TaqMan quantitative-PCR detection of Xylella taiwanensis in Taiwan

Authors: SU, CHIOU-CHU - Taiwan Agricultural Chemicals And Toxic Substances Research Institute; FUNG, JIE-AN - Taiwan Agricultural Chemicals And Toxic Substances Research Institute; CHANG, RUEY-JANG - Taiwan Agricultural Chemicals And Toxic Substances Research Institute; CHANG, CHUNG-JAN - University Of Georgia; JAN, FUH-JYH - National Chung-Hsing University; SHIH, HSIEN-TZUNG - Taiwan Agricultural Research Institute; Chen, Jianchi

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/4/2023
Publication Date: 3/8/2023
Citation: Su, C., Fung, J., Chang, R., Chang, C., Jan, F., Shih, H., Chen, J. 2023. TaqMan quantitative-PCR detection of Xylella taiwanensis in Taiwan. Plant Disease. https://doi.org/10.1094/PDIS-01-23-0064-RE.
DOI: https://doi.org/10.1094/PDIS-01-23-0064-RE

Interpretive Summary: Xylella taiwanensis (Xt) is a recently described, nutritionally fastidious bacterial pathogen causing pear leaf scorch disease (PLSD) in Taiwan. The disease causes early defoliation, weakening of tree vigor and reduction of fruit yield and quality. No curing method is available. The occurrence of Xt outside Taiwan is not known. Early and accurate diagnosis of PLSD is important for disease management, which depends on PCR detection of Xt. This study developed five Xt specific TaqMan Q-PCR systems. Analyses using GenBank database, DNA samples of Xt and other bacterial strains, and PLSD plant samples collected from Taiwan showed that the TaqMan Q-PCR systems were highly specific and sensitive. The TaqMan Q-PCR systems provide new tools to meet current demands for Xt detection by plant pathology researchers and regulatory agencies.

Technical Abstract: Xylella taiwanensis (Xt) is a nutritionally fastidious bacterial pathogen causing pear leaf scorch disease (PLSD) in Taiwan. The disease causes early defoliation, weakening of tree vigor and reduction of fruit yield and quality. No curing method is available. Early and accurate diagnosis of PLSD is important for disease management, which depends on PCR detection of Xt. Currently, only one primer set, PLS-F/R, in the standard PCR format (agarose gel electrophoresis-based) is available. Utilizing the recent genome sequencing and analysis technologies, this study developed and evaluated five Xt specific TaqMan Q-PCR systems (primers/probe sets). The Q-PCR systems targeted three conserve genomic loci commonly used for bacterial pathogen detection: 16S rRNA gene (rrs), 16S-23S intergenic transcribed sequence (16S-23S rRNA ITS), and a DNA gyrase gene (gyrB). BLAST analysis using GenBank nr sequence database showed that all primer and probe sequences were highly specific to Xt and separated from X. fastidiosa (Xf), the phylogenetically closely related bacterial species. Unique nucleotide variations of Xt played key roles in primer/probe specificity. The TaqMan Q-PCR systems were evaluated using DNA samples of pure cultures from 17 Xt strains and one Xf strain, one Xanthomonas campestris strain, and 66 PLSD plant samples collected from 23 pear orchards in four counties in central Taiwan where PLSD was endemic. The two-copy sequences (rrs and 16S-23S rRNA ITS)-based primers-probe sets (Xt803F-R, Xt731F-R, and Xt16SF-R) showed higher detection sensitive than the two single-copy gyrB-based sets (XtgB1F-R and XtgB2F-R). The calculated detection limit in PLSD plant samples could reach 0.002 ng of Xt DNA (~1,000 cells/genome copies). Metagenomic analysis revealed that PLSD plant sample could harbor other proteobacteria in addition to Xt and interfere with interpretation of high Ct (>30). This should be taken into consideration in PLSD diagnosis practice.