Olive Fruit Fly Symbiont Population: Impact of Metamorphosis.

The current symbiotic view of the organisms also calls for new approaches in the way we perceive and manage our pest species. The olive fruit fly, the most important olive tree pest, is dependent on an obligate bacterial symbiont to its larvae development in the immature fruit. This symbiont, Candidatus (Ca.) Erwinia dacicola, is prevalent throughout the host life stages, and we have shown significant changes in its numbers due to olive fruit fly metamorphosis. The olive fruit fly microbiota was analyzed through 16S metabarcoding, at three development stages: last instar larvae, pupae, and adult. Besides Ca. E. dacicola, the olive fruit flies harbor a diverse bacterial flora of which 13 operational taxonomic units (grouped in 9 genera/species) were now determined to persist excluding at metamorphosis (Corynebacterium sp., Delftia sp., Enhydrobacter sp., Kocuria sp., Micrococcus sp., Propionibacterium sp., Pseudomonas sp., Raoultella sp., and Staphylococcus sp.). These findings open a new window of opportunities in symbiosis-based pest management.

Hidden Genetic Variability, Can the Olive Moth Prays oleae (Lepidoptera: Yponomeutidae or Praydidae?) be a Species' Complex?

Prays oleae is the second most important pest in Mediterranean olive groves, causing substantial damage on olive production. We used mitochondrial [cytochrome c oxidase subunit I (COI), and NADH dehydrogenase subunit 5 (nad5)] and nuclear [ribosomal protein S5 (RpS5)] amplicons to assess the population variability in five main olive producing regions from Tunisia, to support or dismiss the existence of two non-monophyletic groups within the species, as found within Portugal. Our phylogenetic analysis with cytochrome c oxidase subunit I (COI) indeed displayed two distinct and well-supported clades of P. oleae, which were corroborated by the haplotype network reconstructed with both mitochondrial and nuclear amplicons. We were also able to dismiss the hypothesis that one of the clades would not develop on olive fruits. No correlation was observed between clades differentiation and geographic distribution. The existence of cryptic species can impact on the management of agroecosystems and on the perception of how these moths responds to environmental changes.

Effect of Long-Term Fungicide Applications on Virulence and Diversity of Colletotrichum spp. Associated to Olive Anthracnose.

In this study, the presence and variability of Colletotrichum spp. was evaluated by comparing fungal isolates obtained from olive trees under long-time phytosanitary treatments with trees without any phytosanitary treatments (treated and untreated, respectively). Olive fruits of trees of the highly susceptible 'Galega vulgar' cultivar growing in the Alentejo region were used as samples. From the 210 olive trees sampled (half from treated and half from untreated orchards), 125 (59.5%) presented Colletotrichum spp., with a significant lower number of infected trees in treated (39) when compared to untreated orchards (86). The alignment and analysis of beta-tubulin (tub2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), chitin synthase (CHS-1) and histone H3 (HIS-3) gene sequences allowed the identification of all 125 isolates as belonging to the C. acutatum complex. The vast majority of the isolates (124) were identified as C. nymphaeae and one isolate, from an untreated tree, was identified as C. godetiae. Isolates were divided into five different groups: Group A: 39 isolates from treated trees matched in 100% with C. nymphaeae sequences from the database; Group B: 76 isolates from untreated trees matched in 100% with C. nymphaeae sequences from the database; Group C: one isolate from untreated trees presenting a single nucleotidic difference in the HIS-3 sequence; Group D: eight isolates from untreated trees presenting differences in two nucleotides in the tub2 sequences that changed the protein structure, together with differences in two specific nucleotides of the GAPDH sequences; Group E: one isolate, from untreated olive trees, matched 100% with C. godetiae sequences from the database in all genes. Considering the similarities of the sampled areas, our results show that the long-time application of fungicides may have caused a reduction in the number of olive trees infected with Colletotrichum spp. but an increase in the number of fruits positive to Colletotrichum spp. within each tree, which may suggest different degrees of virulence of Colletotrichum isolates from trees growing different management regimes. It is imperative that the fungicides described as causing resistance are applied at appropriate times and intervals, since their efficiency decreases when applied incorrectly and new and more virulent species may arise.

A Re-Evaluation of Olive Fruit Fly Organophosphate-Resistant Ace Alleles in Iberia, and Field-Testing Population Effects after in-Practice Dimethoate Use.

The management of the olive fruit fly (Bactrocera oleae, Dacus oleae) is traditionally based upon the use of organophosphate insecticides, mainly dimethoate. In this evolutionary arms race between man and pest, the flies have adapted a pesticide resistance, implying two point-mutations of the Ace gene -I214V and G488S- and a 9bp deletion -Δ3Q. We revisited 11 Iberian locations to evaluate this adaptation of organophosphate (OP)-resistant alleles through amplicon sequencing. Screening for populations where the wild type is prevalent allows an identification of hotspots for targeted mitigation measures; we have hence refined the scale to the region with the lowest OP-resistant alleles frequency 71 locations were sampled and individuals checked using a fast and low-cost allele-specific-primer polymerase chain reaction (ASP-PCR) method]. An increase in Ace gene point-mutations was observed, and the Δ3Q mutation remains undetected. The lowest frequencies of the OP-resistant alleles remain in the west, underlining the hypothesis of an introduction of resistance from eastern Mediterranean areas. A field test was performed by sampling the fly population before and after in-practice dimethoate application. A clear reduction in olive fruit fly numbers was observed, with no relevant changes in the genotypic frequencies of the resistance alleles. The findings are discussed in frame of the type and intensity of the selection pressure that has led to the adaptation to resistance and its consequences from the producer perspective.

Spatial and temporal variation of fungal endophytic richness and diversity associated to the phyllosphere of olive cultivars.

Fungal endophytes are micro-organisms that colonize healthy plant tissues without causing disease symptoms. They are described as plant growth and disease resistance promoters and have shown antimicrobial activity. The spatial-temporal distribution of endophytic communities in olive cultivars has been poorly explored. This study aims to investigate the richness and diversity of endophytic fungi in different seasons and sites, within the Alentejo region, Portugal. Additionally, and because the impact of some pathogenic fungi (e.g. Colletotrichum spp.) varies according to olive cultivars; three cultivars, Galega vulgar, Cobrançosa and Azeiteira, were sampled. 1868 fungal isolates were identified as belonging to 26 OTUs; 13 OTUs were identified to the genera level and 13 to species level. Cultivar Galega vulgar and season autumn showed significant higher values in terms of endophytic richness and diversity. At site level, Elvas showed the lowest fungal richness and diversity of fungal endophytes. This study reinforces the importance of exploring the combined spatio-temporal distribution of the endophytic biodiversity in different olive cultivars. Knowledge about endophytic communities may help to better understand their functions in plants hosts, such as their ecological dynamics with pathogenic fungi, which can be explored for their use as biocontrol agents.

Uncovered variability in olive moth (Prays oleae) questions species monophyly.

The olive moth -Prays oleae Bern.- remains a significant pest of olive trees showing situation dependent changes in population densities and in severity of damages. The genetic variability of olive moth was assessed on three main olive orchards regions in Portugal by three different markers (COI, nad5 and RpS5), suggesting high species diversity albeit with no obvious relation with a regional pattern nor to an identified ecological niche. Selected COI sequences obtained in this study were combined with those available in the databases for Prays genus to generate a global dataset. The reconstruction of the Prays phylogeny based on this marker revealed the need to revise Prays oleae to confirm its status of single species: COI data suggests the co-existence of two sympatric evolutionary lineages of morphologically cryptic olive moth. We show, however, that the distinct mitochondrial subdivision observed in the partial COI gene fragment is not corroborated by the other DNA sequences. There is the need of understanding this paradigm and the extent of Prays variability, as the disclosure of lineage-specific differences in biological traits between the identified lineages is fundamental for the development of appropriate pest management practices.

Antagonistic activity of fungi of Olea europaea L. against Colletotrichum acutatum.

Fungi naturally present in olive trees were identified and tested for their antagonistic potential against Colletotrichum acutatum. A total of 14 isolates were identified, 12 belonged to genera Alternaria, Epicoccum, Fusarium, Aspergillus, Anthrinium, Chaetomium, Diaporthe, Nigrospora, one to family Xylariaceae and one was unclassified. All fungal isolates showed some inhibitory action over the growth of C. acutatum during dual culture growth, however, when agar-diffusible tests were performed only five fungal isolates caused C. acutatum growth inhibition: Alternaria sp. isolate 2 (26.8%), the fungus from Xylariaceae family (14.3%), Alternaria sp. isolate 1 (10.7%); Diaporthe sp. (10.7%), Nigrospora oryzae (3.5%). Volatile substances produced by these isolates were identified through gas-chromatography techniques, as phenylethyl alcohol, 4-methylquinazoline, benzothiazole, benzyl alcohol, lilial, galaxolide, among others. These inhibitory volatiles could play a significant role in reduction of C. acutatum expansion in olive and their study as potential biocontrol agents should be further explored.

The mitochondrial genome of Prays oleae (Insecta: Lepidoptera: Praydidae).

Prays oleae is one of the most important olive tree pests and a species of interest in evolutionary studies, as it belongs to one of the oldest extant superfamilies of Ditrysian Lepidoptera. We determined its mitogenome sequence, and found it has common features for Lepidoptera, e.g. an >80% A + T content, an apparent CGA start codon for COX1 and an ATAGA(T)n motif in the control region, which also contains several copies of a 163-164 bp repeat. Importantly, the mitogenome displays the Met-Ile-Gln tRNA gene order typical of Ditrysia, consistent with the hypothesis that this is a synapomorphy of that clade.

Marked Genetic Differentiation between Western Iberian and Italic Populations of the Olive Fly: Southern France as an Intermediate Area.

The olive fly, Bactrocera oleae, is the most important pest affecting the olive industry, to which it is estimated to cause average annual losses in excess of one billion dollars. As with other insects with a wide distribution, it is generally accepted that the understanding of B. oleae population structure and dynamics is fundamental for the design and implementation of effective monitoring and control strategies. However, and despite important advances in the past decade, a clear picture of B. oleae's population structure is still lacking. In the Mediterranean basin, where more than 95% of olive production is concentrated, evidence from several studies suggests the existence of three distinct sub-populations, but the geographical limits of their distributions, and the level of interpenetration and gene flow among them remain ill-characterized. Here we use mitochondrial haplotype analysis to show that one of the Mediterranean mitochondrial lineages displays geographically correlated substructure and demonstrate that Italic populations, though markedly distinct from their Iberian and Levantine counterparts are more diverse than previously described. Finally, we show that this distinction does not result from extant hypothetical geographic limits imposed by the Alps or the Pyrenees nor, more generally, does it result from any sharp boundary, as intermixing is observed in a broad area, albeit at variable levels. Instead, Bayesian phylogeographic analysis suggests the interplay between isolation-mediated differentiation during glacial periods and bi-directional dispersal and population intermixing in the interglacials has played a major role in shaping current olive fly population structure.

Mitochondrial haplotypes reveal olive fly (Bactrocera oleae) population substructure in the Mediterranean.

The olive fly (Bactrocera oleae) is the most important olive tree (Olea europaea) pest. In the Mediterranean basin, where 98 % of its main hosts are concentrated, it causes major agricultural losses, due to its negative effect on production and quality of both olive and olive oil. Previous phylogeographic analyses have established that Mediterranean olive fly populations are distinct from other Old World populations, but did not agree on the specific population substructure within this region. In order to achieve a higher resolution of the diversity of olive fly populations, particularly in Central and Western Mediterranean (home to 70 % of the world production), we comparatively analyzed a set of samples from Portugal in the context of published mitochondrial sequences across the species' worldwide range. Strong evidence of population substructure was found in the Central and Western Mediterranean area, with two clearly separate phylogenetic branches. Together with previously published data, our results strongly support the existence of at least three distinct Mediterranean populations of the olive fly, raise the possibility of additional regional substructure and suggest specific avenues for future research. This knowledge can be instrumental in the development of better management and control strategies for a major pest of Mediterranean agriculture.