Absolute and relative quantities of 16SrII and 16SrIX group phytoplasmas determined by qPCR in plant and insect samples tested in this study.

<p>Absolute and relative quantities of 16SrII and 16SrIX group phytoplasmas determined by qPCR in plant and insect samples tested in this study.</p

Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors

<div><p>Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (<i>Sesamum indicum</i> L.). Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan^® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 10² and 1.6 x 10² DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease.</p></div

Sequence alignment of the 16S ribosomal gene region used for amplification and specific detection of sesame 16Sr group II and IX phytoplasmas.

<p>Sequences of the primers and probes designed in this study are shown in underlined and italic letters, respectively. Forward primer SPHY-16SrII-IX-F (upper arrow, red), probe 16SrII (dotted line, blue) and 16SrIX (solid line, green), and reverse primer SPHY-16SrII-IX-R (lower arrow, red) are noted. The sequence differences between 16SrII and 16SrIX in the probe regions were shown in boxed letters.</p

Slope, intercept, correlation coefficients (R²), and efficiencies (E) of qPCR assay.

<p>Slope, intercept, correlation coefficients (R²), and efficiencies (E) of qPCR assay.</p

Multiplex qPCR detection of 16SrII and 16SrIX group phytoplasmas in plant and insect samples collected from different locations of Antalya province in Turkey.

<p>Multiplex qPCR detection of 16SrII and 16SrIX group phytoplasmas in plant and insect samples collected from different locations of Antalya province in Turkey.</p

Standard curves generated from the multiplex qPCR amplification of seven 10-fold dilution series of 16SrII (A) and 16SrIX (B) group standards used to convert Ct values to the phytoplasma DNA copies present in the samples.

<p>The amplification mixture contained phytoplasma and sesame primers, as well as 16SrII, 16SrIX, and sesame probes. Each dilution series was added to 10 ng of uninfected sesame DNA per reaction as part of the inhibition experiments. Log₁₀ values of the initial copies of phytoplasma DNA are plotted against corresponding Ct values.</p

Multiplex qPCR amplification of seven 10-fold serial dilutions of 16SrII (A) and 16SrIX (B) group phytoplasma DNA (purified PCR products).

<p>The amplification mixture contained phytoplasma and sesame primers and probes. Each dilution series was mixed with a constant 10 ng (2 μl) of healthy sesame plant DNA in each reaction to determine whether PCR inhibitors were present. Each line represents one of the experiment made in triplicate. Delta Rn (ΔRn) values show the fluorescence emission accumulation normalized to the background emission. Cycle threshold (Ct) values indicate specific cycles at which ΔRn passes the threshold value.</p

Primers and TaqMan^® probes designed for multiplex qPCR detection of 16SrII and 16IX phytoplasmas and sesame DNA.

<p>Primers and TaqMan^® probes designed for multiplex qPCR detection of 16SrII and 16IX phytoplasmas and sesame DNA.</p