Development of improved methods for phytoplasma diagnostics

Phytoplasmas are plant pathogens that are small prokaryotic, wall-less bacteria causing significant diseases in hundreds of plant species globally. They have been named as distinct ‘Candidatus phytoplasma’ species and belong to the Mollicutes. These microorganisms are non-cultivable plant pathogens (obligate), so their diagnoses are primarily achieved by molecular techniques. In infected plants, they inhabit phloem tissues (sieve cells) and they are transmitted between plants by insects (phloem feeders) such as those in the orders Cicadellidae, Psyllidae and Fulgoridae. Phytoplasmas have extremely small genomes and also have very low levels of the nucleotides cytosine and guanine. Polymerase Chain Reaction (PCR) has been developed and employed for investigating phytoplasmas in their hosts, and such techniques have been advanced through the use of nested PCR and real-time PCR to improve the diagnosis of phytoplasmas with low titre in their different hosts, and also to be able to identify the different taxonomic groups of the pathogens. In addition, real-time PCR can be used to quantify phytoplasmas in the infected plants or insects. The purpose of this project was to concentrate initially on 16S rRNA, leucyl tRNA synthetase and secA based PCR assays and test these on periwinkle plants containing phytoplasmas from six taxonomic groups, 16SrI, II, III, V, VI and X. Work also focussed on transmitting the six phytoplasma strains between periwinkle plants and other host plants such as Napier grass, oil palm, and tomato plants through dodder shoots. Cuttings were taken from mother plants, that are all from the same periwinkle genetic background, to enable the propagation of new plants containing isolates from the six groups. These were treated with different potential control strategies, and conventional and real-time PCR and (Loop-mediated isothermal amplification) LAMP were used to test the levels of phytoplasma in these plants following treatments to determine the efficacy of the different control treatments. In more detail, the control strategies being tested included altered nitrogen fertilization regimes, treatment with salicylic acid, and UV-C pulse treatments. As a consequence, phytoplasma titre of different phylogenetic groups were affected by the application of different nitrogen fertilizers and different amounts, and this showed the potential for use of various nitrogen sources as a control method for phytoplasma diseases. Furthermore, this research has shown that UV-C light can be applied to phytoplasma-infected plants to enhance the resistance characteristics of plants against phytoplasma and reduce phytoplasma titre within diseased plants. Through applying different control approaches against phytoplasma infected plants, it has been shown that low concentrations of salicylic acid application had an optimal significant effect on phytoplasma quantity and reduced infection on diseased periwinkle plants. However, a high concentration of the same solution produced a negative effect and phytotoxicity on periwinkle plants