Protocol

Centre : Central Citrus Research Institute, Nagpur
Name of the Protocol
Development of Phytophthora nicotianae specific primers from extracellular cystatin-like cysteine protease inhibitor (EPIC1) gene
Nature : Novel
Crop / Pathogen : Citrus / Phytophthora nicotianae

Inventors

1. Das, A.K.
2. Nerkar, S.G.

Objectives

1. To design extracellular cystatin-like cysteine protease inhibitor (EPIC1) gene-based PCR primers and to validate its use as a diagnostic tool for the detection of P. nicotianae

Procedure / Summary

Amplifications were performed on a BIORAD T100TM Thermal Cycler PCR machine. PCR reactions were executed in a total volume of 25 ìl containing 1X PCR buffer (Fermentas Inc. Maryland, USA), 0.2 mM of each dNTPs (Fermentas Inc. Maryland, USA), 2 mM MgCl2 (Fermentas Inc. Maryland, USA), 0.5 µM of each (FEPIC1F/FEPIC1R) primers (Integrated DNA Technologies, Coralville, USA), 1 U of Taq DNA polymerase (Invitrogen, Life Technologies Corporation, USA) and 1 ìl template DNA (20 to 40 ng/ìl). The thermal cycling conditions used was: initial denaturation at 94°C for 3 min followed by 25 cycles of denaturation, annealing and elongation steps respectively at 94°C for 30 , 58°C for 30 sec and 72°C for 1 min and a final extension step of 72°C for 10 min.

Applications

1. This technique is a reliable, simple and rapid technique to detect P. nicotianae in mycelia cultures as well as in infected plant materials and gives an extension to a range of DNA-based markers to simplify P. nicotianae detection.

Publications

1. Nerkar, S.G. and Das, A.K. (2017) Extracellular Cystatin-like Protease Inhibitor (EPIC1) Gene Based PCR Primers for Specific Detection of Phytophthora nicotianae Infecting Citrus. Plant Pathology Journal. DOI: 10.3923/ppj.2017.