The dynamics of Xylella fastidiosa infections in the context of the endophytic microbiome was studied in field-grown plants of the susceptible and resistant olive cultivars Kalamata and FS17. Whole metagenome shotgun sequencing (WMSS) coupled with 16S/ITS rRNA gene sequencing was carried out on the same trees at two different stages of the infections: In Spring 2017 when plants were almost symptomless and in Autumn 2018 when the trees of the susceptible cultivar clearly showed desiccations. The progression of the infections detected in both cultivars clearly unraveled that Xylella tends to occupy the whole ecological niche and suppresses the diversity of the endophytic microbiome. However, this trend was mitigated in the resistant cultivar FS17, harboring lower population sizes and therefore lower Xylella average abundance ratio over total bacteria, and a higher α-diversity. Host cultivar had a negligible effect on the community composition and no clear associations of a single taxon or microbial consortia with the resistance cultivar were found with both sequencing approaches, suggesting that the mechanisms of resistance likely reside on factors that are independent of the microbiome structure. Overall, Proteobacteria, Actinobacteria, Firmicutes, and Bacteriodetes dominated the bacterial microbiome while Ascomycota and Basidiomycota those of Fungi.

Philaenus spumarius (Linnaeus, 1758) (Hemiptera, Aphrophoridae) was recently classified as a pest due to its ability to act as a vector of the phytopathogen Xylella fastidiosa. This insect has been reported to harbour several symbiotic bacteria that play essential roles in P. spumarius health and fitness. However, the factors driving bacterial assemblages remain largely unexplored. Here, the bacteriome associated with different organs (head, abdomen, and genitalia) of males and females of P. spumarius was characterized using culturally dependent and independent methods and compared in terms of diversity and composition. The bacteriome of P. spumarius is enriched in Proteobacteria, Bacteroidota, and Actinobacteria phyla, as well as in Candidatus Sulcia and Cutibacterium genera. The most frequent isolates were Curtobacterium, Pseudomonas, and Rhizobiaceae sp.1. Males display a more diverse bacterial community than females, but no differences in diversity were found in distinct organs. However, the organ shapes the bacteriome structure more than sex, with the Microbacteriaceae family revealing a high level of organ specificity and the Blattabacteriaceae family showing a high level of sex specificity. Several symbiotic bacterial genera were identified in P. spumarius for the first time, including Rhodococcus, Citrobacter, Halomonas, Streptomyces, and Providencia. Differences in the bacterial composition within P. spumarius organs and sexes suggest an adaptation of bacteria to particular insect tissues, potentially shaped by their significance in the life and overall fitness of P. spumarius. Although more research on the bacteria of P. spumarius interactions is needed, such knowledge could help to develop specific bacterial-based insect management strategies.

The endosymbiotic bacteria that live within the body of insects are involved in many aspects of the host physiology, including reproduction and defense. Thus, the exploitation of these microorganisms may have practical applications for the management of vector-borne diseases. In the Mediterranean area, Philaenus spumarius was identified as the main vector of Xylella fastidiosa. This xylem-restricted bacterium is responsible for several diseases in a variety of agricultural crops of high importance, for which there are not any effective control methods. Thus, in this work we evaluate different media types for the isolation and growth of bacteria living within P. spumarius adults, for their potential exploitation in the management of X. fastidiosa. Specifically, we compared the effect of minimal (Luria Bertani—LB) and complex (Modified Melin-Norkrans—MMN) media, with and without fetal bovine or gelling agents, on the number and diversity of bacteria. The collection of isolates obtained was further genetically characterized by repetitive element–based PCR method (BOX-PCR) and sequencing of the 16S ribosomal RNA (rRNA) gene. The results showed no significant differences in the abundance and diversity of bacteria among the two media tested (LB and MMN). The addition of fetal bovine to the media leads to a slight increase in bacterial abundance, in particular in MMN medium. While the liquid media led to a significant increase in abundance, the solid media facilitated the growth of more diverse bacterial taxa. The comparison between BOX-PCR and 16S rRNA gene sequencing for the analysis of 57 bacterial isolates revealed a greater discriminatory power of the former, allowing the differentiation of the bacteria even at the intra-species level. The clustering of the isolates using BOX-PCR fingerprinting was different to that obtained from the 16S rRNA gene phylogenetic tree. The 16S rRNA gene sequencing method proved to be more suitable in phylogenetic evaluations, generally grouping isolates belonging to the same genus.

Olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), is the most important olive pest, with major economic importance in olive production worldwide. Different olive cultivars exhibit different propensities to fruit fly infestation and the causes are still unclear. Here, we want to disclose the potential role of olive-associated endophytes in conferring such susceptibility differences. Accordingly, the endophytic microbial composition of infested and non-infested fruits from cultivars Madural (susceptible to olive fly) and Cobrançosa (less susceptible) were studied. A culture-dependent approach was used, the isolates being identified by sequencing of their internal transcribed spacer (for fungi) and 16S rRNA gene regions (for bacteria). Overall, there was a larger consortium of bacteria associated with olives than fungi. The bacterial communities were predominantly composed of Proteobacteria and Actinobacteria phyla, while the fungal isolates belonged to the Ascomycota and Basidiomycota. Both host cultivar and level of fly infestation had a negligible effect on fungal and bacterial community composition. Despite this, a clear positive association of microbial consortia with the resistant cultivar (Kocuria sp., Actinobacterium sp., Rhodococcus sp., Pseudomonas citronellolis, Aspergillus flavus, Cladosporium sp., and Meristemomycetes arctostaphylos) and non-infested fruits (Kocuria sp., Stereum sp., and Vishniacozyma victoria) was found. Their functional roles in host cultivar susceptibility/resistance to the fruit fly is a topic that requires further studies.