Development of Nucleic Acid-Based Methods for the Specific Detection of Phytoplasma Subgroups 16Sr IX-B ('Candidatus Phytoplasma phoenicium’) and 16Sr IX-C

Abstract

During the early 1990s, a disease affecting stone fruits was reported in Lebanon and Iran. The disease being both invasive and destructive quickly escalated into an epidemic that destroyed hundreds of thousands of almonds, peaches, and nectarine trees. The disease caused several symptoms, of which the most characteristic was the proliferation of shoots and the appearance of witches’ broom; hence, it was termed almond witches’ broom (AlmWB) disease. The causal agent was eventually identified to be ‘Candidatus Phytoplasma phoenicium’ (‘Ca. P. phoenicium’), a phytoplasma belonging to subgroup 16SrIX-B. Transmission of the phytoplasma occurs persistently via several leafhopper and psyllid species, by grafting, and through propagative tissue. There are currently no effective curative treatments, thus making eradication of infected trees the only remaining option. Therefore, stringent quarantine measures and potent detection tools are imperative for effective prevention of AlmWB phytoplasma. While serological techniques are faster and more cost-effective than other detection tests, there are currently no commercially available phytoplasma detection serological tests. This is primarily due to ineffectiveness in culturing them axenically and due to difficulties in expressing their membrane proteins. Conversely, there are several PCR-based assays for the detection of ‘Ca. P. phoenicium’ as well as other phytoplasmas. Development of such assays has received quite attention, and as a result, several existing methods offer a variety in detection capabilities. This thesis attempted to assess six PCR-based assays for the detection of AlmWB phytoplasma: using primers targeting the 16S-ITS-23S region, or the inmp gene, amplification using generic phytoplasma primers followed by Taq1 digestion, DNA barcoding followed by sequencing, 16SrIX-B specific and non-specific real-time PCR (qPCR). Evaluations were made based on accuracy, diagnostic sensitivity, diagnostic specificity, and reproducibility. For the detection of AlmWB phytoplasma, targeting the ITS region generated by far the best overall indexes followed by 16SrIX-B specific and non-specific real time PCR. Nested PCR of the inmp gene gave acceptable results while the remaining assays demonstrated unsatisfactory results with DNA barcoding having the worst indexes. In another test, two personally designed multiplex primer pairs were investigated for their detection ability of 4 16SrIX-B (AlmWB) and 16SrIX-C (Picris echioides yellows, PEY) phytoplasmas. These primers proved to be very efficient for the specific and sensitive detection of phytoplasmas and generated reproducible results. The multiplex assay detected the mixed infections of both phytoplasmas up to a sensitivity of 0.1 ng/μl total plant DNA. The development of this PCR technique may be very valuable in quarantine services for early specific detection of AlmWB phytoplasma (16SrIX-B), a quarantine pathogen and for differentiation from its closely related 16SrIX-C phytoplasma, which is widespread and not considered a quarantine pathogen.