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

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

Objetivos

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 examination 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%).

Atividades

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;

 

Resultados

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.