Spatial and temporal analysis of microbial community in Pine Wilt Disease (MicroNema)

This proposal aims to integrate advanced technologies for the study of bacterial communities in the elucidation of biological

interactions related to pine wilt disease (PWD), a problem of great economic importance in Portugal, with a major impact on forest

management. The approach is innovative, brings together a team with complementary skills and scientific outcomes are expected to

be useful for the identification of new lines of attack to the disease.

Pine wilt disease is perceived as a complex biological interaction between the pine wood nematode (PWN) (Bursaphelenchus

xylophilus), an insect-vector (Monochamus galloprovinciallis ) and the susceptible host. Since the 1980s, some Japanese and mainly

Chinese researchers have claimed that bacteria associated to the nematode may have a significant role of in the development of the

disease. In fact, bacteria are good candidates as helper organisms in the invasion of the plant tissues by nematodes, a process that

may involve degradation of cell walls; constituted mainly of cellulose, hemicelluloses and pectin, cell walls are good substrates for a

panoply of bacterial lytic enzymes. Some authors have pointed for the possibility of a symbiotic association between nematodes and

specific groups of bacteria. From another point of view, bacteria are also good candidates for the control of nematode proliferation.

The MicroNema proposal focuses on the elucidation of structure, composition and dynamics of the microbial community in the Pine

Wilt Disease complex. For that, three distinct bio-geographical locations, considered as severe foci of the disease in Portugal by

Portuguese Forestry Authorities, will be under study. Sampling is designed in order to allow complete analysis of bacterial

communities in the propagative stage of PWN and interaction with host pine and insect-vector; symptomatic pine trees will be

randomly selected and marked for continuous study. The bacterial communities will be assessed by two different and complementary

approaches: culturable methods and metagenomic analysis using non-culturable methods with input of data obtained by massive

sequencing of relevant samples. The culturable bacterial fraction will constitute a collection of isolates that will be characterized and

identified by phenotypic and molecular methods.

The team joins researchers from two Portuguese universities, Aveiro and Evora: the research group from Evora has long experience in

studies of pine wilt disease; its leader, Manuel Mota was responsible for the detection of the disease in Portugal, in 1999. The team

from Aveiro works in microbiology, with a strong emphasis in environmental microbiology, and assessment of microbial diversity by

culturable and non-culturable methods.

It is expected that MicroNema will obtain solid information on which bacteria are associated to each point of the life cycle and whether

there are preferable associations between bacterial phylogenetic groups and the nematode. The relevance and the role of the insect

vector on the establishment of those associations will also be elucidated.

Considering the economic negative impact of the disease, we expect to provide a solid contribution to the establishment of new lines

of action against the nematode.