Duplex PCR for simultaneous detection of Begomovirus and Phytoplasma from naturally infected tomato



Symptoms associated with the tomato leaf curl disease (ToLCD) were observed in tomato fields from Southern Karnataka, India affected with the big bud phytoplasma. Surveys indicated incidence of tomato leaf curl disease from 27.5 to 74.3%, tomato big bud phytoplasma disease from 5.7 to 13.9% and mixed infection of leaf curl and big bud from 1.2 to 7.2% in different fields. Total DNA extracted from symptomatic and asymptomatic tomato was subjected to uniplex and duplex PCR by using Begomovirus and 16S-23SrRNA specific phytoplasma primers. Duplex PCR products of 1.2kb were obtained for 101/151 of leaf curl symptomatic tomato, 17/151 mixed infection as well as 0.5kb amplicons for 33/151 of big bud symptomatic tomato and 17/151 of mixed infection. No PCR products were observed for asymptomatic plants of 44/195. A full-length clone of Begomovirus was isolated from mixed infection and sequence analysis showed that the genome organization of this virus was found to be similar to those of other old world begomoviruses. The DNA-A molecule (2753 nt) sequences showed the highest levels of nucleotide sequence identity of 89.4 to 98.5% with the DNA-A of Tomato leaf curl New Delhi virus (ToLCNDV) isolates and less 70.2 to 86.3% identity with known tomato-infecting begomoviruses from Indian subcontinent. The 16S-23s rRNA sequences of TBB showed highest nucleotide identity of 97.6 to 99% with those of members of group 16SrII and Ca. Phytoplasma australasia, where as less than 96% with other phytoplasma groups. Duplex PCR enabled the simultaneous detection and differentiation of two different diseases. Using the duplex technique time was saved and quantity of reagents used was reduced, which translated into reduced cost of the diagnostics.


Begomovirus; Duplex PCR; mixed infection; phytoplasma; ToLCNDV; Tomato

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