect to the bacterial concentrations used. Transcripts of the phlD and hcnC genes were detected for all five bacterial concentrations, but the phlD transcript copy numbers detected were lower than those detected for hcnC, regardless of the initial bacterial concentration or sampling date. For quantifying a low number of transcripts, the relative method was more reliable than the absolute method. This study demonstrates for the first time the use of a relative quantification approach to quantifying microbial gene transcripts from field soil using an exogenous spike-in control.

Verticillium dahliae alters Pseudomonas spp. populations and HCN gene expression in the rhizosphere of strawberry.

The production of hydrogen cyanide (HCN) by beneficial root-associated bacteria is an important mechanism for the biological control of plant pathogens. However, little is known about the biotic factors affecting HCN gene expression in the rhizosphere of plants. In this study, real-time reverse transcription PCR (qRT-PCR) assays were developed to investigate the effect of the plant pathogen Verticillium dahliae on hcnC (encoding for HCN biosynthesis) gene expression in Pseudomonas sp. LBUM300. Strawberry plants were inoculated with Pseudomonas sp. LBUM300 and (or) V. dahliae and grown in pots filled with nonsterilized field soil. RNA was extracted from rhizosphere soil sampled at 0, 15, 30, and 45 days following inoculation with V. dahliae and used for qRT-PCR analyses. Populations of V. dahliae and Pseudomonas sp. LBUM300 were also monitored using a culture-independent qPCR approach. hcnC expression was detected at all sampling dates. The presence of V. dahliae had a significant stimulation effect on hcnC gene expression and also increased the population of Pseudomonas sp. LBUM300. However, the V. dahliae population was not altered by the presence of Pseudomonas sp. LBUM300. To our knowledge, this study is the first to evaluate the effect of a plant pathogen on HCN gene expression in the rhizosphere soil.