Microscopic localization of grapevine phytoplasmas: an exciting challenge or a losing battle?

Phytoplasma localization in grapevine by microscopic techniques has always been a big challenge for many reasons, but particularly for their very low concentration in this plant species that makes their finding an almost impossible task, at least by transmission electron microscopy (TEM) (Faoro, 2005). A literature survey, since the discovery of phytoplasmas associated with Flavescence dore\ue8 (FD) and other grapevine yellows (GY) in the sixties, shows that only three papers have been published up to now on this subject (Granata et al., 1991; Meignoz et al., 1992; Credi, 1994), in spite of the huge number of reports dealing with the presence of these prokaryotes in infected grapevine plants and detected by PCR techniques. Indeed, by the advent of molecular biology in the eighties, microscopic visualization of phytoplasmas in infected plants was regarded as a useless diagnostic tool, in any case providing very little information on these microorganisms, particularly from the taxonomic point of view. For all the above reasons microscopic investigations on grapevine yellows were completely abandoned. However, in the last decade the numerous efforts that have been carrying out to study the interaction of phytoplasmas with grapevine tissues, particularly in case of the recovery phenomenon, have shown that the precise localization of the these prokaryotes in the tissues would be determinant to understand the underlying mechanisms. Recovery, i.e. the spontaneous remission of symptoms in diseased plants, has often been observed in FD- and GY-affected grapevines (Caudwell et al.,1961; Osler et al., 1999). This phenomenon may or may not involve the elimination of the pathogen from the host. Physiological mechanisms and possible biological factors involved in recovery are still not clear, though increased hydrogen peroxide level in the phloem of recovered plants has been observed (Musetti et al., 2007), together with the activation of systemic acquire resistance related genes (SAR) (Albertazzi et al., 2009). Moreover, other researchers hypothesized that endophytic microorganisms (bacteria, fungi, and mycorrhiza) associated with plant tissues can take a part in the recovery phenomenon (Romanazzi et al., 2009, Bulgari et al., 2011b). While studying the role of endophytic bacteria in inducing recovery we have faced the need of verifying the distribution of both phytoplasmas and bacteria in grapevine tissues to shed light on their interaction and, in particular, to exclude their direct competition in the phloem cells (Bulgari et al., 2011a). For this reason we resumed microscopic techniques, such as TEM, coupled with fluorescent in situ hybridization (FISH), a method we previously successfully applied to co-localize grapevine phytoplasmas and endophytic bacteria in the host plants Catharanthus roseus (Bulgari et al., 2011a). Materials and Methods Leaf midribs from healthy, FD-diseased and recovered grapevine plants (cv. Cabernet Sauvignon) were collected in summer 2010 and 2011 and processed for conventional TEM analysis and FISH, as previously described (Faoro et al 1991; Bulgari et al. 2011a). Portion of the samples were also analyzed by PCR to confirm the presence of 16SrV phytoplasmas and/or endophytic bacteria. To localize phytoplasmas with FISH, a probe targeting 16SrV group, labeled with FAM (Primm, Milan Italy) or Marina Blue (MB) (Invitrogen, Milan Italy) at 5\u2019 terminus was used. Both these dyes, emitting respectively at 518 nm and 459 nm were tested, to find out the appropriate wavelengths that interfere at least with leaf auto-fluorescence. Endophytic bacteria localization was performed with a universal probe targeting bacterial 16S rDNA (but not phytoplasmal DNA), labeled with a fluorophore (Cy-5) emitting in the far-red (670 nm) (Bulgari et. al 2011a). Labeled sections were observed with a videoconfocal microscope (Nikon, Vico, Italy). Results and Discussion Tem analysis, in spite of the nowadays ameliorated procedure in specimen preparation, confirmed that is not a suitable technique for phytoplasma visualization in grapevine tissues. In fact, phytoplasma detection in thin sections was an almost extraordinary event, also in heavily infected grapevine plants (Fig. 1), in spite of the severe ultrastructural alterations present in the phloem tissue. This is possibly due to the ease with which phytoplasma disruption occurs in grapevine deranged phloem. Even endophytic bacteria localization was not easy by TEM, because these prokaryotes were scattered throughout different tissues and not grouped in enclaves. FISH technique encountered some problems in phytoplasma end endophytic bacteria visualization, mainly due to phloem autofluorescence which was particularly heavy in infected plants (Fig. 2), because of polyphenols deposition. These compounds emitted in both the wavelengths of FAM and MB, thus they were difficult to differentiate from the probes. Only Cy5 was shown to be an excellent reporter molecule for in situ hybridization analysis in grapevine tissues, as it emission was in a band far away from that of polyphenols. With the Cy5 probe it was possible to localize endophytic bacteria in all the examined samples (healthy, diseased and recovered), mostly in the xylem but also in the phloem tissues as scattered spots (Fig. 3), suggesting their random distribution, without large accumulation in specific cells. Instead, phytoplasma probes gave less clear cut results and only in a few cases it was possible to observe specific fluorescence of the probe in the phloem of diseased plants (Fig. 2), but not in healthy or recovered ones. From these results, although too preliminary to drawn any suggestion on phytoplasma-endophytic bacteria interaction in grapevine tissues, it can be concluded that FISH remains the only microscopic technique that have some chance in localizing the very few phytoplasmas present in diseased plants, thus allowing to study their interaction with endophytic bacteria. However, further investigations are needed to improve the technique, particularly for the reduction of tissue autofluorescence that would permit to label probes with a larger set of fluorophores and to co-localize phytoplasma and endophytic bacteria together in the same section