Protocol

Centre : Central Tuber Crops Research Institute(CTCRI),TVM
Name of the Protocol
Rapid and sensitive detection of Phytophthora colocasiae responsible for the taro leaf blight using conventional and real-time PCR assay
Nature : Modification of existing protocol
Crop / Pathogen : Taro/ Phytophthora colocaisae

Inventors

1. Nath V. S.
2. Hegde V. M.
3. Jeeva M. L.
4. Misra R. S.
5. Veena S. S.
6. Raj M.
7. Unnikrishnan S.K.
8. D. S. Sankar

Objectives

1. To design species specific primers to amplify Phytophthora colocaisae genes
2. To develop species specific detection of Phytophthora colocaisae through PCR based technique

Procedure / Summary

A set of three primer pairs was designed from regions of the RAS-related protein (Ypt1), G protein alpha-subunit (GPA1) and phospho-ribosylanthranilate isomerase (TRP1) genes. Amplifications were performed in an Agilent sure cycler 8800 (Agilent Technologies). The PCRs were conducted in a volume of 25 µL, which included 100 ng of template DNA, 100 µM each deoxynucleotide triphosphate, 10 ng of each primer, 1.5 mM MgCl2, 19 Taq buffer (10 mM Tris-HCl pH 9.0, 50 mM KCl, 0.01% gelatin), 1 U of Taq DNA polymerase (Merck GeNei, India). The following optimized PCR regime was used: denaturation at 95 °C for 2 min, followed by 35 cycles of 95 °C for 30 s, 55 °C for 45 s, 72 °C for 1 min and a final extension at 72 °C for 8 min.

Applications

1. The developed PCR assay proved to be a robust and reliable technique to detect P. colocasiae in taro planting material and for assessing the distribution of pathogen within fields

Publications

1. Nath V. S., Hegde V. M., Jeeva M. L., Misra R. S., Veena S. S., Raj M., Unnikrishnan S.K., D. S. Sankar (2014) Rapid and sensitive detection of Phytophthora colocasiae responsible for the taro leaf blight using conventional and real-time PCR assay. FEMS microbiology letters. 352: 174–183. DOI: 10.1111/1574-6968.12395