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- Bacterial dynamics and exchange in plant-insect interactionsPublication . Cameirão, Cristina; Pereira, José Alberto; Tavares, Rui; Lino-Neto, Teresa; Baptista, PaulaIn nature, plants and insects engage in intricate interactions. Despite the increasing knowledge of the microbiomes of plants and insects, the extent to which they exchange and alter each other’s microbiomes remains unclear. In this work, the bacterial community associated with nymphs of Philaenus spumarius (Hemiptera: Aphrophoridae), the stems of Coleostephus myconis where the nymphs were feeding, and the foam produced by the nymphs, were studied by culture-dependent and -independent approaches, with an attempt to elucidate the exchange of bacteria between plants and insects. The results suggest that both approaches complement each other, as many bacterial genera identified by metabarcoding were not detected by culturing, and vice versa. Overall, stems and foam exhibited higher bacterial diversity than nymphs, with all the samples showing enrichment in bacteria known to provide diverse benefits to their host. Stems and foam were the most similar in bacterial composition, but Burkholderiaceae and Moraxellaceae dominated the stems, whereas Rhizobiaceae and Sphingobacteriaceae dominated the foam. Nymphs exhibit the most distinct bacterial composition, yet more similar to that found in the stem compared to the foam. Indeed, nymphs were enriched on endosymbiotic bacteria, mostly Candidatus Sulcia and Sodalis, not found in the stem and foam. Nevertheless, during feeding, nymphs appeared to exchange several bacteria genera with C. myconis, with a significant number being incor- porated into the bacteriome of the nymph. The genera Curvibacter, Cutibacterium, Methylobacterium, Pseudomonas and Rhizobium are likely the most exchanged. Nymphs also appear to exchange bacteria to the foam, notably species from the Enhydrobacter, Pseudomonas, Rhizobium and Roseomonas genera. More studies to infer the functions of the shared bacteria between P. spumarius-C. myconis are needed.
- Exploitation of Coleostephus myconis-associated endophytic fungi repel or attract Xylella fastidiosa vectorPublication . Cameirão, Cristina; Fernandes, Gisela Mariana; Martins, Fátima; Pinto, Maria Beatriz; Pereira, J.A.; Baptista, PaulaXylella fastIdiosa is a bacterium transmitted by insectvectors that cause high losses in a variety ofagriculturally important crops. In Europe, an outbreak ofthe disease was recently detected in olive trees, being Philaenus spumarius só far identífied as the only vector involved in this outbreak. Thus, vector contrai by using bait plants and/or insect repellents that emit volatile compounds produced by microorganisms that colonize host plants [endophyte] may help to reduce the dissemination ofthe disease. In this work the endophytic fungal community of Coleostephus myconis, a ground cover plant present in the olive graves and commonly infested by P. spumarius, was studied. Fungi were isolated from stems, leaves and inflorescences ofplants with three leveis of P. spumarius infestation, collected in an olive grove located in Mirandela. The fungal isolates obtained were identified by sequencing the ITS region of rDNA the partial tefl. A total of 121 species/genera were identified, bemg Alternaria, Chalastospora and Valsa the most abundant genera. The diversity and abundance of endophytic fungi was higher in the leaf and inflorescences than in the stem. The composition and diversity of endophytic fungal community was different between infested and non-infested plants, especially at the levei of their leaves. Endophyte community similarity between the different plant organs was low, especially between stem and inflorescences. Altogether, the results show that P. spumaríus affects the composition and structure ofendophytic fungal community associated to C. myconis, especially in their leaves. The effect of this change in the fungal community in attractiveness/ repulsion of P. spumarius should be studied in the future.
- Differences in the endophytic microbiome of olive cultivars infected by xylella fastidiosa across seasonsPublication . Giampetruzzi, Annalisa; Baptista, Paula; Morelli, Massimiliano; Cameirão, Cristina; Neto, Teresa Lino; Costa, Daniela; D’Attoma, Giusy; Kubaa, Raied Abou; Altamura, Giuseppe; Saponari, Maria; Pereira, J.A.; Saldarelli, PasqualeThe 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.
- Characterization of the endophytic fungal communities associated to Coleostephus myconis: looking for potential repellent/attractant compounds towards Philaenus spumariusPublication . Cameirão, Cristina; Fernandes, Gisela Mariana; Martins, Fátima; Pereira, J.A.; Baptista, PaulaPhilaenus spumarius is a xylem feeding insect identified so far as the only vector involved in the first European outbreak of the phytobacterium Xylella fastidiosa, affecting and devastating olive groves in Italy. One approach to reduce X. fastidiosa transmission could rely in the vector control through the use of baits to attract and/or repel de insect. Fungal endophytes inhabiting plants provide a source of candidate repellent/attractant compounds. In this work, the endophytic fungi community associated to the weed Coleostephus myconis, was studied in order to identified potential fungi that can be used as attractants or repellents of P. spumarius. Coleostephus myconis is common in olive groves and preferentially used by P. spumarius for the nymph development. Fungi were isolated from the stems, leaves and inflorescences from plants with three levels of P. spumarius infestation (i.e. no infestation, moderate and high infestation). The fungal isolates obtained were identified by sequencing the ITS region of rDNA and the partial tef1. Overall, was obtained 138 taxa belonging to 37 different genera. The endophytic community composition differs between infested and non-infested plants, solely at stems level, where the P. spumarius foam nests are present. The fungal genera, Cytospora, Diaporthe and Penicillium, which are well-known with producers of volatiles compounds repellent activity against insect, were found only on non-infested plants. Botrytis was restricted to infested plants. The behavioral responses and preferences of P. spumarius for C. myconis plants colonized by these endophytes should be investigated in the future.
- Análise preliminar da comunidade bacteriana em diferentes fases do processo de compostagem de bagaço de azeitonaPublication . Royer, Ana Caroline; Nancassa, Inês; Cameirão, Cristina; Figueiredo, Tomás de; Figueiredo, Daniel; Hernández, Zulimar; Baptista, PaulaO bagaço de azeitona (BA) é um subproduto húmido gerado na produção de azeite, com alta carga orgânica, e elevada toxicidade devido à sua composição em polifenóis. A sua produção pode, assim, constituir um problema ambiental para os países produtores de azeite, como Portugal. A compostagem poderá ser uma forma de valorizar este subproduto, sendo os microrganismos os principais agentes deste processo de degradação aeróbica. O objetivo do presente estudo foi estudar a evolução da comunidade bacteriana ao longo do processo de compostagem de BA e isolar e identificar bactérias com a finalidade de explorá-las no futuro como inóculos de compostagem. A compostagem foi realizada em quatro pilhas de escala industrial com diferentes proporções de BA, fonte de azoto, agente estruturante e reviramentos. Amostras compósitas foram coletadas nas pilhas em três momentos: fase inicial, fase mesófila e fase termófila. Os isolados bacterianos, obtidos através de diluições seriadas, foram identificados por sequenciação da região 16S do rRNA. Os resultados de composição e diversidade, mostraram diferenças significativas entre as fases de compostagem e entre pilhas, sugerindo uma mudança contínua de espécies bacterianas ao longo das diferentes fases do processo de compostagem do BA.
- Exploitation of plant microbiota in the development of new approaches to manage Xylella fastidiosa and its insect vectorsPublication . Cameirão, Cristina; Baptista, PaulaXylella fastidiosa is a plant pathogen transmitted by insect vectors that cause high losses in a variety of agriculturally important crops. In Europe, an outbreak of X. fastidiosa was first detected in 2013 in olive trees in Southern Italy, being Philaenus spumarius (L.) identified as the vector involved in this outbreak. In this work, as a first approach, we explored the endophytic microbial community inhabiting plants in order to design new approaches to manage the X. fastidiosa in the olive tree and its insect vector. Nymphs of P. spumarius feeds commonly on stems of Coleostephus myconis (L.), which is a common weed found in olive groves. A better understand of the feeding effect on the fungal community residing in this weed and of the role of these fungi in feeding choice could assist in the manipulation of P. spumarius populations. For this, the endophytic fungal community of stems, leaves and inflorescences of C. myconis with three levels of P. spumarius infestation was studied by cultural dependent method. Feeding was showed to affect the fungal communities in the stem and leaf but not the ones in the inflorescence. A set of fungi was identified to be associated to either infested or non-infested plants, suggesting playing a key role on the feeding process. As a second approach, it was observed, in the outbreak, the presence of olive cultivars resistant (cv. FS17) or susceptible (cv. Kalamata) to X. fastidiosa. The study of the xylem microbiome of these two cultivars, was performed through Illumina amplicon sequencing in order to determine whether susceptibilities differences are linked to its core microbiome. Overall, the core microbiome was dominated by members of the Proteobacteria (81% of the total bacteria reads) and Ascomycota (98% of the total fungal reads). In non-infected trees, the core microbiome was found to differ between susceptible and resistant cultivars. The presence of X. fastidiosa showed to induce greater variation in microbial composition and diversity of the susceptible cultivar than of the resistant cultivar. Specific fungal/bacterial signatures were detected to either the presence or the absence of X. fastidiosa in the xylem vessels, suggesting an important role of these microorganisms in pathogen establishment/development. Our study provides microbial candidates to further evaluate their role in the management of P. spumarius and X. fastidiosa.
- Potential natural biocontrol agents of Aphrophoridae eggsPublication . Reis, Carlos; Villa, Maria; Rodrigues, Isabel; Cameirão, Cristina; Baptista, Paula; Pereira, J.A.Xylella fastidiosa is a phytopathogenic bacterium detected recently in Europe. It causes important economical losses in different crops, mainly in olive, almond and vineyard. Up to now the Aphrophoridae Philaenus spumarius L. (1758) is the main identified vector in Europe. The information about Aphrophoridae biology is insufficient and the knowledge about its natural enemies reduced. Nymphs excrete spumes remaining protected from natural enemies. Therefore, eggs are likely the most vulnerable stage to the natural enemies action. In this work, the potential action of parasitoids, predators and fungi against eggs of Aphrophoridae was studied. For that, at the end of the winter and before the egg hatching, rests of the remaining vegetation in the ground cover, mainly Poaceae, were collected. In the laboratory leaves, particularly the interior part of the blade which is a common oviposition location for spittlebugs, were observed under binocular stereoscope. Viable eggs were introduced in petri dishes until hatching. A total number of eggs of 3841 in 322 eggs laying and with a mean of 12 eggs per egg laying was recorded. From them, 603 eggs were intact and 3238 had sings of natural control agent action. A potential action of predators (in 183 eggs laying), parasitoid (in 198 eggs laying) and entomopathogenic fungi (in 48 eggs laying) was observed. Additionally, 321 parasitoids emerged from 43 of the eggs laying. These results indicate that the eggs mortality due to beneficial organisms, particularly parasitoids, may have a high importance for the control of X. fastidiosa vectors
- Endophytic fungal community succession in reproductive organs of two olive tree cultivars with contrasting anthracnose susceptibilitiesPublication . Martins, Fátima; Cameirão, Cristina; Mina, Diogo; Benhadi-Marín, Jacinto; Pereira, J.A.; Baptista, PaulaThe factors shaping endophytic fungal assemblages in reproductive organs have been less evaluated than in vegetative organs. Here, the temporal dynamics of fungal endophytes in inflorescences and fruits of olive tree cultivars with contrasting anthracnose susceptibilities was assessed, starting before flowers open until fruit ripening. Fungal diversity assessed by rRNA sequencing of cultivable isolates revealed a higher richness and abundance of endophytes in inflorescences than in fruits. Endophytes from Sordariomycetes were dominant in inflorescences, while in fruits they were from Dothideomycetes. The overall assemblages of inflorescences and fruits were shaped mainly by the plant organ, followed by the phenological stage and lastly, by the plant cultivar. The importance of cultivar as a shaping factor varied according to the plant organ, being only significant in inflorescence-associated endophytes. A set of fungal taxa were positively associated with a particular cultivar and phenological stage, likely contributing to the plant anthracnose resistance.
- Diversity, Composition, and Specificity of the Philaenus spumarius BacteriomePublication . Cameirão, Cristina; Costa, Daniela; Rufino, José; Pereira, J.A.; Lino-Neto, Teresa; Baptista, PaulaPhilaenus 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 effect of Philaenus spumarius feeding on the endophytic fungi community of Coleostephus myconis and its possible applicability in the insect biocontrolPublication . Cameirão, Cristina; Fernandes, Gisela Mariana; Martins, Fátima; Pereira, J.A.; Baptista, PaulaPhilaenus spumarius was recently confirmed as the European vector for the ongoing Xylella fastidiosa outbreak in the olive orchards of Southern Italy. In the northeast region of Portugal, P. spumarius is widespread and common in olive groves. In these groves was observed that their nymphs feed preferentially on the weed Coleostephus myconis, displaying in the same time period and area different levels of infestation. This work aims to assess the correlation between the fungal endophytic communities inhabiting C. myconis with the feeding preference of P. spumarius. Fungi were isolated from stems, leaves and inflorescences of plants with three levels of P. spumarius infestation (no infestation, moderate and high infestation), collected in an olive grove located in Mirandela. The fungal isolates were further identified by sequencing the ITS region of rDNA and the partial tef1. A total of 138 OTUs belonging to 37 genera were isolated, being Alternaria, Valsa, Cladosporium and Chalastospora the most abundant genera. An increase in overall endophytic fungal abundance and richness was observed across infestation levels, in particular of isolates belonging to Chalastospora and Cladosporium. In contrast, Biscogniauxia, Chaetomium and Stemphylium, decreased across the levels of infestation. Differences in fungal composition between non-infested and infested plants were particularly observed in stems, where the P. spumarius foam nests are present. Some genera were exclusively found either in non-infested or in infested plants. Our survey provides fungal candidates to further evaluate their role in the repellency/attraction of P. spumarius in order to develop new management tools against this insect.