Development of serological detection methods for ‘Candidatus Phytoplasma phoenicium’.

Abstract

‘Candidatus Phytoplasma phoenicium’ has been suggested as the causal agent of Almond Witches’ Broom disease (AlmWB), a disease that has devastated hundreds of thousands of stone fruit trees in Lebanon for the past three decades. Thus far, the disease has been identified only in Iran and Lebanon. AlmWB is invasive, suspected of being transmitted by many leafhopper and psyllid species as well as propagative tissue or grafting. This raises the need for strict quarantine measures as the damage of the disease severely impacts yield and kills trees. To date, specific detection of ‘Ca. P. phoenicium’ is done by PCR and variants of PCR such as qPCR or nested PCR. Although reliable, these methods are more time consuming, more expensive, and with comparable sensitivity to serological detection methods. Phytoplasmas have not been yet grown in axenic cultures and the expression of their membrane proteins was reported to be very difficult, therefore, no commercial serological tests are available for phytoplasma detection. This thesis attempted to express 8 previously identified integral membrane proteins in different E. coli expression systems and expression vectors; but ultimately no protein expression was observed. Modifications were made to express only the non-transmembrane regions of the targeted proteins with no success. The methodology was shifted towards trying a cell-free expression system which was tested with two transmembrane proteins and one truncated protein excluding the transmembrane region. While all potential possibilities of this system were tried, no expression of the target proteins was obtained. Finally, we tested three anti-peptide polyclonal antibodies, designed against antigenic and exposed sites of the membrane proteins. Different serological assays were conducted such as Tissue Blot Immunoassay (TBIA), Dot Blot Immunoassay (DBIA), and Western Blot. Only one of the antibodies tested positive in TBIA but similar results were obtained in negative control samples conf