Phytoplasma axenic cultivation was recently achieved using as a source micropropagated phytoplasma infected periwinkle shoots from the collection established more than twenty years ago. Following these results, further work was carried out to verify the possibility of achieving axenic phytoplasma cultivation from field collected phytoplasma-infected samples. Shoots showing typical symptoms of phytoplasma infection, together with asymptomatic ones of the same species, were employed. After phytoplasma identification by PCR/RFLP analyses, midribs stripped from fresh leaves were selected for phytoplasma cultivation. From each sample two midribs were surface sterilized for 1 min in 1% NaClO; ends were then discarded and two half midribs per sample were used for tube inoculation following reported procedures. Uninoculated tubes and tubes inoculated with midribs from healthy shoots were also processed under the same conditions. Colonies were obtained only from tubes inoculated with symptomatic plant material and purified by filtering three times through 0.8 µm filters. Several purified colonies were separately collected from three plates per strain, and subjected to nucleic acid extraction by DNeasy Plant Minikit (Qiagen, Germany). At the same time nucleic acid was also extracted from the corresponding tubes containing pure cultures by a phenol/chloroform based method. Phytoplasma identification was carried out by specific PCR assays on 16S rDNA gene with general and group specific phytoplasma primers. Identification of detected phytoplasmas was done using RFLP analyses with appropriate restriction enzymes (in accord with phytoplasma isolated), and allowed the confirmation of phytoplasma nucleic acid presence. The profiles obtained by restriction of amplicons from liquid cultures and from purified colonies were identical to the ones of the original strains. The results of this study clearly confirm that phytoplasmas can grow independently from the plant host(s) and can also be isolated from field infected plants, with little modification to the procedure employed for phytoplasma isolation from strains maintained in micropropagated periwinkle collection.
Contaldo N., E. Satta, A. Bertaccini, D. Windsor (2014). Methods for isolation by culture, and subsequent molecular identification, of phytoplasmas from plants sourced in the field. Blumeanu, Brazil : FURB.
Methods for isolation by culture, and subsequent molecular identification, of phytoplasmas from plants sourced in the field
CONTALDO, NICOLETTA;SATTA, ELEONORA;BERTACCINI, ASSUNTA;
2014
Abstract
Phytoplasma axenic cultivation was recently achieved using as a source micropropagated phytoplasma infected periwinkle shoots from the collection established more than twenty years ago. Following these results, further work was carried out to verify the possibility of achieving axenic phytoplasma cultivation from field collected phytoplasma-infected samples. Shoots showing typical symptoms of phytoplasma infection, together with asymptomatic ones of the same species, were employed. After phytoplasma identification by PCR/RFLP analyses, midribs stripped from fresh leaves were selected for phytoplasma cultivation. From each sample two midribs were surface sterilized for 1 min in 1% NaClO; ends were then discarded and two half midribs per sample were used for tube inoculation following reported procedures. Uninoculated tubes and tubes inoculated with midribs from healthy shoots were also processed under the same conditions. Colonies were obtained only from tubes inoculated with symptomatic plant material and purified by filtering three times through 0.8 µm filters. Several purified colonies were separately collected from three plates per strain, and subjected to nucleic acid extraction by DNeasy Plant Minikit (Qiagen, Germany). At the same time nucleic acid was also extracted from the corresponding tubes containing pure cultures by a phenol/chloroform based method. Phytoplasma identification was carried out by specific PCR assays on 16S rDNA gene with general and group specific phytoplasma primers. Identification of detected phytoplasmas was done using RFLP analyses with appropriate restriction enzymes (in accord with phytoplasma isolated), and allowed the confirmation of phytoplasma nucleic acid presence. The profiles obtained by restriction of amplicons from liquid cultures and from purified colonies were identical to the ones of the original strains. The results of this study clearly confirm that phytoplasmas can grow independently from the plant host(s) and can also be isolated from field infected plants, with little modification to the procedure employed for phytoplasma isolation from strains maintained in micropropagated periwinkle collection.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
