Abstract
Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR) assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO) with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays.
Highlights
‘Candidatus Phytoplasma’ (PP) and Xylella (XL) spp. are phloem- and xylem-limited fastidious bacteria, respectively
Discussion quantitative real-time PCR (qPCR)-C2004/C2013 targeting of the 16S rDNA of PP [9,10] can result in non-specific amplification from Acholeplasma spp. (Table 3), which are found in plant tissues and surfaces [27]. qPCR protocol by Li et al (qPCR-L) targeting of the 16S rDNA of X. fastidiosa [14] can result in clear non-specific amplification from X. albilineans (Table 4)
The multiplex qPCR will be helpful for plant quarantine screening in countries that restrict the importation of PP- and XL-infected plants
Summary
‘Candidatus Phytoplasma’ (PP) and Xylella (XL) spp. are phloem- and xylem-limited fastidious bacteria, respectively. PP and XL have wide host ranges, with the ability to infect over 1,000 and 359 plants, respectively [1,2]. They cause economically important diseases in many plants, woody plants such as citrus, grapevine, peach, and pear. XL causes Pierce’s disease in grapevine, citrus variegated chlorosis, plum leaf scald, and pear leaf scorch [3,4]. These bacteria can severely affect host plants by reducing crop production and killing the plants. Outbreaks of new diseases caused by these bacteria have emerged an
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