CP GENE SPECIFIC DRT-PCR: A FAST AND COST-EFFECTIVE DIAGNOSTIC TOOL TO DETECT A VIRAL CO-INFECTION IN POTATO

Lalit Kharbikar, a scientist at ICAR - Central Research Institute for Jute and Allied Fibres, Barrackpore, India, who analyzed the results of this study and also wrote the manuscript suggests that primers PVACPF3/ PVACPR3 and PVMCPF2/PVMCPR2 designed to use in a duplex RT-PCR optimized in the present study offers a reliable diagnostic tool for the simultaneous detection of PVA and PVM in seed certification and quarantine programmes.

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Jul 01, 2017
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Potato, a tuber crop is the third most important source of staple food, after rice and wheat, for an ever-increasing population in the world. This crop is infected by many pathogens, including bacteria, fungi and viruses, causing a significant reduction in yield and quality of the tubers. Among these pathogens, viruses sometimes may pose a serious threat since they alone can cause up to 50 % reduction in tuber yield. Etiological observations would suggest that coinfection of two or more viruses is very frequent in potato. Among the viruses, Potato virus A (PVA, genus Potyvirus) and Potato virus M (PVM, genus Carlavirus) are of economic concern since they significantly reduce the tuber yield. PVM alone can reduce the tuber yield by 15 to 45 %. The reduction could be 100 %, in some potato growing regions of the world, if the virus incidence is high due to early or severe infection.

Both PVA and PVM are transmitted by aphids and spread in a non-persistent manner in the host. These viruses infect the potato crop either singly or simultaneously (coinfection). When infected singly, PVA shows very mild foliar symptoms of curling, vein deepening and leaf bronzing that are difficult to character­ize. Sometimes the crop even remains symptomless. As a consequence, growers who save the tubers, from such symptomless crop with latent PVA infection, for planting in the next growing season may bear huge losses. PVA has been reported to cause yield losses of up to 40% in potato. Upon coinfection of PVA and PVM in potato, severe foliar symptoms appear that can be easily characterized as a mosaic, mottling, crinkling and rolling of leaves. However, these symptoms are always attributed only to high PVA incidence; since this virus is commonly found to infect potato crop worldwide. Despite spraying systemic insecticides to control the vectors transmitting PVA, the growers bear huge losses in tuber yield. This happens because PVM, which also transmits mechanically and coinfects with PVA remains undetected. Consequently, growers who use systemic insecticides to kill its vector could not get good control of the disease.

Disease diagnostic and management strategies increasingly require fast and accurate methods for the detection of multiple pathogenic microorganisms from complex samples. Detection of several viruses individually by polymerase chain reaction (PCR) is expensive and time-consuming. Therefore, detection of at least two viruses in a single PCR assay (duplex PCR, D-PCR) would be more economical and time-saving. However, the success of duplex PCR mainly depends on the specificity of primers used in the assay. Coat protein (CP) gene-specific primers are known to be more sensitive than any other primers; since they can detect the virus in micro plants and dormant potato tubers harvested from infected plants. This is because CP gene is the most conserved gene in geminiviruses.

Plant health experts from the Central Potato Research Institute (CPRI), Shimla, India have developed a fast and cost-effective D-PCR tool that is cheaper than existing uniplex PCR method. This tool can simultaneously detect PVA and PVM in an optimized Real-Time - PCR (RT - PCR). Published in the Journal of Pharmacognosy and Phytochemistry, CPRI’s research demonstrated for the first time that CP gene-specific primers can be used to detect PVA and PVM simultaneously. First time used in biomedical research to detect the dystrophin gene in human, this method has now been successfully adapted to simultaneously detect and indicate the presence of PVA and PVM in potato.

Different primer pairs have been developed for the detection of potato viruses through amplification of their cDNAs. However, most of these primers lacked specificity for new variants of some potato viruses and hence could not satisfactorily differentiate all the isolates, such as in case of PVY. Since CP gene is the most conserved gene in geminiviruses the primers specific to this gene can detect most plant viruses not only from infected potato leaves but also from dormant tubers harvested from infected plants. This indicates that the CP gene-specific primers are more sensitive than any other primers. Therefore, in the present study different primer pairs specific to CP genes of PVA and PVM were designed and used for further amplification of cDNAs of these viruses in a duplex PCR.

PCR can be used for the detection of both DNA and RNA viruses. Since, most plant viruses are RNA viruses, amplifying RNA instead of DNA from the infected plant would increase the chances of their accurate detection. The RNA-dependent DNA polymerase (reverse transcriptase) is used to amplify an RNA molecule in a variant of PCR called RT-PCR. RT-PCR offers an easy, sensitive and reliable approach for the diagnosis of viruses in plants. The possibility of simultaneous detection of two or more viruses in a single RT-PCR has speed-up the process of diagnosis. Hence, detection of viruses through RT-PCR is not only efficient but also faster. Considering these and several other advantages of RT-PCR over the other virus diagnostic methods, a duplex RT-PCR for simultaneous detection of potato viruses A and M has been optimized in the present study. In RT-PCR oligo(dT) is generally used to prepare cDNA from mRNA. However, in the present study random primers were used for this purpose. Oligo(dT) can only be used if the viral RNAs contain polyadenylated tail. This means, in order to obtain cDNAs from viruses the mRNAs should be intact in terms of size and shape and their numbers must be known. In contrast, random primers can be used for the reverse transcription of all RNAs, irrespective of their size, shape and numbers.

Lalit Kharbikar, a scientist at ICAR - Central Research Institute for Jute and Allied Fibres, Barrackpore, India, who analyzed the results of this study and also wrote the manuscript suggests that primers PVACPF3/ PVACPR3 and PVMCPF2/PVMCPR2 designed to use in a duplex RT-PCR optimized in the present study offers a reliable diagnostic tool for the simultaneous detection of PVA and PVM in seed certification and quarantine programmes.

The research was conducted at the CPRI, Shimla, India by P.N. Meena, Ravinder Kumar, Baswaraj R and Jeevalatha A and facilitated by the Director of the institute. For more information visit: http://www.phytojournal.com/archives/?year=2017&vol=6&issue=4&part=X&ArticleId=1551

Go to the profile of Lalit Laxman Kharbikar

Lalit Laxman Kharbikar

Scientist, Indian Council of Agricultural Research

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