Recently the proof that phytoplasmas can be grown on laboratory media was provided employing specific commercially available media and 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 from field collected phytoplasma-infected samples. Shoots from trees 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. Uninoculated tubes and tubes inoculated with midribs from healthy shoots were also processed under the same conditions. Phytoplasma colonies were obtained in 24 to 72 hours only from tubes inoculated with symptomatic plant material and purified by filtering. Several purified colonies were separately collected from three plates per strain, and subjected to nucleic acid extraction by commercial kits. At the same time nucleic acid was also extracted from the corresponding tubes containing cultures by a phenol/chloroform based method. Phytoplasma identification was carried out by PCR assays on phytoplasma 16S rDNA gene with general and group specific primers. Identification of detected phytoplasmas was done using RFLP analyses with appropriate restriction enzymes and/ or sequencing; the real and/or virtual profiles obtained from liquid cultures and from purified colonies were identical to the ones of the original strains.
Morphological and molecular identification of phytoplasmas in culture from plants sourced in the field
CONTALDO, NICOLETTA;SATTA, ELEONORA;ZAMBON, YURI;PALTRINIERI, SAMANTA;BERTACCINI, ASSUNTA
2014
Abstract
Recently the proof that phytoplasmas can be grown on laboratory media was provided employing specific commercially available media and 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 from field collected phytoplasma-infected samples. Shoots from trees 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. Uninoculated tubes and tubes inoculated with midribs from healthy shoots were also processed under the same conditions. Phytoplasma colonies were obtained in 24 to 72 hours only from tubes inoculated with symptomatic plant material and purified by filtering. Several purified colonies were separately collected from three plates per strain, and subjected to nucleic acid extraction by commercial kits. At the same time nucleic acid was also extracted from the corresponding tubes containing cultures by a phenol/chloroform based method. Phytoplasma identification was carried out by PCR assays on phytoplasma 16S rDNA gene with general and group specific primers. Identification of detected phytoplasmas was done using RFLP analyses with appropriate restriction enzymes and/ or sequencing; the real and/or virtual profiles obtained from liquid cultures and from purified colonies were identical to the ones of the original strains.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
