MSc

Vanilla, often quoted as the ‘Princess of Spices’, owing to its unique flavour and aroma is the second most expensive spice on the world market and a valuable source of foreign exchange for several countries. Viruses are known to cause significant yield reduction in many vanilla growing countries of the world. Vanilla mosaic disease was first reported by Wisler et al. (1987) from French Polynesia. Recently mosaic disease has been reported from South India (Thomas, 2002). The virus was identified to be a potyvirus and is sap transmissible. It is known that viruses can be readily transmitted through tissue culture process. To produce virus free plantlets, the source plants for tissue culture must be completely free of virus. Considering the nature of spread and damage that can be caused by the virus, it is essential that we should take effective control measures in the initial stage itself. Recent advances in biotechnology and molecular biology have played a significant role in development of rapid, specific and sensitive assays for detection of plant viruses. RT-PCR and PCR are popular techniques for detection and identification of RNA and DNA plant viruses respectively. The procedures are extremely sensitive, fairly inexpensive and require minimal skill to perform and it has the potential to be an extremely sensitive alternative to ELISA. In the present study an attempt was made to develop a reverse transcription polymerase chain reaction assay to detect vanilla mosaic virus (VanMV) infecting vanilla.
Mosaic and necrosis infected vanilla plants collected from different locations of Kerala were used to inoculate the test plants viz., healthy tissue culture-derived vanilla. Biological indexing of the virus on petunia (Petunia hybrida) and cowpea (Vigna ungiculata subsp. sesquipedalis) seedlings, which are the indicator plants of potyvirus and cucumber mosaic virus (CMV) respectively, clearly indicated that the infection is due to potyvirus, not due to CMV.
RNA was isolated from mosaic infected plants collected from different locations of Kerala, artificially inoculated vanilla samples and also from healthy tissue culture-derived vanilla plants and this was used for cDNA synthesis with oligo dT primers using MMuLV RT-PCR kit.
A set of degenerate primers was designed. Of these two sets were designed based on published data (RKJ3 and HRP52; PotyF1 and PotyR1) and the other one (PVF1 and PVR1) was based on the conserved sequence of coat protein genes of potyviruses, and these were used in PCR. Primer pair RKJ3 and HRP52 yielded a product of lower size [approximately 300 bp than expected (850 bp)]. On sequencing the amplified product gave information on 240 bp and sequence analysis using blastn revealed identify with chloroplast genome. This could be due to nonspecific amplification. Hence based on the present study this primer pair cannot be recommended for detection of poty viruses infecting vanilla. Attempts made to obtain amplification with primers PotyF1 and PotyR1 did not prove successful. Hence this primer pair was not used for further experiments.
Amplification of two bands (approximately 350 bp and 149 bp) could be observed with primer combination PVF1 and PVR1 invariably with all the isolates collected from different locations. The similar bands were even yielded with all the artificially inoculated vanilla samples, confirming that it was the coat protein gene of potyvirus that was amplified. It was not found with the analysis of healthy control suggesting that the characteristic bands are of virus coat protein gene. Two bands yielded may be due to binding of primers at an internal site of potyvirus sequence. Reamplification of the upper band gave two bands of 350 bp and 149 bp, corresponding to the bands obtained in PCR analysis and the lower band gave an intact band of 149 bp. Hence it could be concluded that the amplified product is a part of coat protein gene of vanilla mosaic potyvirus. Therefore, RT-PCR assay using PVF1 and PVR1 primers could be used for rapid and easy detection of potyviruses infecting vanilla. However, sequence information of the amplified fragment is necessary before this method is recommending on a large scale for the detection of potyviruses infecting vanilla.