|1086-1||Endophytic Microbial Community in Pine Trees in Areas Subject to Pine Wilt Disease - Complexity of Bacteria and Archaea|
|Autores:||Diogo Neves Proença (IMAR-CMA - Instituto do Mar) ; Romeu Francisco (IMAR-CMA - Instituto do Mar) ; Gabriel Paiva (IMAR-CMA - Instituto do Mar) ; Susana S. Santos (IMAR-CMA - Instituto do Mar) ; Isabel M.o. Abrantes (IMAR-CMA - Instituto do Mar / DCV - Departamento de Ciências da Vida) ; Paula V. Morais (IMAR-CMA - Instituto do Mar / DCV - Departamento de Ciências da Vida) |
Pine wilt disease (PWD), native to North-America, has spread into Asia and recently into Europe. The first report in Portugal was in 1999 but in 2008 spread to the Center-North of the country. Bursaphelenchus xylophilus, the pinewood nematode (PWN), has been thought to be the only causal agent of PWD. However, bacteria have been reported as play a role on PWD. This study aimed to characterize the endophytic microbial community structure from infected and non-infected pine trees, Pinus pinaster, based on culture isolates and molecular profiling. Pine trees samples from Arganil and Oliveira do Hospital, consisted of pinewood cross-sections or wood obtained by drilling under aseptic conditions. All samples were screened for nematodes. The endophytic microbial community was studied by cultivable (R2A at 25ºC) and molecular methods (Denaturing gradient gel electrophoresis - DGGE). 293 endophytic strains (representing all RAPD groups) were identified by 16S rRNA gene sequencing. The classes Actinobacteria, Alphaproteobacteria, Bacilli, Betaproteobacteria, Flavobacteria, Gammaproteobacteria and Sphingobacteria were found in both sampling areas. Flavobacteria was only found in Arganil. The relative percentage of each class in the endophytic community was different according to sampling area. The most abundant bacteria were identified as belonging to Gammaproteobacteria in both sampling areas (34% in Arganil and 59% in Oliveira do Hospital). In Oliveira do Hospital, the Gammaproteobacteria included mostly strains from the families Xanthomonadaceae and Enterobactereaceae. DGGE profiles were not able to produce a specific pattern for PWD. From DGGE gels, 85 bands were cloned and sequenced. Compared to the cultivable methods, DGGE detected the presence of endophytes belonging to 4 additional classes: Acidobacteria, Bacteroidia, Deinococci and Spirochaetes. Furthermore, Archaea were found as part of the endophytic community (DGGE) and were identified as belonging to "Methanomicrobia", Thermococci, Thermoprotei or Crenarchaeota. This is the first report of Archaea as endophytes in trees. The presence of Bacteria and Archaea as part of endophytic community in P. pinaster could potentiate the ability to adapt to changing in the habitat.
Palavras-chave: Archaea, Endophytic Microbial diversity, DGGE, Pine wilt disease