Cultivation of several phytoplasmas from a micropropagated plant collection

The recognition that certain plant diseases were associated with mycoplasmalike structures in the sieve-tubes was first reported by Doi and coworkers in 1967 who extended this observation by confirming possible disease causation, and their similarity to mycoplasmas, by showing that disease symptoms could be ameliorated by treatment with tetracycline (Ishiie et al., 1967). Hitherto, these diseases - referred to as the aster yellows group - had been thought to be caused by viruses. In animals and humans, mycoplasma infections had similarly often been regarded as viral in nature due partly to their filterability through membranes of small pore size but also due to the inability to culture the infectious organism in common bacteriological media. Many workers, mainly plant pathologists rather than mycoplasmologists, took up the challenge to culture these organisms. An early outcome of this effort was the culture of spiroplasmas – microorganisms derived from insects capable of causing plant diseases following infection of the sieve tubes. This distinct group of organisms grew readily in mycoplasma media formulations whereas the mycoplasma-like objects (phytoplasmas) did not and a possible reason was that a closer alignment of media to sieve-tube sap composition was required. Morphologically the two groups were quite distinct, spiroplasmas having a spiral structure as the name suggests whereas phytoplasmas were generally pleomorphic. Some reports of successful phytoplasma culture were published (Lombardo and Pignattelli, 1970; Lin et al., 1970; Giannotti and Vago, 1971; Ghosh et al., 1975) but none proved repeatable and as the years of failure increased, hope of cultivation decreased to such an extent that some plant workers came to consider that phytoplasmas were indeed uncultivable (reviewed in Maramorosch, 2011). Over forty years had elapsed since Doi’s publication but there were workers who believed culture to be possible and a collaboration was initiated in 2008 between Bologna University and Mycoplasma Experience. Early success was definitely not anticipated and a lengthy process of medium modification/alignment to sieve-tube sap composition was envisaged.