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.

Lack of knowledge on ecological determinants and cryptic lifestyles hinder our understanding of Terfezia diversity.

Developing below the soil surface desert, truffles are hard to find. Within Terfezia genus, at least 18 species are described and many are endemic to the Mediterranean basin. Ecological and geographic information are key factors for species diagnosis, and so far Terfezia species are believed to be linked to either acidic or basic soils or to specific plant hosts. Thus, we have looked at Terfezia diversity within a relatively homogeneous geographical area in Portugal that is suitable for these species and that covered different soils and different dominant host species. We analyzed the observed intraspecific variability within the context of species ecological preferences (e. g. edaphic and putative host). One of our major findings was the discovery of T.grisea in acid soils in association with Tuberariaguttata, a puzzling information since, until now, this species was only found in alkaline soils. We also report on the linkage of different Terfezia lineages within species and ecologic parameters such as soil texture, soil pH and plant host. Additionally, by placing the collected specimens on the most recent genus phylogeny based on the ITS region, we also updated the number of known Terfezia species occurring in Portugal from three to ten. Terfeziadunensis is here reported for the first time for Portugal. Overall, our results show that the exploration of undersampled sites reveals itself as a good strategy to disclose unknown aspects of desert truffle diversity and ecology. These aspects are of prime importance when considering the economic value of the desert truffles for rural populations in the Mediterranean basin.

Olive fruit fly and its obligate symbiont Candidatus Erwinia dacicola: Two new symbiont haplotypes in the Mediterranean basin.

The olive fruit fly, specialized to become monophagous during several life stages, remains the most important olive tree pest with high direct production losses, but also affecting the quality, composition, and inherent properties of the olives. Thought to have originated in Africa is nowadays present wherever olive groves are grown. The olive fruit fly evolved to harbor a vertically transmitted and obligate bacterial symbiont -Candidatus Erwinia dacicola- leading thus to a tight evolutionary history between olive tree, fruit fly and obligate, vertical transmitted symbiotic bacterium. Considering this linkage, the genetic diversity (at a 16S fragment) of this obligate symbiont was added in the understanding of the distribution pattern of the holobiont at nine locations throughout four countries in the Mediterranean Basin. This was complemented with mitochondrial (four mtDNA fragments) and nuclear (ten microsatellites) data of the host. We focused on the previously established Iberian cluster for the B. oleae structure and hypothesised that the Tunisian samples would fall into a differentiated cluster. From the host point of view, we were unable to confirm this hypothesis. Looking at the symbiont, however, two new 16S haplotypes were found exclusively in the populations from Tunisia. This finding is discussed in the frame of host-symbiont specificity and transmission mode. To understand olive fruit fly population diversity and dispersion, the dynamics of the symbiont also needs to be taken into consideration, as it enables the fly to, so efficiently and uniquely, exploit the olive fruit resource.

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.

Transcriptome Analysis of Wheat Roots Reveals a Differential Regulation of Stress Responses Related to Arbuscular Mycorrhizal Fungi and Soil Disturbance.

Symbioses with soil microorganisms are central in shaping the diversity and productivity of land plants and provide protection against a diversity of stresses, including metal toxicity. Arbuscular mycorrhizal fungi (AMF) can form extensive extraradical mycelial networks (ERM), which are very efficient in colonizing a new host. We quantified the responses of transcriptomes of wheat and one AMF partner, Rhizoglomus irregulare, to soil disturbance (Undisturbed vs. Disturbed) and to two different preceding mycotrophic species (Ornithopus compressus and Lolium rigidum). Soil disturbance and preceding plant species engender different AMF communities in wheat roots, resulting in a differential tolerance to soil manganese (Mn) toxicity. Soil disturbance negatively impacted wheat growth under manganese toxicity, probably due to the disruption of the ERM, and activated a large number of stress and starvation-related genes. The O. compressus treatment, which induces a greater Mn protection in wheat than L. rigidum, activated processes related to cellular division and growth, and very few related to stress. The L. rigidum treatment mostly induced genes that were related to oxidative stress, disease protection, and metal ion binding. R. irregulare cell division and molecular exchange between nucleus and cytoplasm were increased by O. compressus. These findings are highly relevant for sustainable agricultural systems, when considering a fit-for-purpose symbiosis.

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.

Symbiosis in Sustainable Agriculture: Can Olive Fruit Fly Bacterial Microbiome Be Useful in Pest Management?

The applied importance of symbiosis has been gaining recognition. The relevance of symbiosis has been increasing in agriculture, in developing sustainable practices, including pest management. Insect symbiotic microorganisms' taxonomical and functional diversity is high, and so is the potential of manipulation of these microbial partners in suppressing pest populations. These strategies, which rely on functional organisms inhabiting the insect, are intrinsically less susceptible to external environmental variations and hence likely to overcome some of the challenges posed by climate change. Rates of climate change in the Mediterranean Basin are expected to exceed global trends for most variables, and this warming will also affect olive production and impact the interactions of olives and their main pest, the obligate olive fruit fly (Bactrocera oleae). This work summarizes the current knowledge on olive fly symbiotic bacteria towards the potential development of symbiosis-based strategies for olive fruit fly control. Particular emphasis is given to Candidatus Erwinia dacicola, an obligate, vertically transmitted endosymbiont that allows the insect to cope with the olive-plant produced defensive compound oleuropein, as a most promising target for a symbiosis disruption approach.

What is in a name? Terfezia classification revisited.

Desert truffles (mycorrhizal hypogeous Ascomycota) are found in arid and semi-arid areas of the globe and have great ecological and economic importance. Terfezia is undoubtedly the most diversified of all desert truffle genera, but its taxonomy is far from resolved. Specifically, the large number of newly described species plus the high intraspecific morphological variability observed within some Terfezia lineages as rendered the use of molecular techniques mandatory for specimen's discrimination. But the subsequent increasing amount of sequence data produced also a huge number of undescribed taxa that required determination. We compiled and used the public available ITS data on Terfezia spp. on the custom-curated UNITE database to reconstruct the genus phylogeny. We found at least 17 distinct lineages within the genus and successfully resolved some of the more pressing taxonomic issues, namely the T. leptoderma/olbiensis complex and some misapplied synonymy. Based on this resolved phylogeny, and motivated by the recent new described species, we proposed an identification key to Terfezia genus highlighting the importance of morphological and ecological characterization.

Symbiosis Specificity of the Preceding Host Plant Can Dominate but Not Obliterate the Association Between Wheat and Its Arbuscular Mycorrhizal Fungal Partners.

The symbiosis established between arbuscular mycorrhizal fungi (AMF) and roots of most land plants plays a key role in plant nutrient acquisition and alleviation of environmental stresses. Despite the ubiquity of the symbiosis, AMF and host species display significant specificity in their interactions. To clarify preferential associations between wheat (Triticum aestivum) and AMF, we characterized root AMF communities in the transition from two first host species, ryegrass (Lolium rigidum) and yellow-serradella (Ornithopus compressus), grown separately or together, to a second host (wheat), by sequencing the large subunit ribosomal DNA (LSU rDNA) gene. The response of AMF communities in wheat to prior soil disturbance - and consequently of the mycelial network [intact extraradical mycelium (ERM) vs. disrupted mycelium] established with either of the first hosts - was also investigated. Since the outcome of a specific host-symbiont interaction depends on the molecular responses of the host plant upon microbial colonization, we studied the expression of six key symbiosis-related genes in wheat roots. AMF communities on L. rigidum and O. compressus roots were clearly distinct. Within an undisturbed ERM, wheat AMF communities were similar to that of previous host, and O. compressus-wheat-AMF interactions supported a greater growth of wheat than L. rigidum-wheat-AMF interactions. This effect declined when ERM was disrupted, but generated a greater activation of symbiotic genes in wheat, indicating that plant symbiotic program depends on some extent on the colonizing symbiont propagule type. When a mixture of L. rigidum and O. compressus was planted, the wheat colonization pattern resembled that of O. compressus, although this was not reflected in a greater growth. These results show a lasting effect of previous hosts in shaping wheat AMF communities through an efficient use of the established ERM, although not completely obliterating host-symbiont specificity.

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.

Symbiotic flagellate protists as cryptic drivers of adaptation and invasiveness of the subterranean termite Reticulitermes grassei Clément.

Changes in flagellate protist communities of subterranean termite Reticulitermes grassei across different locations were evaluated following four predictions: (i) Rural endemic (Portugal mainland) termite populations will exhibit high diversity of symbionts; (ii) invasive urban populations (Horta city, Faial island, Azores), on the contrary, will exhibit lower diversity of symbionts, showing high similarity of symbiont assemblages through environmental filtering; (iii) recent historical colonization of isolated regions-as the case of islands-will imply a loss of symbiont diversity; and (iv) island isolation will trigger a change in colony breeding structure toward a less aggressive behavior. Symbiont flagellate protist communities were morphologically identified, and species richness and relative abundances, as well as biodiversity indices, were used to compare symbiotic communities in colonies from urban and rural environments and between island invasive and mainland endemic populations. To evaluate prediction on the impact of isolation (iv), aggression tests were performed among termites comprising island invasive and mainland endemic populations. A core group of flagellates and secondary facultative symbionts was identified. Termites from rural environments showed, in the majority of observed colonies, more diverse and abundant protist communities, probably confirming prediction (i). Corroborating prediction (ii), the two least diverse communities belong to termites captured inside urban areas. The Azorean invasive termite colonies had more diverse protist communities than expected and prediction (iii) which was not verified within this study. Termites from mainland populations showed a high level of aggressiveness between neighboring colonies, in contrast to the invasive colonies from Horta city, which were not aggressive to neighbors according to prediction (iv). The symbiotic flagellate community of R. grassei showed the ability to change in a way that might be consistent with adaptation to available conditions, possibly contributing to optimization of the colonization of new habitats and spreading of its distribution area, highlighting R. grassei potential as an invasive species.

Wild Carrot Differentiation in Europe and Selection at DcAOX1 Gene?

By definition, the domestication process leads to an overall reduction of crop genetic diversity. This lead to the current search of genomic regions in wild crop relatives (CWR), an important task for modern carrot breeding. Nowadays massive sequencing possibilities can allow for discovery of novel genetic resources in wild populations, but this quest could be aided by the use of a surrogate gene (to first identify and prioritize novel wild populations for increased sequencing effort). Alternative oxidase (AOX) gene family seems to be linked to all kinds of abiotic and biotic stress reactions in various organisms and thus have the potential to be used in the identification of CWR hotspots of environment-adapted diversity. High variability of DcAOX1 was found in populations of wild carrot sampled across a West-European environmental gradient. Even though no direct relation was found with the analyzed climatic conditions or with physical distance, population differentiation exists and results mainly from the polymorphisms associated with DcAOX1 exon 1 and intron 1. The relatively high number of amino acid changes and the identification of several unusually variable positions (through a likelihood ratio test), suggests that DcAOX1 gene might be under positive selection. However, if positive selection is considered, it only acts on some specific populations (i.e. is in the form of adaptive differences in different population locations) given the observed high genetic diversity. We were able to identify two populations with higher levels of differentiation which are promising as hot spots of specific functional diversity.

Misannotation Awareness: A Tale of Two Gene-Groups.

Incorrectly or simply not annotated data is largely increasing in most public databases, undoubtedly caused by the rise in sequence data and the more recent boom of genomic projects. Molecular biologists and bioinformaticists should join efforts to tackle this issue. Practical challenges have been experienced when studying the alternative oxidase (AOX) gene family, and hence the motivation for the present work. Commonly used databases were screened for their capacity to distinguish AOX from the plastid terminal oxidase (also called plastoquinol terminal oxidase; PTOX) and we put forward a simple approach, based on amino acids signatures, that unequivocally distinguishes these gene families. Further, available sequence data on the AOX family in plants was carefully revised to: (1) confirm the classification as AOX and (2) identify to which AOX family member they belong to. We bring forward the urgent need of misannotation awareness and re-annotation of public AOX sequences by highlighting different types of misclassifications and the large under-estimation of data availability.

Oligocene Termite Nests with In Situ Fungus Gardens from the Rukwa Rift Basin, Tanzania, Support a Paleogene African Origin for Insect Agriculture.

Based on molecular dating, the origin of insect agriculture is hypothesized to have taken place independently in three clades of fungus-farming insects: the termites, ants or ambrosia beetles during the Paleogene (66-24 Ma). Yet, definitive fossil evidence of fungus-growing behavior has been elusive, with no unequivocal records prior to the late Miocene (7-10 Ma). Here we report fossil evidence of insect agriculture in the form of fossil fungus gardens, preserved within 25 Ma termite nests from southwestern Tanzania. Using these well-dated fossil fungus gardens, we have recalibrated molecular divergence estimates for the origins of termite agriculture to around 31 Ma, lending support to hypotheses suggesting an African Paleogene origin for termite-fungus symbiosis; perhaps coinciding with rift initiation and changes in the African landscape.

Stress-Induced Accumulation of DcAOX1 and DcAOX2a Transcripts Coincides with Critical Time Point for Structural Biomass Prediction in Carrot Primary Cultures (Daucus carota L.).

Stress-adaptive cell plasticity in target tissues and cells for plant biomass growth is important for yield stability. In vitro systems with reproducible cell plasticity can help to identify relevant metabolic and molecular events during early cell reprogramming. In carrot, regulation of the central root meristem is a critical target for yield-determining secondary growth. Calorespirometry, a tool previously identified as promising for predictive growth phenotyping has been applied to measure the respiration rate in carrot meristem. In a carrot primary culture system (PCS), this tool allowed identifying an early peak related with structural biomass formation during lag phase of growth, around the 4th day of culture. In the present study, we report a dynamic and correlated expression of carrot AOX genes (DcAOX1 and DcAOX2a) during PCS lag phase and during exponential growth. Both genes showed an increase in transcript levels until 36 h after explant inoculation, and a subsequent down-regulation, before the initiation of exponential growth. In PCS growing at two different temperatures (21°C and 28°C), DcAOX1 was also found to be more expressed in the highest temperature. DcAOX genes' were further explored in a plant pot experiment in response to chilling, which confirmed the early AOX transcript increase prior to the induction of a specific anti-freezing gene. Our findings point to DcAOX1 and DcAOX2a as being reasonable candidates for functional marker development related to early cell reprogramming. While the genomic sequence of DcAOX2a was previously described, we characterize here the complete genomic sequence of DcAOX1.

Can functional hologenomics aid tackling current challenges in plant breeding?

Molecular plant breeding usually overlooks the genetic variability that arises from the association of plants with endophytic microorganisms, when looking at agronomic interesting target traits. This source of variability can have crucial effects on the functionality of the organism considered as a whole (the holobiont), and therefore can be selectable in breeding programs. However, seeing the holobiont as a unit for selection and improvement in breeding programs requires novel approaches for genotyping and phenotyping. These should not focus just at the plant level, but also include the associated endophytes and their functional effects on the plant, to make effective desirable trait screenings. The present review intends to draw attention to a new research field on functional hologenomics that if associated with adequate phenotyping tools could greatly increase the efficiency of breeding programs.

Intra and Inter-Spore Variability in Rhizophagus irregularis AOX Gene.

Arbuscular mycorrhizal fungi (AMF) are root-inhabiting fungi that form mutualistic symbioses with their host plants. AMF symbiosis improves nutrient uptake and buffers the plant against a diversity of stresses. Rhizophagus irregularis is one of the most widespread AMF species in the world, and its application in agricultural systems for yield improvement has increased over the last years. Still, from the inoculum production perspective, a lack of consistency of inoculum quality is referred to, which partially may be due to a high genetic variability of the fungus. The alternative oxidase (AOX) is an enzyme of the alternative respiratory chain already described in different taxa, including various fungi, which decreases the damage caused by oxidative stress. Nevertheless, virtually nothing is known on the involvement of AMF AOX on symbiosis establishment, as well on the existence of AOX variability that could affect AMF effectiveness and consequently plant performance. Here, we report the isolation and characterisation of the AOX gene of R. irregularis (RiAOX), and show that it is highly expressed during early phases of the symbiosis with plant roots. Phylogenetic analysis clustered RiAOX sequence with ancient fungi, and multiple sequence alignment revealed the lack of several regulatory motifs which are present in plant AOX. The analysis of RiAOX polymorphisms in single spores of three different isolates showed a reduced variability in one spore relatively to a group of spores. A high number of polymorphisms occurred in introns; nevertheless, some putative amino acid changes resulting from non-synonymous variants were found, offering a basis for selective pressure to occur within the populations. Given the AOX relatedness with stress responses, differences in gene variants amongst R. irregularis isolates are likely to be related with its origin and environmental constraints and might have a potential impact on inoculum production.

Complementary symbiont contributions to plant decomposition in a fungus-farming termite.

Termites normally rely on gut symbionts to decompose organic matter but the Macrotermitinae domesticated Termitomyces fungi to produce their own food. This transition was accompanied by a shift in the composition of the gut microbiota, but the complementary roles of these bacteria in the symbiosis have remained enigmatic. We obtained high-quality annotated draft genomes of the termite Macrotermes natalensis, its Termitomyces symbiont, and gut metagenomes from workers, soldiers, and a queen. We show that members from 111 of the 128 known glycoside hydrolase families are represented in the symbiosis, that Termitomyces has the genomic capacity to handle complex carbohydrates, and that worker gut microbes primarily contribute enzymes for final digestion of oligosaccharides. This apparent division of labor is consistent with the Macrotermes gut microbes being most important during the second passage of comb material through the termite gut, after a first gut passage where the crude plant substrate is inoculated with Termitomyces asexual spores so that initial fungal growth and polysaccharide decomposition can proceed with high efficiency. Complex conversion of biomass in termite mounds thus appears to be mainly accomplished by complementary cooperation between a domesticated fungal monoculture and a specialized bacterial community. In sharp contrast, the gut microbiota of the queen had highly reduced plant decomposition potential, suggesting that mature reproductives digest fungal material provided by workers rather than plant substrate.