Reverse transcription polymerase chain reaction-based detection of Arabis mosaic virus and Strawberry latent ringspot virus in vector nematodes

In a survey of rose and lily plantations in experimental fields of the Institute of Himalayan Bioresource Technology, Palampur, a number of symptoms characteristic of nepoviruses were observed. Symptomatic rose and lily leaves were tested for the presence of nepoviruses. Strawberry latent ringspot virus (SLRSV) and Arabis mosaic virus (ArMV) were detected by ELISA and RT–PCR using virus-specific antibodies and primers respectively, in rose cultivars Raktgandha, Landora and Sonia. Only SLRSV was detected in lily cultivars Galeili, Star Gazer Max and White Merostar. To ascertain the presence of viruliferous nematodes, soil samples were collected from around the roots of rose and lily cultivars. Xiphinema diversicaudatum and Longidorous macrosoma isolated from soil were placed separetely into 5–10 μl each of RNAlater™ and sterile water. Total RNA from these nematodes was isolated using three different RNA isolation kits, viz. RNAqueous™, RNeasy Plant mini kit and RNAwiz™. RT–PCR was performed using virus-specific primers for SLRSV and ArMV. Out of the three RNA isolation kits used, only RNAaqueous™ gave reproducible results. Nematodes isolated and stored in RNAlater™ gave amplification of expected size (over a period of one month storage at -20°C) for both the viruses, while nematodes stored in water did not give any amplification (even after one week of storage) with RT–PCR. Cucumis sativus was used as bait plant for confirming the nematode transmission of these viruses. In the present study RNAlater™ was found to be the best nematode storage solution and RNAqueous™ the best RNA isolation kit for virus detection in viruliferous nematodes by RT–PCR.