Molecular characterization of a phytoplasma causing Phyllody in Clover and other herbaceous hosts in Northern Italy

Abstract

Red clover (Trifolium pratense) and Ladino clover (Trifolium repens) plants showing phytoplasma-associated symptoms (yellowing/reddening, virescence and phyllody) have been recovered in Friuli-Venezia Giulia, Italy. Using AluI RFLP analysis of PCR amplified 16S rDNA we showed that the disease can be caused independently by two phylogenetically distinct phytoplasmas. One of them showed the very typical 16S rDNA RFLP pattern of the agent of Clover Phyllody in Canada (CCPh). The 16S rDNA of the other phytoplasma (Italian Clover Phyllody phytoplasma, ICPhp) has been PCR amplified, cloned and sequenced. The sequence revealed high similarity (>98%) with phytoplasmas belonging to the X disease cluster, which includes organisms not reported to cause phyllody on their hosts. The analysis by AluI RFLP of the PCR amplified pathogen 16S rDNA from other herbaceous plants (Crepis biennis, Taraxacum officinale, Leucanthemum vulgare) collected nearby with phytoplasma-associated symptoms showed similar patterns. Southem blot hybridization of their EcoRI digested total DNA revealed identical RFLP patterns, suggesting that the causative agent may be the same organism.

Genetic diversity among phytoplasmas infecting Opuntia species: virtual RFLP analysis identifies new subgroups in the peanut witches'-broom phytoplasma group

“…While the two rrn operons may be identical in some phytoplasma strains, apparent interoperon sequence heterogeneity exists in other strains (Lee et al, 1993;Firrao et al, 1996;Liefting et al, 1996;Davis & Sinclair, 1998;Jomantiene et al, 2002). Thus, in assignments of phytoplasma strains to 16S rRNA gene RFLP subgroups, the rrn interoperon sequence heterogeneity of a strain must be addressed.…”

Geminivirus-related extrachromosomal DNAs of the X-clade phytoplasmas share high sequence similarity

“…This was the only variation in the EC-DNA hybridization pattern which was observed during this work on the plants kept in the greenhouse: none of the strains maintained by grafting onto periwinkle for over 4 years showed a change in the EC-DNA banding pattern. clade and ICPh phytoplasmas may infect the same plant (Osler et al, 1994;Firrao et al, 1996) and insect host (S. Palmano & G. Firrao, unpublished) and multiple infections occur (Firrao et al, 1996). Hybridizations with probe pSA45 showed that the AY-clade phytoplasmas VE and LEO containcd EC-DNA molecules, but they did not hybridize with EC-DNA from the VAC phytoplasma.…”

“…The DNAs of the various IICPh strains, which were indistinguishable by chromo$omal RFLP (Firrao et al, 1996;Rekab, 1998), showed 'different banding patterns when hybridized to the EC-DNA probes pVAC79 and pVAC131 in Southern blots ( Fig. 1; and data not shown).…”

“…No differences in the RFLP patterns were obtained although three restriction enzymes were used (Firrao et al, 1996;Rekab, 1998). T h e isolates from different plant species were therefore thought to represent strains of a single phytoplasma, which w a s named the Italian clover phyllody phytoplasma (ICPh), since it was first detected in Trifolium repens (Firrao et al, 1996).…”

“…No differences in the RFLP patterns were obtained although three restriction enzymes were used (Firrao et al, 1996;Rekab, 1998). T h e isolates from different plant species were therefore thought to represent strains of a single phytoplasma, which w a s named the Italian clover phyllody phytoplasma (ICPh), since it was first detected in Trifolium repens (Firrao et al, 1996). In this paper, w e report the analysis of extrachromosomal D N A s (EC-DNAs) which were found t o be associated with the ICPh strains collected in Cavazzo Carnico a n d with the phylogenetically related Vaccinium witches' b r o o m (VAC) a n d walnut witches' broom (WWB) phytoplasmas.…”