CRISPR/Cas12a-based diagnosis of potato purple top disease complex associated with infection by 'Candidatus Phytoplasma trifolii'-related strains

Authors: WHEATLEY, MATTHEW - Pennsylvania State University; WANG, QIN - Pennsylvania State University; Wei, Wei; Bottner, Kristi; Zhao, Yan; YANG, YINONG - Pennsylvania State University

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/18/2022
Publication Date: 3/29/2022
Citation: Wheatley, M.S., Wang, Q., Wei, W., Bottner-Parker, K.D., Zhao, Y., Yang, Y. 2022. CRISPR/Cas12a-based diagnosis of potato purple top disease complex associated with infection by 'Candidatus Phytoplasma trifolii'-related strains. Plant Disease. https://doi.org/10.1094/PDIS-09-21-2119-RE.
DOI: https://doi.org/10.1094/PDIS-09-21-2119-RE

Interpretive Summary: 'Candidatus Phytoplasma trifolii' is a small plant-pathogenic bacterium capable of infecting many agriculturally important crop species and causing significant economic losses. The bacterium is responsible for repeated outbreaks of potato purple top, potato witches'- broom, and other vegetable crop diseases that occurred along the Pacific Coast of the United States and Mexico. Early detection of the phytoplasma is key to effective management of these crop diseases. In collaboration with scientists at the Pennsylvania State University, ARS researchers at the Beltsville Agricultural Research Center applied cutting edge genome editing technology to phytoplasma detection for the first time. The team identified molecular target unique to 'Candidatus Phytoplasma trifolii' and developed a highly sensitive diagnostic tool for rapid and specific detection of the phytoplasma. This article will be of interest to research scientists, plant disease diagnosticians, and extension personnel who are interested in plant disease management and in development of advanced disease diagnostic tools.

Technical Abstract: 'Candidatus Phytoplasma trifolii' is a cell wall-less phytopathogenic bacterium that infects many agriculturally important plant species including leguminous, solanaceous, and brassicas crops. The phytoplasma is responsible for repeated outbreaks of potato purple top (PPT), potato witches'- broom (PWB), and other vegetable crop diseases that occurred along the Pacific Coast of the United States and Mexico, inflicting significant economic losses. To effectively manage these phytoplasmal diseases, it is important to develop diagnostic tools for specific, sensitive and rapid detection of the pathogens. Here we report the development of a Cas12a-based DETECTR (DNA endonuclease targeted CRISPR trans reporter) assay that couples isothermal amplification and Cas12a trans-cleavage of fluorescent oligos for highly specific and sensitive detection of 'Candidatus Phytoplasma trifolii'-related strains responsible for PPT and PWB. The DETECTR assay was capable of specifically detecting the phytoplasma 16S rDNAs from PPT- and PWB-diseased samples at the attomolar sensitivity level, superior to most polymerase chain reaction (PCR)-based assays. Furthermore, the DETECTR strategy allows flexibility to capture assay outputs with fluorescent microplate reader or lateral flow assay for potentially high-throughput and/or field-deployable disease diagnostics.