Abstract
Many efforts have been made to develop a rapid and sensitive method for phytoplasma and virus detection. Taking our cue from previous works, different rapid sample preparation methods have been tested and applied to Candidatus Phytoplasma prunorum (‘Ca. P. prunorum’) detection by RT-qPCR. A duplex RT-qPCR has been optimized using the crude sap as a template to simultaneously amplify a fragment of 16S rRNA of the pathogen and 18S rRNA of the host plant. The specific plant 18S rRNA internal control allows comparison and relative quantification of samples. A comparison between DNA and RNA contribution to qPCR detection is provided, showing higher contribution of the latter. The method presented here has been validated on more than a hundred samples of apricot, plum and peach trees. Since 2013, this method has been successfully applied to monitor ‘Ca. P. prunorum’ infections in field and nursery. A triplex RT-qPCR assay has also been optimized to simultaneously detect ‘Ca. P. prunorum’ and Plum pox virus (PPV) in Prunus.
Highlights
Phytoplasmas are uncultured wall-less bacteria, which live in the phloem of their host plants and are transmitted by insect vectors belonging to the Homoptera order [1]
We have developed a rapid and inexpensive crude sap extraction method, which exploits Nylon membrane discs and can be applied to a new TaqMan1 based reverse transcriptase (RT)-qPCR protocol for specific 16S rRNA amplification of ‘Ca. P. prunorum’
The designed probe for European stone fruit yellows (ESFY) detection differs by two mismatches from ‘Ca. P. mali’ sequence and three mismatches from ‘Ca. P. pyri’ sequence within group 16SrX and shows even more point mutations in comparison with phytoplasma sequences belonging to other groups
Summary
Phytoplasmas are uncultured wall-less bacteria (class Mollicutes), which live in the phloem of their host plants and are transmitted by insect vectors belonging to the Homoptera order [1]. These microorganisms are associated with more than 300 diseases in several hundred plant species worldwide [2]. The apple proliferation group (16SrX) includes quarantine species responsible for major economic losses in Europe, such as ‘Ca. P. pyri’ associated with pear decline (PD), ‘Ca. P. prunorum’ associated with European stone fruit yellows (ESFY), ‘Ca. P. mali’ associated with apple proliferation (AP) and peach yellow leafroll (PYLR). ESFY is mainly known in Europe, but has been reported in Turkey and PLOS ONE | DOI:10.1371/journal.pone.0146515 January 7, 2016
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