Phytoplasmas (Candidatus Phytoplasma) are among the most important plant pathogens worldwide and are known to infect more than a thousand plant species, including many fruit trees and economically valuable crops. They can spread through vegetative propagation methods such as cuttings and grafting, as well as through parasitic dodder and various insect vectors including leafhoppers, planthoppers, and psyllids. Infected plants often exhibit symptoms such as phyllody, stunting, yellowing, and witches’ broom, leading to substantial agricultural losses. Since phytoplasmas cannot be cultured in vitro, their detection and identification remain highly challenging. With increasing demands for plant quarantine and disease prevention, establishing internationally aligned detection and diagnostic methods for phytoplasmas is crucial for crop disease diagnosis and surveys of seed-associated pathogens in Taiwan. In the first year of this project, representative materials of the two major phytoplasma groups found in Taiwan were collected and established. These include the Periwinkle leaf yellowing (PLY) phytoplasma (16Sr I-B, aster yellows group) representing Group I (16Sr I) and the Peanut witches’ broom (PnWB) phytoplasma (16Sr II-A) representing Group II (16Sr II), collected from peanut fields in Tai-si and Tuku, Yunlin County. This material library provides an essential foundation for developing detection methods. Two nucleic acid extraction systems, TANBead and taco, were compared, and DNA purity was evaluated using A260/280 and A260/230 ratios. Results indicated that TANBead yielded higher DNA purity (1.79-1.91) and could be reliably applied to various suspected hosts, including Ixeris chinensis , Emilia sonchifolia , Arachis duranensis, and Cynodon dactylon and semi-nested PCR systems were established. The primer pair P1A/P7A was selected for the first PCR (approximately 1.8 kb), and P1A/16S-Sr for the semi-nested PCR (approximately 1.5 kb). Both PLY and PnWB phytoplasmas successfully produced the expected. Amplicons were sequenced, and the resulting sequences were compared using the BLAST tool in the NCBI (National Center for Biotechnology Information, https://www.ncbi.nlm.nih.gov/) database. Sequence identities were all above 99.2%, confirming that the samples correspond to PLY and PnWB phytoplasmas, respectively.

▲Figure 1. General Characteristics of Phytoplasmas (Candidatus Phytoplasma).

▲Figure 2. Establishment of Detection Techniques for Phytoplasmas.