Title : Real-time quantitative PCR based method for quantification of Xanthomonas axanopodis pv punicae in pomegranate
Abstract:
Pomegranate is one of the important commercial fruit crops of India, known for its high nutritive and remunerative value. Cultivation of pomegranate is severely affected by bacterial blight caused by Xanthomonas axanopodis pv punicae (Xap). Under epidemic condition bacterial blight drastically reduces the yield and deteriorates the quality crop produce up to 80% under field condition. Breeding for developing resistant lines is the most appropriate strategies for effective management of plant disease. Therefore screening of a large genotype is very necessary for identification of resistant or tolerant type for resistant breeding. However, existing protocols for screening of different genotype green house and field often results in high experimental error leading to inconsistent disease scoring. In post-genomic era, early diagnosis and accurate quantification of pathogen is very crucial in identification of resistant cultivar. for identification of candidate gene inferring resistant. Hence, in the present study, we screened a large number of pomegranate accession for identification of resistant or tolerant lines. Further, accessions exhibiting highest disease tolerance were quantified using Xanthmonas specific primer XOPQ. The relative pathogen quantification was successfully conducted on 7 tolerant line IC318762, IC318735, IC318724, IC318734, IC318707, ACC8, IC318706 and two susceptible wild type Nana, Daru and one highly susceptible commercial cultivar Bhagwa selected from screened lines. The primer found to be specific to Xanthmonas, the sensitivity was analyzed using PCR coupled with agarose gel electrophoresis (PCR-AGE), PCR coupled with capillary electrophoresis (PCR-CE) and Real Time quantitative Polymerase Chain Reaction. The Conventional PCR-AGE useful in quantifying the pathogen with detection limit of 10 pg, followed by High resolution PCR-CE and RT-qPCR with 100fpg and 10 fg respectively. This approach can be employed for screening large number of pomegranate accession for breeding programmers to differentiate quantative resistant among genotypes. Key words: Pathogen quantification, Pomegranate Bacterial blight, Real Time quantitative Polymerase Chain Reaction (RT-qPCR), Capillary gel free electrophoresis system.
