Projetos em Curso

Cofinanciado por:
Acrónimo | PineEnemy
Designação do projecto | Estudo das interacções nemátode-fungos na doença da murchidão do pinheiro
Código do projecto | ALT20-03-0145-FEDER-028724
Objetivo principal | Reforçar a Investigação, o desenvolvimento tecnológico e a inovação

Região de intervenção | Alentejo, Lisboa

Entidade beneficiária | INIAV - Instituto Nacional de Investigação Agrária e Veterinária(líder), Universidade de Évora(parceiro)

Data de aprovação | 12-07-2018
Data de inicio | 15-10-2018
Data de conclusão | 14-10-2021
Data de prorrogação | 14-10-2022

Custo total elegível | 230234 €
Apoio financeiro da União Europeia | FEDER - 104637 €
Apoio financeiro público nacional/regional | República Portuguesa - 125596 €

Objetivos, atividades e resultados esperados/atingidos


PineEnemy will study the fungal community occurring in pine trees infected by B. xylophilus in order to elucidate the importance of this Kingdom in the biological interactions occurring in the pine wilt disease complex. All PWD components (PWN, insect-vector and host tree) can harbour a range of microorganisms, changing both temporally and spatially. The work in PineEnemy will characterize the mycobiota present in the different phases of the PWN life-cycle, insect-vector and pine trees using two complementary approaches: culture methods and the emerging technique of barcoded pyrosequencing of the ITS rDNA region combined with the Illumina MiSeq platform (Schmidt et al. 2013), hence providing a robust analysis of the fungal community present in all phases of PWD progress. Three bio-geographical locations, considered severe foci for the disease in Portugal by Portuguese Forestry Authorities, will be used as field sites: the Setúbal Peninsula (Centre/South) (S1), Gois (Centre/North) (S2) and Madeira Island (S3). These locations have been chosen to complement ongoing research on the bacterial communities associated with PWN, selected in the project FCT-MICRONEMA.

 Task 1 (T1) comprises sample collection from the three PWD sites in the first year of the project. At least 15 symptomatic pine trees will be selected along with the same number of healthy trees of the same age, chosen at random, at each site. Trees will be felled and cut into stem discs for analysis. The sampling protocol will allow complete analysis of the mycoflora of both PWN affected pines and healthy trees. Sampling will begin at the beginning of 2018 and continue until April of the same year. At the same time, pinewood nematodes will be extracted from wood samples using the Baerman funnel technique and, after surface sterilisation, sliced in two longitudinally to release the gut contents. These contents will be maintained in suspension, in PCR tubes with a drop of water for immediate processing or stored at -20°C. Following T1, samples will be analysed by the two different and complementary approaches: metagenomic analysis (non-culturable methods) and culture-dependent methods.

In Task 2 (T2), the large-scale sequencing will provide an extremely powerful tool to determine the true level of phylogenetic diversity in the fungal communities associated with the pinewood nematode. Total genomic DNA will be extracted directly from pine infected and healthy wood tissues (used as a negative control), and from the intestinal contents obtained from the nematodes. High throughput metabarcoding using fungal barcode marker ITS rDNA, combined with an Illumina MiSeq platform, will be particularly useful for the analysis of high numbers of samples. Pyrosequencing using these markers provides an exhaustive characterization of the mycological community in the samples and allows the less-abundant taxa to be determined through large scale sequencing, hence enabling analysis and comparison of the changes in the mycological diversity of pines affected by the pinewood nematode. In the same way, the fungal community used as nutrition by the nematode will be characterized.

In Task 3 (T3), the culturable fungi, paying particular attention to putative blue-stain fungi (Ophiostomatales), will be obtained from each sample (both wood and the nematode gut) in selective artificial media, to provide a collection of isolates for full characterization and identification using phenotypic and molecular methods. Pure cultures of each fungus will be obtained and comprehensively characterized and identified based on i) cultural and morphological features and growth studies on different media; ii) mating studies ? the isolates will be crossed, based on the production of anamorph structures and geographical location of isolation; the cultures will be examined regularly for the presence of ascomata; iii) extraction of DNA from fungal isolates, followed by PCR and DNA sequencing for species identification using a multigene approach employing ribosomal and protein-coding genetic markers. Four markers genes will be amplified by PCR for sequencing and phylogenetic analysis: the ITS2 and partial large subunit (LSU) of the rDNA, the actin gene, the ?-tubulin gene and translation elongation factor-1-alpha (TEF-1?).

Task 4 (T4) is focused on inoculations of Pinus pinaster seedlings and other pine tissues with the blue-stain species identified in T3, both with and without simultaneous PWN inoculations in order to determine which fungi are important in nematode development in the various life cycle stages. Over a period of 2 months, wilting symptoms will be monitored for each treatment; nematodes will be extracted from the inoculated plants and enumerated. Correlations between wilting symptoms, fungal presence and nematode development will be made. Temporal developmental stages of Bursephalenchus xylophilus will be examined, including assessment of life cycle stages and numbers of individuals produced on pine branch segments inoculated with the different isolates of blue-stain fungi under laboratory conditions.

To investigate the putative transport of fungi by the insect-vector of PWN, Monochamus galloprovincialis, in Task 5 (T5), beetles will be obtained from the network of multi-funnel traps lured with Galloprotect 2D-Plus, the most efficient commercially available pheromone for this species, already installed in each sampling site. Traps will be placed on site at the beginning of June 2016 and left until the end of September 2016. Traps will be visited weekly to collect the insects that are caught. Beetles will be subject to xamination using the methods described in in T2 and T3: their external mycobiota will be recovered by washing and plating techniques, with culturing. Morphological as well as NGS assessment of Monochamus borne fungal diversity by direct amplification of ITS sequences from the washings of the insects will also be performed. PineEnemy, therefore, will provide robust information on the species of fungi associated with each element of the pine wilt disease complex and moreover, will determine whether there are preferred associations between specific fungal phylogenetic groups and PWN. The project brings together Portuguese researchers from INIAV and University of Évora (ICAAM), and a research scientist experienced in next generation sequencing and bioinformatics from the University of Tuscia IT and a forest pathologist of worldwide reputation (Univ Aberdeen UK) as consultants. 

The INIAV team is composed of entomologists (E Sousa and P Naves) and a mycologist (H Bragança) and a Superior Technician (A Fontes). F Nóbrega is expert in molecular identification of fungi and nematodes. The PI (ML Inacio) has long experience in interactions between biotic agents, particularly in the assessment of fungal diversity in diverse matrices (wood, insects, and nematodes) by culturable methods, with vast know-how and skills in project development and team coordination. In addition, she has academic qualifications in both mycology and nematology, giving her the experience required to supervise the interdisciplinary tasks and ensure their timely completion, through promotion of communication between the different partners. She is also highly experienced in the dissemination of knowledge through reports, publications in SCI journals and conferences organization. ML Inacio is fully committed to the work in this project as a PI (45%).


T1 - Wood sampling and nematode collection

T2 - Identification of PWD-associated fungal communities through metagenomic analysis

T3 - Characterization of candidate fungi (Ophiostomatales) for PWN feeding

T4 - Testing the effect of PWN specific associated fungi on the nematode life-cycle

T5 - Investigation of the putative transport of blue-stain fungi by the insect-vector Monochamus galloprovincialis

T6 - Scientific activity spreading actions


- Ações de divulgação de cultura científica;

- Ações Promoção e disseminação do conhecimento;

- Publicações técnicas/científicas;

- Conferências, seminários ou fóruns;

- Ações junto dos sectores alvo;


As a preliminary result, the insect vector of the PWN was found to transport fungi putatively involved in the nematode-insect attraction thus facilitating disease dispersal. This result and further doubts, coupled with the lack of research in this area, both in national and international level, led to the build up of the present project PineEnemy. This preliminary study revealed a vast mycobiota associated with the pine wilt disease complex, namely with the insect vector (Inácio et al. 2015). This result further pointed out the interest of studying the mycobiota associated with the nematode and the affected host trees. A very interesting project both in what concerns the different tasks as the connections with other reseachers coming from different countries and deeply concerned about PWN spreading all over Europe. Survey of PWN procedures and exceptional skills for the study of this quarantine organism.

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