PHYTOPLASMAS-ENDOPHYTES INTERACTIONS: THE CASE STUDY OF GRAPEVINE YELLOWS RECOVERY

This PhD project aims to study the interactions between endophytic bacteria and phytoplasmas associated with grapevines to clarify the role of endophytic bacteria in grapevine yellows (GY) recovery and discuss the possibility to apply these bacteria for GY management. Diversity of bacterial endophytes associated with grapevine leaf tissues was analyzed by cultivation and cultivation-independent methods. To identify bacterial endophytes directly from metagenome, a protocol for bacteria enrichment and DNA extraction was optimized. Library analysis of a PCR-amplified 16S rDNA fragment showed best sequence matches with Proteobacteria, family Enterobacteriaceae, with a dominance of the genus Pantoea. Nucleotide sequences of 16S rDNA from bacteria isolated through cultivation showed best sequence matches with Curtobacterium, Stenotrophomonas, Methylobacterium, Pectobacterium, Enterobacter, Brevundimonas, Agrobacterium, Brevibacillus, Staphylococcus, Sphingomonas, Acaricomes, and Enterococcus. Length Heterogeneity-PCR (LH-PCR) electrophoretic peaks from single bacterial clones were used to setup a database representing the bacterial endophytes identified in association with grapevine tissues. Furthermore, composition and structure of endophytic bacterial community were examined in healthy, phytoplasma-diseased and recovered grapevine plants. LH-PCR of total DNA from grapevine leaves was used to generate amplicon profiles that were analyzed with univariate and multivariate statistical methods. Jaccard analyses highlighted that microbial diversity and structure is different in healthy, diseased and recovered grapevine plants. Multivariate analyses confirmed this trend and showed that three LH-PCR peaks determined the variation in microbial composition. Furthermore, LH-PCR database were used to monitor the distribution of bacterial endophytes in total DNAs from analyzed plants. Bacterial community associated with healthy plants was characterized by a greater richness (higher number of LH-PCR peaks) than that present in diseased and recovered plants. Observed low bacterial richness and different microbial composition in infected and recovered plants suggest that phytoplasma infection could directly and/or indirectly restructure bacterial community selecting endophytic strains that are able to elicit plant defense response. Moreover, we investigated the influence of Flavescence dor\ue9e phytoplasmas (FDp) on endophytic bacterial community by studying the seasonal fluctuation of bacterial species associated with healthy, FDp-diseased and recovered plants during phytoplasma infection process. Statistical analysis indicated that, before phytoplasma titre inside diseased plant tissues becomes detectable, endophytic bacterial community is similar to that associated with healthy plants and differs from that associated with recovered plants. Moreover, it seems that a change in microbial composition could be determined when phytoplasmas start to replicate. LH-PCR database showed a seasonal fluctuation of some bacteria identified in the analyzed grapevines; these fluctuations are also related to the presence/absence of the pathogen. On the basis of these evidences we hypothesized that phytoplasma replication determines a change in microbial composition selecting few endophytes that could induce recovery mediated by priming response. Finally, in order to study in which way phytoplasmas interact with endophytes and host plant we developed and tested two different microscopy techniques. Fluorescence in situ hybridisation (FISH) allowed to localise, as expected, phytoplasmas in Catharanthus roseus L. (G. Don) phloem tissues, and also to visualize endophytes in phloem, xylem and parenchyma in the same leaf tissues. On the other hand, immune-confocal laser scanning microscopy was applied for demonstrating that SAP11 (a phytoplasma effector protein) can be unloaded from the phloem by itself. These techniques will be used in combination for clarifying if phytopasmas co-localise with ISR bacterial-inducers and for studying proteins involved in phytoplasmas-endophytes-host cross-talk