Protocol

Centre : Central Citrus Research Institute, Nagpur
Name of the Protocol
Development of SCAR markers for species-specific detection and identification of Phytophthora nicotianae
Nature : Novel
Crop / Pathogen : Citrus / Phytophthora nicotianae

Inventors

1. Das, A.K.
2. Gawande, N.D.
3. Nerkar, S.G.
4. Thakre, N.
5. Kumar. A.

Objectives

1. To develop species-specific PCR primers for Phytophthora nicotianae outside the intergenic sequences of ribosomal genes, using Sequence-Characterized Amplified Region (SCAR) markers Phytophthora nicotianae.

Procedure / Summary

Primer pair was designed from the Sequence Characterized Amplified Region (SCAR) obtained by screening 20 RAPD primers (OPA1 to OPA20). The polymorphic band associated only with Phytophthora nicotianae was sequenced and specific primers (SCAR4F/SCAR4R) were designed. PCR reactions were carried out in a volume of 25µl, consisting of 1X PCR buffer (Thermo Fisher Scientific Inc., USA), 2 mM MgCl2 (Fermentas Inc. Maryland, USA), 0.2 mM each dNTP (Fermentas Inc. Maryland, USA), 0.5 µM of each SCAR primer (SCAR4F/ SCAR4R) (Integrated DNA Technologies, Coralville, USA), 1 U of Taq DNA polymerase (Bioline USA Inc, Taunton, MA) and 1 µl of template DNA (20 - 40 ng/ìl). Amplifications were performed on a BIORAD T100TM Thermal Cycler using the following cycling protocols: initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation, annealing and elongation steps respectively at 94°C for 45 s , 58°C for 1 min and 72°C for 1 min and a final extension step of 72°C for 10 min.

Applications

1. The developed SCAR-based PCR assay proved to be reliable, simple and rapid technique to detect P. nicotianae in laboratory (mycelial) cultures as well as in infected plant materials.