Fast and sensistive on-site isothermal assay (LAMP) for the detection of three fruit tree phytoplasmas

Candidatus Phytoplasma mali (AP), Candidatus Phytoplasma pyri (PD) and Candidatus Phytoplasma prunorum (ESFY) are three of the major fruit tree pathogens. They are closely related wall‐less predominantly phloem residing prokaryotes sharing more than 97.5% sequence identity in the 16S rRNA
gene. This 16S region has been used in many generic, AP group specific and species specific PCR based diagnostic methods for the detection of AP, PD and ESFY. Over the years, real‐time PCR outflanked endpoint PCR in phytodiagnostics, mainly because of the increase in sensitivity and timesaving aspects of the technique. However, a time consuming 16S rRNA‐based nested PCR method is still
the gold standard for Phytoplasma diagnosis, especially when low titters related to unequal distribution in the host plants are expected. This is also the case for Phytoplasma detection in Malus, Pyrus and Prunus, the three main host plants of AP, PD and ESFY, respectively. The last decade, loop‐mediated isothermal amplification (LAMP) (Notomi et al., 2000) is gaining a lot of interest and is also for phytoplasmas expected to become a fast and reliable diagnostic tool. The
main advantages of this technique over PCR are specificity and sensitivity which also have their impact on less stringent needs for DNA purification. Additionally, the short analysis time and the limited equipment requirements (no thermal cycler needed) makes the LAMP method a reliable and affordable alternative with great point‐of‐care diagnostic potential. So far, specific LAMP primers have been developed for Phytoplasma groups 16SrI, II, III, V, XI, XII and XXII. In this paper, we present a primer set which was developed against the ribosomal group 16SrX, containing the important fruit tree phytoplasmas AP, PD and ESFY. We present the validation of these primers for the detection of apple proliferation, pear decline and European stone fruit yellows phytoplasma and the usefulness in a one hour protocol. We foresee that the LAMP technique and especially the presented detection method for the fruit tree phytoplasms will also have its further application in on site diagnosis during inspections and surveys.