BackgroundSeveral phytoplasmas, wall-less phloem limited plant pathogenic bacteria, have been shown to contain extrachromosomal DNA (EcDNA) molecules encoding a replication associated protein (Rep) similar to that of geminiviruses, a major group of single stranded (ss) DNA plant viruses. On the basis of that observation and of structural similarities between the capsid proteins of geminiviruses and the Satellite tobacco necrosis virus, it has been recently proposed that geminiviruses evolved from phytoplasmal EcDNAs by acquiring a capsid protein coding gene from a co-invading plant RNA virus.ResultsHere we show that this hypothesis has to be rejected because (i) the EcDNA encoded Rep is not of phytoplasmal origin but has been acquired by phytoplasmas through horizontal transfer from a geminivirus or its ancestor; and (ii) the evolution of geminivirus capsid protein in land plants implies missing links, while the analysis of metagenomic data suggests an alternative scenario implying a more ancient evolution in marine environments.ConclusionThe hypothesis of geminiviruses evolving in plants from DNA molecules of phytoplasma origin contrasts with other findings. An alternative scenario concerning the origin and spread of Rep coding phytoplasmal EcDNA is presented and its implications on the epidemiology of phytoplasmas are discussed.

Sequence analysis of five of the six endopolygalacturonase-encoding genes (Bcpg1, Bcpg2, Bcpg3, Bcpg4, Bcpg5) from 32 strains of Botrytis cinerea showed marked gene to gene differences in the amount of among-strains diversity. Bcpg4 was almost invariable in all strains; Bcpg3 and Bcpg5 showed a moderate variability, similar to that of non-pathogenicity-associated genes examined in other studies. Conversely, Bcpg1 and Bcpg2 were highly variable and were shown to be under positive selection based on the McDonald-Kreitman test and likelihood ratio test. The evolution of the five endopolygalacturonase genes is explained by their different ecophysiological role. Diversification and balancing selection, as detected in Bcpg1 and Bcpg2, can be used by the pathogen to escape recognition by the host and delay plant reaction in the early phases of infection. The analysis of the polymorphisms and the location of the sites with high probability of being positively selected highlighted the relevance of variability of the BcPG1 and BcPG2 proteins at their C-terminal end. By contrast, the absence of variability in Bcpg4 suggests that the efficiency of the product of this gene is critical for B. cinerea growth in late phases of infection or during intraspecific competition, thus markedly affecting strain fitness.

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Notwithstanding the availability of several different real time PCR protocols for "Candidatus Phytoplasma mali", it is still unclear how informative is the estimation of the concentration of phytoplasma cells in the leaves of apple proliferation infected trees, and how the reliability of the estimations may be affected by an erratic and uneven distribution of the pathogen in the host. Here we investigated these issues systematically and showed that phytoplasma concentration varies significantly among seasons, but not between two cultivars that appeared to have different degree of susceptibility on the basis of the symptoms displayed. In fully symptomatic trees sampled at the end of the season the phytoplasmas were detectable in most leaves, but in more than half of the leaves at low concentrations. Both the pattern of colonization of the canopy and the amount of phytoplasmas varied greatly in trees that show symptom remission, although a direct relation between symptom severity and colonization could not be established. The sampling of the apple canopy for the purpose of evaluation of concentration of "Candidatus Phytoplasma mali" should take into consideration the complex pattern of colonization and seasonal variation

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