Potential natural biocontrol agents of Aphrophoridae eggs

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 vectorsinfo:eu-repo/semantics/publishedVersio

Characterization of the endophytic fungal communities associated to Coleostephus myconis: looking for potential repellent/attractant compounds towards Philaenus spumarius

Philaenus 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.This work is funded by national funds through the Foundation for Science and Technology - FCT - in the scope of the project PTDC/AGR-PRO/0856/2014 XF-FREEOLIVE: Multifunctional study of xylem-sap of Portuguese olive cultivars and its relation with susceptibility to infection by Xylella fastidiosa, and by European fund in the scope of the project H2020-SFS-2016-3 XF-ACTORS - Xylella fastidiosa active containment through a multidisciplinary-oriented research strategyinfo:eu-repo/semantics/publishedVersio

Endophytic fungal community succession in reproductive organs of two olive tree cultivars with contrasting anthracnose susceptibilities

The 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.This work is supported by FEDER funds through the COMPETE (Operational Programme for Competitiveness Factors) and by the Foundation for Science and Technology (FCT, Portugal) within the POCI-01-0145-FEDER-031133 (MicOlives) project and FCT/MCTES to CIMO (UIDB/00690/2020). F. Martins thanks the award of a PhD scholarship (ref. SFRH/BD/112234/2015) by FCT.info:eu-repo/semantics/publishedVersio

Differences in the endophytic microbiome of olive cultivars infected by xylella fastidiosa across seasons

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.CIMO -Kansainvälisen Liikkuvuuden ja Yhteistyön Keskus(UIDB/04046/2020

Exploitation of plant microbiota in the development of new approaches to manage Xylella fastidiosa and its insect vectors

Xylella 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.Xylella fastidiosa é um fitopatogénico transmitido por insetos vetores, responsável por perdas elevadas numa grande variedade de culturas agrícolas importantes. O primeiro foco de doença causada por esta bactéria na Europa foi detetado em 2013, em olivais no Sul de Itália, tendo sido Philaenus spumarius (L.) identificado como o vetor envolvido na transmissão da doença. Neste trabalho exploramos a comunidade microbiana endófita presente nas plantas para desenvolver novas abordagens para o controlo de X. fastidiosa na oliveira e no seu inseto vetor. As ninfas de P. spumarius alimentam-se preferencialmente nos caules de Coleostephus myconis (L.), uma infestante muito comum nos olivais. Uma melhor compreensão do efeito do inseto vetor na comunidade fúngica associada à planta de que se alimenta, e o papel destes fungos na preferência do inseto por uma determinada planta, poderá auxiliar na manipulação da população de P. spumarius. Para isso, estudou-se a comunidade fúngica endófita do caule, folha e inflorescência de C. myconis com três níveis de infestação por P. spumarius, recorrendo a métodos culturais. Verificou-se que a alimentação afetou a comunidade fúngica do caule e da folha, mas não da inflorescência. Foi identificado um conjunto de fungos associado a plantas infestadas ou não infestadas, sugerindo terem um papel no processo de alimentação. Na área do foco da doença foi observada a presença de cultivares de oliveiras resistentes (cv. FS17) ou suscetíveis (cv. Kalamata) à X. fastidiosa. O microbioma do xilema destas duas cultivares foi estudado, através da sequenciação Illumina, para determinar se as diferenças de suscetibilidade estão relacionadas com o seu microbioma central (core microbioma). No geral, o microbioma central foi dominado por membros da Proteobacteria (81% do total de reads de bactérias) e Ascomycota (98% do total de reads de fungos). Em árvores não infetadas, o microbioma central foi distinto entre cultivares suscetíveis e resistentes. A presença de X. fastidiosa induziu uma maior variação na composição e diversidade microbiana da cultivar suscetível do que na resistente. Foi identificado um conjunto de fungos/bactérias associados à presença ou ausência de X. fastidiosa nos vasos xilémicos, sugerindo terem um papel importante no estabelecimento/desenvolvimento do patogénico. No geral, este estudo permitiu identificar um conjunto de microrganismos candidatos para o controlo de P. spumarius e X. fastidiosa, e cuja ação deverá ser analisada em trabalhos futuros. Palavras-chave: Xylella fastidiosa; Philaenus spumarius; endófitos; planta hospedeira; xilema; microbioma; controlo biológico.This work is funded by FEDER funds through COMPETE (Programa Operacional Fatores de Competitividade) and by national funds through the Foundation for Science and Technology - FCT - in the scope of the project PTDC/AGR-PRO/0856/2014 “XF-FREEOLIVE: Multifunctional study of xylem-sap of Portuguese olive cultivars and its relation with susceptibility to infection by Xylella fastidiosa”, and by European funds in the scope of the project H2020-SFS-2016-3 “XF-ACTORS - Xylella fastidiosa active containment through a multidisciplinary-oriented research strategy”

The effect of Philaenus spumarius feeding on the endophytic fungi community of Coleostephus myconis and its possible applicability in the insect biocontrol

Philaenus 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.info:eu-repo/semantics/publishedVersio

Diversity, Composition, and Specificity of the <i>Philaenus spumarius</i> Bacteriome

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

Preliminary analysis of the bacterial community in different phases of the olive pomace composting process

O 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.Olive pomace (OP) is a wet by-product generated in olive oil production, with a high organic load, and high toxicity due to its polyphenol composition. Its production can therefore be an environmental problem for olive oil-producing countries, such as Portugal. Composting may be a way to valorise this by-product, and the microorganisms are the main agents of this aerobic degradation process. The aim of the present study was to study the evolution of the bacterial community along the OP composting process and to isolate and identify bacteria with the purpose of exploring them in the future as composting inoculums. Composting was carried out in four industrial-scale piles with different proportions of OP, nitrogen source, structuring agent, and turnings. Compost samples were collected from the piles at three-time points: initial phase, mesophilic phase, and thermophilic phase. The bacterial isolates, obtained through serial dilutions, were identified by sequencing the 16S rRNA region. The results of composition and diversity, showed significant differences among the composting phases and among piles, suggesting a continuous change of bacterial species throughout the different phases of the OP composting process.Os autores agradecem o suporte do projeto “BIOMA - Soluções integradas de BIOeconomia para a Mobilização cadeia Agroalimentar” (POCI-01-0247-FEDER-046112)info:eu-repo/semantics/publishedVersio

Exploitation of Coleostephus myconis-associated endophytic fungi repel or attract Xylella fastidiosa vector

Xylella 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.info:eu-repo/semantics/publishedVersio

Microrganismos aerotransportados e material particulado em ambiente hospitalar

A qualidade do ar interior em ambiente hospitalar é um fator essencial no controlo de infeções nosocomiais. Este estudo teve por objetivo determinar as concentrações de microrganismos e material particulado (PM) em suspensão no ar interior de um Bloco operatório e identificar, por métodos moleculares, os microrganismos presentes. Utilizou-se a técnica de impacto em meio sólido para determinar as concentrações de bactérias e fungos e paralelamente utilizou-se um espectrómetro portátil para medir as partículas em suspensão. As menores concentrações de bactérias e de fungos foram observadas nas salas de cirurgia e as maiores na antecâmara. Os géneros bacterianos com maior prevalência foram Staphylococcus, Micrococcus e Bacillus e os fúngicos foram Penicillium, Cladosporium, Rhodotorula e Aspergillus. Verificou-se uma correlação forte (r ≥ 0,9) entre as concentrações médias de bactérias, fungos e a concentração de material particulado, expressa em número de partículas por centímetro cúbico, demonstrando que o material particulado pode ser usado de forma indireta como indicador de qualidade do ar interior, uma vez que estes parecem estar associados aos microrganismos aerotransportados. A ausência de ventilação mecânica na antecâmara demonstrou ter importância na acumulação de poluentes. Compreender as condições do ambiente avaliado é estratégia fundamental na aplicação de medidas de controlo.Os autores agradecem à Fundação para a Ciência e Tecnologia (FCT, Portygal) e FEDER ao abrigo do Programa PT2020 pelo apoio financeiro ao CIMO [UID/AGR/00690/2019]. Os autores agradecem igualmente à equipa médica, de enfermagem e funcionários do hospital pela cooperação neste estudoinfo:eu-repo/semantics/publishedVersio