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Volume :29 Issue : 2 2002      Add To Cart                                                                    Download

Molecular and Microscopical Detection of Phytoplasma Associated with Yellowing Disease of Date Palms Phoenix dactylifera L. in Kuwait

Auther : HUSA1N A. AL-AWADHI, ASMA HANIF, PATRICE SULEMAN, &

Department of Biological Sciences, Facility of Science, Kuwait University, P.O.Box
5969
, Safat  13060
, State of Kuwait. E-mail: alawadhi@kuc01.kuniv.edu.kw

ABSTRACT

Polymerase chain reaction (PCR) and electron and fluorescence microscopy were used to detect the phytoplasma (wall-less prokaryotes previously termed mycoplasma-like organisms or MLOs) associated with yellowing disease of the dale palm Phoenix dactylifera L. in Kuwait. An accumulation of DNA was observed by fluorescence microscopy in phloem tissues of diseased palms. Electron microscopy showed that phytoplasma cells were primarily confined to the phloem-sieve elements of tissue samples collected from infected mature palms from the field. Tetracycline therapy was carried out as a further evidence for the presence of the phyloplasma and as an attempt to control the disease pathogen. Infected young palms treated with tetracycline-HCl at early stages of the phytoplasmal infection showed remission of the yellowing symptoms. The pathogen was identified on the basis of molecular analysis using universal and specific nested primers in PCR amplifications. Amplifications for detecting the prokaryotic 16Sr DNA gene sequence region using universal primers (Rml6F/Rml6R) resulted in a DNA size of about 1.5 kbp resolved by gel electrophoresis. Nested PCR with primer pair R16F2nR16R2n resulted in DNA amplification of about 1.2 kbp. A phytoplasmal DNA specific diagnostic test (using primers LYF/LYR designed for lethal yellowing DNA sequence) produced a sufficient size of 1 kbp, confirming a similar but not identical strain of lethal yellowing. Restriction fragment length polymorphism (RFLP) analysis of nested PCR-amplified 16S rDNA-gene sequence with the restriction endonuclease enzyme TaqI proved that the phytoplasma strain associated with yellows in date palms in Kuwait belongs to the same sub-group 16SrIV-A of coconut lethal yellows. RFLP analysis provided a simple, reliable and rapid means for characterisation of the phytoplasma. This is the first report of a phytoplasmal rDNA gene identified in the presumed causal pathogen of yellows in date palms in the Arabian Gulf region.

 Keywords: date palm; diagnostic techniques: phytoplasma; PCR; yellows.

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