A comparison of different phytoplasma DNA extraction methods using competitive PCR

The primer pair 16endF-Tmod, specific for the16S/23S rDNA spacer region, amplifies different length fragments when the target DNA belongs to phytoplasmas of the X-clade and AP-clade in compared with phytoplasmas of the AY-clade. The cloned 16S/23S spacer of the tagete witches’-broom phytoplasma (TWB, AY-clade) was used in a competitive PCR assay to quantify the DNA of the apple proliferation phytoplasma (AP, AP-clade) and the clover phyllody phytoplasma (CP, X-clade) present in the nucleic acids extracted from infected periwinkle plants. Thus, four methods, normally used for the extraction of phytoplasma DNA from infected plant tissue, were compared to determine which was the most efficient in recovering phytoplasma nucleic acids. The different methods detected relatively minor differences in the yields obtained. It was concluded that the sampling and detection protocols are more critical than the DNA extraction method as far as sensitivity is concerned

miRVIT: A Novel miRNA Database and Its Application to Uncover Vitis Responses to Flavescence dorée Infection

Micro(mi)RNAs play crucial roles in plant developmental processes and in defense responses to biotic and abiotic stresses. In the last years, many works on small RNAs in grapevine (Vitis spp.) were published, and several conserved and putative novel grapevine-specific miRNAs were identified. In order to reorganize the high quantity of available data, we produced “miRVIT,” the first database of all novel grapevine miRNA candidates characterized so far, and still not deposited in miRBase. To this aim, each miRNA accession was renamed, repositioned in the last version of the grapevine genome, and compared with all the novel and conserved miRNAs detected in grapevine. Conserved and novel miRNAs cataloged in miRVIT were then used for analyzing Vitis vinifera plants infected by Flavescence dorée (FD), one of the most severe phytoplasma diseases affecting grapevine. The analysis of small RNAs from healthy, recovered (plants showing spontaneous and stable remission of symptoms), and FD-infected “Barbera” grapevines showed that FD altered the expression profiles of several miRNAs, including those involved in cell development and photosynthesis, jasmonate signaling, and disease resistance response. The application of miRVIT in a biological context confirmed the effectiveness of the followed approach, especially for the identification of novel miRNA candidates in grapevine. miRVIT database is available at http://mirvit.ipsp.cnr.it.Highlights: The application of the newly produced database of grapevine novel miRNAs to the analysis of plants infected by Flavescence dorée reveals key roles of miRNAs in photosynthesis and jasmonate signaling