Development of a Real-Time Microchip PCR System for Portable Plant Disease Diagnosis

Rapid and accurate detection of plant pathogens in the field is crucial to prevent the proliferation of infected crops. Polymerase chain reaction (PCR) process is the most reliable and accepted method for plant pathogen diagnosis, however current conventional PCR machines are not portable and require additional post-processing steps to detect the amplified DNA (amplicon) of pathogens. Real-time PCR can directly quantify the amplicon during the DNA amplification without the need for post processing, thus more suitable for field operations, however still takes time and require large instruments that are costly and not portable. Microchip PCR systems have emerged in the past decade to miniaturize conventional PCR systems and to reduce operation time and cost. Real-time microchip PCR systems have also emerged, but unfortunately all reported portable real-time microchip PCR systems require various auxiliary instruments. Here we present a stand-alone real-time microchip PCR system composed of a PCR reaction chamber microchip with integrated thin-film heater, a compact fluorescence detector to detect amplified DNA, a microcontroller to control the entire thermocycling operation with data acquisition capability, and a battery. The entire system is 25 × 16 × 8 cm(3) in size and 843 g in weight. The disposable microchip requires only 8-µl sample volume and a single PCR run consumes 110 mAh of power. A DNA extraction protocol, notably without the use of liquid nitrogen, chemicals, and other large lab equipment, was developed for field operations. The developed real-time microchip PCR system and the DNA extraction protocol were used to successfully detect six different fungal and bacterial plant pathogens with 100% success rate to a detection limit of 5 ng/8 µl sample

Characterization of Five ECF Sigma Factors in the Genome of <em>Pseudomonas syringae</em> pv. syringae B728a

<div><p><i>Pseudomonas syringae</i> pv. syringae B728a, a bacterial pathogen of bean, utilizes large surface populations and extracellular signaling to initiate a fundamental change from an epiphytic to a pathogenic lifestyle. Extracytoplasmic function (ECF) sigma (σ) factors serve as important regulatory factors in responding to various environmental signals. Bioinformatic analysis of the B728a genome revealed 10 ECF sigma factors. This study analyzed deletion mutants of five previously uncharacterized ECF sigma factor genes in B728a, including three FecI-type ECF sigma factors (ECF5, ECF6, and ECF7) and two ECF sigma factors placed in groups ECF11 and ECF18. Transcriptional profiling by qRT-PCR analysis of ECF sigma factor mutants was used to measure expression of their associated anti-sigma and outer membrane receptor proteins, and expression of genes associated with production of extracellular polysaccharides, fimbriae, glycine betaine and syringomycin. Notably, the B728aΔ<i>ecf7</i> mutant displayed reduced swarming and had decreased expression of CupC fimbrial genes. Growth and pathogenicity assays, using a susceptible bean host, revealed that none of the tested sigma factor genes are required for <i>in planta</i> growth and lesion formation.</p> </div

Expression analysis in low iron media of putative iron-responsive genes in B728aΔ<i>ecf5</i>, B728aΔ<i>ecf6</i>, and B728aΔ<i>ecf7</i> mutant strains as compared to B728a.^a

a<p>Values represent the average fold differences of three technical replicates of three biological samples. Gene expression was normalized to the <i>16s-rRNA</i> and <i>recA</i> internal control genes. Negative values indicate a decrease in expression levels as computed by taking the negative inverse of a fold change value less than 1.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Expression analysis in strain B728a of three FecI-like ECF σ factor genes (<i>ecf5</i>, <i>ecf7</i>, and <i>ecf6</i>) and associated putative iron-responsive genes in low and high iron media.^a

a<p>Values represent the average fold differences of three technical replicates of three biological samples. Gene expression was normalized to the <i>16s-rRNA</i> and <i>recA</i> internal control genes. Fold changes for expression in iron-limited HMM were compared to cells grown in HMM supplemented with 10 µM iron. Fold changes for expression in HMM supplemented with 100 µM iron were compared to cells grown in HMM supplemented with 10 µM iron. Negative values indicate a decrease in expression levels as computed by taking the negative inverse of a fold change value less than 1.</p

Quantitative real-time PCR analysis of Type I fimbrial gene expression in Δ<i>ecf5</i> and Δ<i>ecf7</i> mutants of B728a.

<p>The pilus assembly/fimbrial biogenesis gene cluster (Psyr_1131-Psyr_1134) analyzed is located in close proximity to the <i>ecf7</i> sigma factor gene. Values represent the average fold differences in gene expression from parental strain B728a; results are averages of three technical replicates (to measure reproducibility from a single source) from each of three biological samples grown in NBY liquid medium at 25°C (∼5×10⁸ CFU/ml final concentrations). Gene expression levels were normalized to the <i>16S-rRNA</i> and <i>recA</i> internal control genes, and standard deviations from the mean are denoted by the error bars; asterisks denote greater than a 2-fold change in gene expression. A Student’s <i>t</i>-test was performed using 95% confidence interval to calculate <i>p</i>-values between biological replicates. Negative values indicate a decrease in expression levels as computed by taking the negative inverse of a fold change value less than 1.</p

Putative pilus assembly/fimbrial genes downstream of <i>ecf7</i> in B728a.^a

a<p>Percent amino acid identities of orthologs are shown in parentheses.</p

Pathogenicity assays to evaluate the contribution of the <i>ecf5</i> and <i>ecf7</i> genes to disease development in a susceptible bean (<i>Phaseolus vulgaris</i> cv. Blue Lake 274) host. (A) Disease symptoms.

<p>Bean leaves were inoculated using vacuum infiltration with cell suspensions containing 10⁶ CFU/ml of strain B728a, ECF sigma factor mutants B728aΔ<i>ecf5</i> and B728aΔ<i>ecf7</i>, and the avirulent B728aΔ<i>gacS</i> as a negative control. Plants were maintained at 25°C in a growth chamber for 6 days. The experiment was performed twice; representative results are shown 4 days after inoculation. <b>(B) Bacterial populations.</b> Bean leaf populations of the four bacterial strains identified in panel A were determined at days 0, 2, 4 and 6 days after inoculation with 10⁶ CFU/ml for each strain. Bacterial populations are shown in terms of the logarithm of CFU/cm² of leaf surface. Values are the average counts from four individual plants sampled at each time point; the experiment was repeated on two occasions. Error bars represent the standard errors (SE) of the respective means.</p

Genomic neighborhood of <i>ecf5</i>, <i>ecf7</i>, <i>ecf6</i>, <i>ecf18</i>, and <i>ecf11</i> sigma factor genes.

<p>The genes for ECF sigma factor genes are shown in red, the anti-sigma factor genes in brown, the FecA-like genes in yellow, a transmembrane (TM) helix protein in light green, and the toxin/antitoxin (Tx/Atx) genes in olive green; other nearby genes are shown in gray. Additional genes are labeled as T-Reg, transcriptional regulator; <i>gapA</i>, glyceraldehyde-3-phosphate dehydrogenase; <i>betA</i>, choline dehydrogenase; <i>nhaA</i>, Na+/H+antiporter NhaA; <i>cysE</i>, serine O-acetyltransferase; and <i>aroA</i>, 5-enolpyruvylshikimate-3-phosphate synthase.</p

ECF sigma factors in the genome of <i>Pseudomonas syringae</i> pv. syringae strain B728a.

a<p>ECF classification system according to Staroń et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058846#pone.0058846-Staro2" target="_blank">[27]</a>.</p>b<p>The closest homologs found in the genomes of <i>P. syringae</i> pv. phaseolicola strain 1448A and <i>P. syringae</i> pv. tomato strain DC3000 using BLASTP at the NCBI website.</p