Efficiency of 'Ghana 813 16S forward' primer in the detection of West African coconut phytoplasma

Abstract

Cape St. Paul Wilt Disease (CSPWD) of coconut in Ghana is believed to be caused by phytoplasma(s) (MLOs). These pathogens are non-culturable, obligate prokaryotes, and therefore cannot be studied by conventional microbiological methods. DNA amplification by PCR using primers based on the nucleotide sequence of the 16S rRNA gene, is routinely used in detecting phytoplasmas. Many of the primers available are universal for mollicutes. Primer Awka SR, however, is specific for the CSPWD-phytoplasma. In combination with Deng & Hiruki P1(universal for mollicutes), Awka SR has been used for specific detection of the CSPWD-phytoplasma. The primer `GH813' was later developed at the Rothamsted Experimental Station, UK. Since the sequence of this `forward' primer was based on the CSPWD-phytoplasma, it was anticipated that in combination with the `reverse' primer Awka SR also based on the same phytoplasma, a primer set with a higher specificity for CSPWD-phytoplasma would be obtained. This study was to verify the efficiency of the GH813 primer. Fourteen mollicute DNAs, from 6 different sources, were screened by PCR using different primer combinations. PCR products were separated on 1 % agarose gel and visualised on the UV transilluminator after ethidium bromide staining. `GH813' showed high specificity in detecting West African coconut phytoplasma. GH813/Awka SR primer set also yielded more amplified product from the phytoplasma, compared with the Deng & Hiruki P1/Awka SR combination. The GH813/Awka SR primer set is hence a better choice for a more efficient routine detection of the pathogen and should give a new impetus to the search for the CSPWD-phytoplasma vector(s) which has remained elusive till now.

JOURNAL OF THE GHANA SCIENCE ASSOCIATION Volume 2 No. 2 (2000) pp. 80-87