Engin Yol

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

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 (Sesamum indicum 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.

Traits for Phenotyping

The term plant phenotyping has been regenerated with the contribution of sensors, system technologies, and algorithms. This new plant describing concept allows multi-trait assessment with automatic measurements. Uniform structure, nondestructive measurements, precise results, and direct storage are the advantages of digital phenotyping. The hyper-spectral spectroradiometers and imaging technologies lead the way of new plant phenotyping applications. This high-throughput technique therefore requires lots of traditional and novel traits to present new characterization. Digital-based phenotyping in plants is new and still a developing area of research. The most often used traits of digital phenotyping are canopy temperature, chlorophyll fluorescence, stomatal conductance, chlorophyll content, leaf water potential, leaf area, fruit color, carbon isotope discrimination, light interception, senescence, and root traits which have been discussed in this chapter together with their advantages, limitations, and plant breeding potentials.

Screening, selection and real-time qPCR validation for phytoplasma resistance in sesame (Sesamum indicum L.)

Phyllody is one of the most destructive diseases of sesame and causes serious yield losses worldwide. The present research was conducted to identify phyllody resistant genotypes in sesame. A total of 542 sesame genotypes were screened for the disease resistance in the field using a disease incidence scale of 1–5 in the year 2012. Three hundred four genotypes showing high disease intensity were eliminated under artificially infected field conditions. In the year 2013, only 30 out of 238 accessions were determined as potential resistant genotypes based on the disease incidence scale. These selected genotypes were further evaluated for confirmation of the resistance in greenhouse conditions using the phytoplasma-infected vector insects under choice and no-choice conditions. Furthermore, real-time qPCR was employed for detection and quantification of phytoplasmas to select true resistant genotypes. The sesame accessions ACS38 and ACS102 were identified as resistant to the disease after evaluation in field, greenhouse and qPCR assays. This work is one of the most comprehensive studies to select genotypes resistant to the diseases caused by phytoplasmas.