A REALTIME PCR ASSAY FOR DETECTION OF COCONUT LETHAL YELLOWING PHYTOPLASMAS OF GROUP 16SrIV SUBGROUPS A D AND E FOUND IN THE AMERICAS
I. Córdova, C. Oropeza, C. Puch-Hau, N. Harrison, A. Collí-Rodríguez, M. Narvaez, G. Nic-Matos, C. Reyes, L. Sáenz
Lethal yellowing (LY) is a fatal disease that affects coconut and other palm species in the Americas. Phytoplasmas associated with this disease belong to group 16SrIV. Reliable detection of group 16SrIV phytoplasmas is important for diagnostic purposes and to increase understanding of pathogen-plant-vector pathosystems. The present study describes the development of a TaqMan/real-time PCR assay for detection and quantification of selected 16SrIV subgroups affecting palms in the Americas. The specificity of the assay was assessed on DNA extracts from LY-infected palms in the Americas, coconut lethal phytoplasma-associated diseases in Africa and other phytoplasma-infected plants. Successful amplification was obtained only with DNA extracts from palms infected by LY phytoplasmas that were sampled in the Americas, belonging to the 16SrIV group, subgroups A, D and E. No amplification was obtained from DNA of palms sampled in Africa and phytoplasma-infected plants of other 16Sr groups. The assay was compared with conventional nested-PCR on DNA extracts from 36 palms. The real-time PCR assay showed higher sensitivity as phytoplasmas were detected in several nested-PCR negative and in all the nested-PCR positive samples. The assay was also used to evaluate accumulation of LY phytoplasma DNA in different tissues of palms showing LY symptoms. The highest concentration was found in the trunk, followed in decreasing order by primary root apex, mature inflorescences -1 and -2, inflorescences -3 to -7, spear leaf, flag leaf and mature leaf. The present TaqMan/real-time PCR assay represents a new alternative for LY phytoplasmas detection and quantification, offering high specificity and improvements in sensitivity.