Project Identification

Project Objectives

Abstract

Outputs

 

Project Identification

Project title:   PlantBioNem - Plant biotechnology in phytonematicidals screening

Project Reference:  PTDC/AGR-CFL/117026/2010

Principal Investigator (PI):  Ana Cristina Silva Figueiredo

Principal Contractor:  Fundação da Faculdade de Ciências da Universidade de Lisboa (FFC/FC/UL)

Partner Institution: Universidade de Évora (NemaLab, ICAAM)

Start date:  02-01-2012     End Date:  01-04-2015

 

Research team

Name

Role

Tasks

Time (%)

Start date

End date

Ana Cristina Silva Figueiredo

Principal investigator

1 to 11

55

02-01-2012

01-04-2015

Jorge Miguel Silva Faria

Researcher

1 to 11

100

02-01-2012

01-04-2015

José Manuel Gonçalves Barroso

Researcher

1-3, 5,6, 9-11

15

02-01-2012

01-04-2015

Lia Maria P. Ascensão S Sousa

Researcher

7, 9-11

15

02-01-2012

01-04-2015

Luís Manuel Gaspar Pedro

Researcher

1-3, 5,6, 9-11

15

02-01-2012

01-04-2015

Manuel Galvão de Melo e Mota

Researcher

4,5, 9-11

15

02-01-2012

01-04-2015

Maria Helena M. Trindade De Donato

Researcher

1, 2, 8-11

15

02-01-2012

01-04-2015

Pedro Miguel de Sousa Barbosa

Researcher

4,5, 9-11

15

02-01-2012

14-09-2012

Inês C. dos S. Carvalho de Sena

Fellowship

1 to 11

100

01-03-2012

28-02-2015

Bruno Emanuel Duarte Ribeiro

Fellowship

1 to 11

100

05-01-2013

01-01-2015

 

 

Project Objectives

Supported on the knowledge previously acquired by the research team and on the pine and potato in vitro cultures already established in the lab, the innovative nature and main goals of this study were to:

1  Establish stable co-culture systems of Pinus spp. with Bursaphelenchus xylophilus and Solanum tuberosum with Meloidogyne chitwoodi , from previously established Pinus spp. shoot cultures and potato hairy roots,

2  Make a real-time assessment of the nematode phytoparasitic infection and proliferation mechanisms in the co-cultures before and after phytonematicidal addition, through culture growth measurement, metabolomic time-course studies and anatomic and cellular observations,

3  Evaluate the response of the host plant to the added phytonematicidals for toxicity and plant biotransformation capacity,

4  Identify phytochemicals or specific compounds, which effectively diminish nematode growth and development in biotechnological model co-cultures that simulate the host-parasite environment,

5  Identify different levels of gene expression following infection and phytonematicidal application using Reverse transcriptase, through RT-PCR techniques, with special emphasis to cDNAs coding for enzymes involved in terpene metabolism (ex. Conifer limonene synthase, among others) for pine and protease-inhibitors for potato, and

6  Establish novel model co-cultures for the screening of phytochemicals to be used in pest control of phytoparasitic nematodes.

 

Abstract

Phytoparasitic nematodes are, nowadays, an alarming, difficult to control, threat to national agriculture and forestry industries. Their life cycle and infection mechanism seems to be dependant on volatile cues, and several phytochemicals have been shown to affect their growth and development.

Bursaphelenchus xylophilus (Pinewood Nematode, PWN) along with the members of the genus Globodera (Potato-Cyst Nematodes), Heterodera (Soybean-Cyst Nematodes) and Meloidogyne (Root-Knot Nematodes) and are among the most damaging, and economically devastating, phytoparasites, to agriculture and forestry. Pest control is often a complex process, which relies, generally, on traps and/or pesticides targeted to the insect vector or on synthetic nematicides that are unspecific, expensive and harmful to the environment. In this context, there is an urgent need to find solutions that are safer for the environment and public health, while less costly.

The approaches commonly used to study nematode-caused diseases seldom evaluate the real time invasive mechanism. Additionally, the determination of potential phytonematicidal compounds relies mostly on phytonematicide-nematode direct-contact assays, that do not take into account the plant response nor do they assess the toxicity to the plant.

Plant in vitro culture is a useful experimental system that can be controlled in terms of nutrition and environmental conditions, providing additional advantages, to conventional systems, in the evaluation of phytochemicals as nematicidals. Additionally, it is an ideal system to study biotransformation based decontamination mechanisms, as well as to investigate the plant-nematode relationships in loco without the need of field assays.

In an attempt to clarify the invasive mechanism of different types of nematodes, and to identify the plant response to nematode and to potential nematicides, viewing at obtaining alternative strategies for nematode control, the current project proposes the use of in vitro plant-nematode co-cultures as testing models that simulate the in vivo infection conditions.

Particularly, and using

- two types of nematodes that adversely affect the national economy, B. xylophilus , responsible for pine wilt disease (PWD), by affecting the aerial parts of plants, and nematodes of the genus Meloidogyne , causative agents of the root galls that destroy the root system,

and

in vitro cultures of micropropagated Pinus spp. and Solanum tuberosum hairy roots already established in the course of the PhD work of one of the team members,

We proposed:

To use two types of in vitro co-culture model systems: co-culture of micropropagated Pinus spp. with B. xylophilus and Solanum tuberosum hairy roots with M. chitwoodi , in order to:

a) characterize the volatile metabolomic profile resulting from nematode infection and proliferation,

b) follow the morpho-anatomical mechanisms of infection,

c) elucidate the host and parasite responses to phytonematicide application, and

d) evaluate the changes in gene expression as a result of nematode invasion and of phytonematicide application.

To successfully carry out the research work, the involved research units will join their skills in the areas related to this study. The main research unit, Instituto de Biotecnologia e Bioengenharia, Centro de Biotecnologia Vegetal, Faculdade de Ciências da Universidade de Lisboa (CBV/FCUL), has a solid knowledge and know-how in the areas of phytochemistry, plant in vitro culture, morphological and anatomical characterization and molecular biology. These abilities coupled with the long experience of the University of Évora (UE), in nematology and assessment of nematicidal activity against B. xylophilus . The main research unit relies on other institutions, as consultants, for certain stages / phases of the research work, namely, the IMAR, Departamento de Ciências da Vida, Universidade de Coimbra (Prof. Dr Isabel Abrantes ) for her scientific knowledge on the genus Meloidogyne , and in whose lab the PhD student already performed a training period on the specific growth conditions of this nematode. The research team will also have the support of Laboratório de Biotecnologia Florestal of Instituto de Tecnologia Química e Biológica (Grupo Pinus/ITQB) (Dra Célia Miguel) currently involved in the transformation of embryogenic Pinus cultures and studying the role of genes potentially involved in response to the nematode.

The results will complement a completed project (PTDC/AGR-AAM/74579/2006) in partnership with UTAD an UE, and will allow the successful conclusion of the PhD of one of the team researchers. Taking advantage of the knowledge acquired to date, the expected results will add more knowledge both to scientific community as well as to the public and will follow the national effort to develop strategies, and implement environmentally friendly control measures for the control of damaging phytoparasitic nematodes.

 

Outputs

Book

[1] Figueiredo A. C., J. G. Barroso, L. G. Pedro (Eds) (2012) 43rd International Symposium on Essential Oils: Program, Book of Abstracts and Participants List , Edição Centro de Biotecnologia Vegetal  FFCUL, Europress, Lisboa, Portugal (ISBN: 978-989-20-3188-0).

 

PhD Thesis

[1] Faria J. M. S. (2015) Essential oils as anti-nematode agents and their influence on in vitro nematode / plant co-cultures. Doutoramento em Biologia, especialidade de Biotecnologia. Universidade de Lisboa. 290 pp.

 

Papers in international scientific journals

[1] Barbosa P., J. M. S. Faria, M. D. Mendes, L. S. Dias, M. T. Tinoco, J. G. Barroso, L. G. Pedro, A. C. Figueiredo, M. Mota (2012) Bioassays against pinewood nematode: assessment of a suitable dilution agent and screening for bioactive essential oils. Molecules 17: 1312-1329.

www.mdpi.com/1420-3049/17/10/12312/pdf

[2] Faria J. M. S., P. Barbosa, R. N. Bennett, M. Mota, A. C. Figueiredo (2013) Bioactivity against Bursaphelenchus xylophilus : nematotoxics from essential oils, essential oils fractions and decoction waters. Phytochemistry 94: 220-228.

http://www.sciencedirect.com/science/article/pii/S003194221300232X

[3] Faria J. M. S., I. Sena, C. M. Maleita, I. Vieira da Silva, L. Ascensão, I. Abrantes, R. N. Bennett, M. Mota, A. C. Figueiredo (2014) In vitro co-culture of Solanum tuberosum hairy roots with Meloidogyne chitwoodi : structure, growth and production of volatiles. Plant Cell Tissue Organ Culture 118: 519530.

http://link.springer.com/article/10.1007%2Fs11240-014-0504-6

[4] Faria J. M. S., I. Sena, I. Vieira da Silva, B. Ribeiro, P. Barbosa, L. Ascensão, R. N. Bennett, M. Mota, A. C. Figueiredo (2015) In vitro co-cultures of Pinus pinaster with Bursaphelenchus xylophilus : a biotechnological approach to study Pine Wilt Disease. Planta (in press)

http://link.springer.com/article/10.1007/s00425-015-2257-9

[5] Faria J. M. S., I. Sena, B. Ribeiro, A. M. Rodrigues, C. M. N. Maleita, I. Abrantes, R. N. Bennett, M. Mota, A. C. Figueiredo (2015a) First report on Meloidogyne chitwoodi hatching inhibition activity of essential oils and essential oils fractions. J. Pest Science (in press)

http://link.springer.com/article/10.1007%2Fs10340-015-0664-0

 

 

Communications

Communications in international meetings

Oral presentations

[1] Figueiredo A. C. (2013) Plant tissue culture and secondary metabolite production . 8th International Symposium on In Vitro Culture and Horticultural Breeding , 2 a 7 de Junho, Coimbra, Portugal, p. 29.

[2] Mota M., P. Barbosa, A. M. Rodrigues, I. Silva, L. Ascensão, M. Nunes da Silva, A. C. Silva Ferreira, M. W. Vasconcelos, J. M. S. Faria, I. Sena, B. Ribeiro, J. G. Barroso, L. G. Pedro, H.Trindade, A. C. Figueiredo (2015) Characterization of the pinewood nematode, Bursaphelenchus xylophilus - Pinus system in Portugal: phytochemical, histopathological, molecular and biotechnological approaches. 47th Annual ONTA (Organization of Nematologists of Tropical America) Meeting , 17 a 22 de Maio, Varadero, Cuba.

 

Poster presentations

[1] Faria J. M. S., Barbosa P., M. Mota, A. C. Figueiredo (2012) Nematoxic effect of essential oils and their fractions against the pinewood nematode, Bursaphelenchus xylophilus . 43rd International Symposium on Essential Oils , 5-8 de Setembro, FCUL, Lisboa, Portugal, p. 72.

[2] Barbosa P., P. Vieira, L. S. Dias, L. G. Pedro, J. G. Barroso, A. C. Figueiredo, M. Mota (2012) Control of the pinewood nematode Bursaphelenchus xylophilus by essential oils and extracts obtained from plants: a review . 43rd International Symposium on Essential Oils , 5-8 de Setembro, FCUL, Lisboa, Portugal, p. 183.

[3] Sena I., J. M. S. Faria, J. Sanches, H. Trindade, L. G. Pedro, J. G. Barroso, A. C. Figueiredo (2012) Essential oil composition from twenty six Eucalyptus taxa from Mata Experimental do Escaroupim (Portugal). 43rd International Symposium on Essential Oils , 5-8 de Setembro, FCUL, Lisboa, Portugal, p. 216.

[4] Faria J. M., I. Sena, C. M. Maleita, I. Abrantes, M. Mota, A. C. Figueiredo (2013) Solanum tuberosum hairy roots /  Meloidogyne chitwoodi co-cultures: growth and volatiles production. 8th International Symposium on In Vitro Culture and Horticultural Breeding , 2 a 7 de Junho, Coimbra, Portugal, p. 222.

[5] Faria J. M. S., P. M. Barbosa, I. Sena, B. Ribeiro, J. G. Barroso, L. G. Pedro, L. Ascensão, H. Trindade, M. Mota, A. C. Figueiredo (2013) Plant in vitro cultures as model systems for evaluating phytoparasitic nematodes invasion mechanism and phytonematicidals screening. XIII Congresso Luso-Espanhol de Fisiologia Vegetal , 24 a 28 de Julho, FCUL Lisboa, Portugal, p. 196.

[6] Rodrigues A. M., P. M. Barbosa, B. Ribeiro, A. C. Figueiredo, M. Mota, L. Ascensão (2013) Secretory structures, essential oil composition and its nematotoxic effects, from the Portuguese endemic Asteraceae species, Santolina impressa . SPMicros Congress ( XLVII Congress of the Portuguese Society for Microscopy ), 9 a 10 de Dezembro, Monte da Caparica, Portugal, p. LSP25.

[7] Ribeiro B., A. M. Rodrigues, J. Faria, I. Sena, J. G. Barroso, L. G. Pedro, M. Mota, A. C. Figueiredo (2014) Nematotoxic activity of essential oils against the Columbia root-knot nematode, Meloidogyne chitwoodi . SEB Manchester 2014 (Society for Experimental Biology), 1 a 4 de Julho, Manchester, UK, pp. 148.

[8] Faria J. M. S., I. Sena, C. M. Maleita, I. Vieira da Silva, L. Ascensão, I. Abrantes, R. N. Bennett, M. Mota, A. C. Figueiredo (2014) Characterization of structure, growth and production of volatiles of Solanum tuberosum hairy roots and Solanum tuberosum hairy roots/Meloidogyne chitwoodi in vitro co-cultures. 62nd International Congress and Annual Meeting of the Society for Medicinal Plant and Natural Product Research (62nd GA) , 31 de Agosto a 4 de Setembro, Braga, Portugal, p. 466-467.

[9] Faria J. M. S., I. Sena, C. M. N. Maleita, I. Abrantes, R. Bennett, M. Mota, A. C. Figueiredo (2014) Essential oils and essential oil fractions hatching inhibition activity against Meloidogyne chitwoodi nematode. 45th International Symposium of Essential Oils (ISEO 2014), 7 a 10 de Setembro, Istambul, Turquia, p. 67.

[10] Barbosa P., A. M. Rodrigues, I. Vieira da Silva, J. M. S. Faria, I. Sena, B. Ribeiro, H. Trindade, L. Ascensão, L. G. Pedro, J. G. Barroso, A. C. Figueiredo, M. Mota (2015) Pine Wilt Disease: evaluation of parasite control and parasite with host interactions. 67th International Symposium on Crop Protection (67th ISCP) , 19 Maio, Ghent, Bélgica.

[11] Figueiredo A. C., J. M. S. Faria, I. Sena, B. Ribeiro, H. Trindade, L. Ascensão, L. G. Pedro, J. G. Barroso, M. Mota (2015) Plant biotechnology in phytonematicidals screening (PlantBioNem) . 46th International Symposium of Essential Oils (ISEO 2015) , 13 a 1 6 de Setembro , Lublin , Poland .

[12] Sena I., H. Trindade, A. C. Figueiredo (2015) a-Pinene synthase gene expression following nematode invasion in Pinus pinaster and P. pinea with Bursaphelenchus xylophilus in vitro co-cultures . 46th International Symposium of Essential Oils (ISEO 2015) , 13 a 1 6 de Setembro , Lublin , Poland .

 

Communications in national meetings

Oral presentations

[1] Faria J. M. S., A. C. Figueiredo (2012) Effect of Bursaphelenchus xylophilus inoculation on Pinus pinaster volatiles: juvenile trees versus in vitro cultures, 3rd Scientific Meeting of the Institute for Biotechnology and Bioengineering , 16-17 Março, IST, Lisboa, Portugal, p. 65.

[2] Mota M., P. Barbosa, J. M. S. Faria, I. Sena, B. Ribeiro, A. M. Rodrigues, I. Silva, L. Mota, J. G. Barroso, L. G. Pedro, L. Ascensão, H. Trindade, A. C. Figueiredo (2013) Breve historial da investigação efectuada no NemaLab (ICAAM) e CBV / FCUL (IBB) sobre o Nemátode da Madeira do Pinheiro (NMP). Mini-Workshop sobre o estado actual do conhecimento em Portugal sobre o Nemátode da Madeira do Pinheiro (NMP), Integrado no âmbito da unidade curricular de Agricultura e Florestas, do Mestrado em Ecologia e Gestão Ambiental Departamento de Biologia Vegetal, Faculdade de Ciências da Universidade de Lisboa, 6 de Maio de 2013, Lisboa, Portugal.

[3] Faria J. M. S., I. Sena, P. Barbosa, B. Ribeiro, J. G. Barroso, L. G. Pedro, L. Ascensão, H. Trindade, M. Mota, A. C. Figueiredo (2013) Óleos essenciais como agentes anti-nemátode e a sua influência em co-culturas in vitro nemátode / planta. Mini-Workshop sobre o estado actual do conhecimento em Portugal sobre o Nemátode da Madeira do Pinheiro (NMP), Integrado no âmbito da unidade curricular de Agricultura e Florestas, do Mestrado em Ecologia e Gestão Ambiental Departamento de Biologia Vegetal, Faculdade de Ciências da Universidade de Lisboa, 6 de Maio de 2013, Lisboa, Portugal.

[4] Barbosa P., A. M. Rodrigues, J. M. S. Faria, M. D. Mendes, A. S. Lima, L. S. Dias, P. Vieira, M. T. Tinoco, R. N. Bennett, L. G. Pedro, J. G. Barroso, A. C. Figueiredo. M. Mota (2014) Plant volatile compounds to control the pinewood nematode in Portugal. 1º Simpósio SCAP (Socieadade de Ciências Agrárias de Portugal) - Novos desafios na proteção das plantas e 7º Congresso da SPF (Sociedade Portuguesa de Fitopatologia), 20 e 21 de Novembro de 2014, Oeiras, Portugal, p. 34.

 

Poster presentations

[1] Faria J. M. S., I. Sena, P. M. Barbosa, B. Ribeiro, J. G. Barroso, L. G. Pedro, L. Ascensão, H. Trindade, M. Mota, A. C. Figueiredo (2013) PlantBioNem A biotecnologia vegetal na avaliação de fitonematicidas para controlo do nemátode da madeira do pinheiro, Bursaphelenchus xylophilus . 7º Congresso Florestal Nacional , Florestas - Conhecimento e Inovação , 5 a 8 de Junho de 2013, Vila Real, Bragança, Portugal, p. 192.