PhD highlights: Diogo Proença

Role of Endophytic Microbial Community in Pine Wilt Disease.

Pine wilt disease (PWD) is native to North America and has spread to Asia and Europe. Mutualistic relationship was suggested between Bursaphelenchus xylophilus, the pinewood nematode (PWN) – the causal nematode agent of PWD, and bacteria. In this work, the microbial communities within the host-plant and carried by PWN, were assessed in order to elucidate the structure and to understand their functional roles.

The endophytic microbial community structure of Pinus pinaster in Portugal was assessed based on cultivation methods and molecular profiling (DGGE). Eight classes were identified in bacterial isolates and additionally six by DGGE.Gammaproteobacteria was the most abundant. DGGE profiles of the microbial endophytic community did not produce a common pattern for PWD indicating that the communities were diverse and variable within diseased pine trees. Furthermore,Archaea was found as part of the endophytic community (DGGE). The presence of nifH,nifJ, nirS and nirK genes in this community supports the possibility of N2 fixation inside the trees and also denitrification, with the production of the metabolic relevant intermediary NO.

The diversity of PWN-associated bacteria in Portugal showed that most isolates belonged to the genus Pseudomonas, Burkholderia or to the family Enterobacteriaceae. The strains were able to produce siderophores, proteases and lipases. In the USA, bacteria carried by PWN mainly belonged to the class Gammaproteobacteria. Strains from the genus Pseudomonas were the only carried by nematodes from all countries, but none of the bacterial species was found in all different countries.

Moreover, 21 Portuguese strains carried by PWN showed capacity to produce nematicidal extracellular products. Genome sequencing of two nematicidal bacterial strains, Serratia sp. M24T3 and Pseudomonas sp. M47T1, showed genes that might be involved in nematotoxicity and in plant-growth promotion. A third strain, Serratia sp. A88copa13 produced a serine protease (70 kDa) identified as the major factor responsible for the toxicity against PWN.

Serratia sp. M24T3 was able to colonize Arabidopsis thaliana and showed potential to be PGPB.

All in all, considering the diversity of the nematode-associated bacteria and their physiological characteristics, we may suggest that they probably play a role in plant physiological response to external factors and we cannot suggest an active role of the bacteria in PWD.


Acknowledgments: This research was partially supported by Direcção Regional de Florestas, Fundo Florestal Permanente and Autoridade Florestal Nacional, through a national project ‘O nemátode-da-madeira-do-pinheiro (NMP), Bursaphelenchus xylophilus’, by FEDER funds through the Programa Operacional Factores de Competitividade – COMPETE and by national funds through the Fundação para a Ciência e a Tecnologia (FCT), Portugal, under the project PTDC/AGR-CFL/115373/2009. D.N.P. was supported by FCT, graduate fellowship SFRH/BD/61311/2009.


Proença D. N., Nobre M. F. & Morais P. V. 2014. Chitinophaga costaii sp. nov., an endophyte of Pinus pinaster, and emended description of Chitinophaga niabensis. Int. J. Syst. Evol. Microbiol. 64: 1237–1243.

Paiva G., Proença D. N., Francisco R., Veríssimo P., Santos S. S., Fonseca L., Abrantes I. M. O. & Morais P. V. 2013. Nematicidal bacteria associated to pinewood nematode produce extracellular proteases. PLoS One 8: e79705. (These authors contributing equally to this work).

Proença D. N., Espírito Santo C., Grass G. & Morais P. V. 2012. Draft genome sequence of Pseudomonas sp. strain M47T1, carried by Bursaphelenchus xylophilusisolated from Pinus pinaster. J. Bacteriol. 194: 4789–4790.

Proença D. N., Espírito Santo C., Grass G. & Morais P. V. 2012. Draft genome sequence of Serratia sp. strain M24T3, isolated from pinewood disease nematodeBursaphelenchus xylophilus. J. Bacteriol. 194: 3764.

Proença D. N., Francisco R., Santos C. V., Lopes A., Fonseca L., Abrantes I. M. O. & Morais P. V. 2010. Diversity of bacteria associated with Bursaphelenchus xylophilusand other nematodes isolated from Pinus pinaster trees with pine wilt disease. PLoS One 5: e15191.

PhD Degree: Biochemistry, specialty in Microbiology

Year of conclusion: 2014

Institution: Institute of Marine Research (IMAR-CMA), University of Coimbra

SupervisorPaula Vasconcelos Morais

Co-Supervisor: Gregor Grass

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