The infected samples were first tested through DAS-ELISA by following the standard protocol. Further from the positive PLRV samples identified through DAS-ELISA, total RNA were extracted from mid rib and major veins of infected leaf using RNA extraction kit and first strand cDNA was synthesized using cDNA synthesis kit (Fermentas) with respective primers. Finally, to confirm the PLRV infection, RT-PCR was performed using PLRV coat protein gene specific primers. Total RNA were extracted from the all possible parts of infected plant (viz., tuber, main shoot, branched shoot and leaf) and electrophoresis analysis of RT-PCR product showed a single amplified fragment of 627 bp (Fig. 1A and B). Further, TA cloning approach was employed to sequence all the amplified cp gene from different part of infected plants.
We used same tubers from infected plant as a seed for next season cropping in controlled environment and all the results were further validated. Our consistent data of four cropping seasons indicate that these tubers act as a reservoir for these viruses and leads to the emergence of unhealthy progeny plants. From infected tubers, all the parts of progeny plants could get infected via systemic translocation of the PLRV (Fig. 1C). Because the aphid population increases exponentially during late winter season, the probability that they will encounter an infected plant is more. Aphids acquire the PLRV into the salivary glands by feeding on infected plants and further continue to transmit the virus to healthy plants for its remaining life and completes the disease cycle (Fig. 1C). Our present study also supports the findings of Leonard and Holbrook (1978) on role of aphid in PLRV pathogenicity. Control depends solely on the use of disease-free seeds for planting [8, 9]. Farmers of indo-gangetic plain of this region import the potato seeds from neighbour states or local companies, where risk of getting unhealthy seeds is very high. As a result, the pressure of production of disease-free seed potato has become a major challenge. The most effective approach to manage the viral diseases is planting of healthy and virus free seed potato. The diagnosis and management of aphids are considered to be an important step towards preventing spread of the viruses in potato. Our lab is also trying to the development of a rapid, reliable and cost-effective protocol to detect simultaneously multiple virus infection in a single assay in plants and tubers (Funded by DBT, Govt. of India).
Although, the study of Wright and Bishop (1981) reveals that potato tubers may act as volunteer source for PLRV and potato virus X [6], but the molecular basis for PLRV aggravation has been not reported so far. Our present study has dissected the mechanism of transmission of PLRV by collective influence of potato tuber and aphids. As PLRV has emerged as devastating disease in the eastern indo-gangetic plain during last 5 years, the small and marginal farmers of this region are suffering with huge economical loss due to inadequate screening of quality of tubers. Till date, there was no reports on how PLRV spreads and affect the crop production particularly in this region, the present study validates the findings of disease biology of PLRV in order to find out an effective and timely management. Our present findings have opened up an opportunity for developing strategies for timely management of PLRV before planting and/or before rising up the aphid populations by breaking out the chain in the peak season of aphid multiplication. Our lab is also involved in development of kit based diagnosis of tubers for early identification and further conducting knockdown study of PLRV, which could help the scientific community in order to undertake long term management against this disease. This work will also have a significant impact on scientific community for developing PLRV resistance plant vis-a-vis agricultural sector to produce disease-free potato seed.